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Genetic improvementofprocessesyieldingmicrobial productsJose L. Adrio1 & Arnold L. Demain2
1Department of Biotechnology, Puleva Biotech, S.A., Granada, Spain; and 2Charles A. Dana Institute for Scientists Emeriti, Drew University, Madison, NJ, USA
Correspondence: Jose L. Adrio, Department
of Biotechnology, Puleva Biotech, S.A.,
Camino de Purchil, 66, 18004 Granada,
Spain. Tel.:134 958 24 02 27; fax:134 958
24 01 60; e-mail: [email protected]
Received 3 March 2005; revised 18 August
2005; accepted 19 August 2005
First published online 17 October 2005.
doi:10.1111/j.1574-6976.2005.00009.x
Editor: Alexander Boronin
Keywords
strain improvement; genetic recombination;
primary metabolites; secondary metabolites;
directed evolution; combinatorial biosynthesis.
Abstract
Although microorganisms are extremely good in presenting us with an amazing
array of valuable products, they usually produce them only in amounts that they
need for their own benefit; thus, they tend not to overproduce their metabolites. In
strain improvement programs, a strain producing a high titer is usually the desired
goal. Genetics has had a long history of contributing to the production of
microbial products. The tremendous increases in fermentation productivity and
the resulting decreases in costs have come about mainly by mutagenesis and
screening/selection for higher producing microbial strains and the application of
recombinant DNA technology.
Introduction
Microorganisms can generate new genetic characters (‘geno-
types’) by two means: mutation and genetic recombination. In
mutation, a gene is modified either unintentially (‘sponta-
neous mutation’) or intentially (‘induced mutation’).
Although the change is usually detrimental and eliminated by
selection, some mutations are beneficial to the microorgan-
ism. Even if it is not beneficial to the organism, but beneficial
to humans, the mutation can be detected by screening and can
be preserved indefinitely. This is indeed what the fermentation
microbiologists did in the strain development programs that
led to the great expansion of the fermentation industry in the
second half of the twentieth century.
It was fortunate that the intensive development of micro-
bial genetics began in the 1940s when the fermentative
production of penicillin became an international necessity.
The early studies in basic genetics concentrated on the
production of mutants and their properties. The ease with
which ‘permanent’ characteristics of microorganisms could be
changed by mutation and the simplicity of the mutation
techniques had tremendous appeal to microbiologists. Thus
began the cooperative ‘strain-selection’ program among work-
ers at the U.S. Department of Agriculture Laboratories in
Peoria, the Carnegie Institution, Stanford University and the
University of Wisconsin, followed by the extensive individual
programs that still exist today in industrial laboratories
throughout the world. It is clear that mutation has been the
major factor involved in the hundred- to thousand-fold
increases obtained in production of microbial metabolites
and that the ability to modify genetically a microbial culture
to higher productivity has been the most important factor in
keeping the fermentation industry in its viable, healthy state.
Applicationsofmutation
Mutation has improved the productivity of industrial cul-
tures (Vinci & Byng, 1999; Parekh et al., 2000). It has also
been used to shift the proportion of metabolites produced in
a fermentation broth to a more favorable distribution,
elucidate the pathways of secondary metabolism, yield new
compounds, and for other functions. The most common
method used to obtain high yielding mutants is to treat a
population with a mutagenic agent until a certain ‘desired’
kill is obtained, plate out the survivors and test each
resulting colony or a randomly selected group of colonies
for product formation in flasks. The most useful mutagens
include nitrosoguanidine (NTG), 4-nitroquinolone-1-
oxide, methylmethane sulfonate (MMS), ethylmethane sul-
fonate (EMS), hydroxylamine (HA) and ultraviolet light
(UV). The optimum level of kill for increased production of
antibiotics is thought to be in the range 70–95% (Simpson &
FEMS Microbiol Rev 30 (2006) 187–214 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
Caten, 1979), although some industrial programs use much
higher levels, e.g. up to 99.99%. It is incorrect to condemn a
mutation and screening procedure because, on average, it
decreases production ability; indeed, this is the case for
successful mutagenesis. One should only be interested in the
frequency of improved mutants.
Although single cells or spores are preferred for mutagen-
esis, non-spore-forming filamentous organisms have been
mutated successfully by mutagenizing mycelia, preparing
protoplasts and regenerating on solid medium (Keller,
1983). Sonication is sometimes used to break up Strepto-
myces mycelia after mutagenesis and before screening for
improved mutants (Takebe et al., 1989). Poorly sporulating
filamentous organisms can be mutagenized after fragmenta-
tion or formation of protoplasts (Kim et al., 1983; Kurzat-
kowski et al., 1986).
More detailed information can be found in several authora-
tive reviews on genetics and especially on mutation in actino-
mycetes (Baltz, 1986, 1995, 1998, 1999; Hopwood, 1999).
Mutantsproducing increasedquantitesofmetabolites
Genetics has had a long history of contributing to the produc-
tion of microbial products. The tremendous increases in
fermentation productivity and the resulting decreases in costs
have come about mainly by mutagenesis and screening for
higher producing microbial strains. At least five different classes
of genes control metabolite production (Malik, 1979): (i)
structural genes coding for product synthases, (ii) regulatory
genes determining the onset and expression of structural genes,
(iii) resistance genes determining the resistance of the producer
to its own antibiotic, (iv) permeability genes regulating entry,
exclusion and excretion of the product, and (v) regulatory
genes controlling pathways providing precursors and cofactors.
Overproduction of microbial metabolites is effected by (i)
increasing precursor pools, (ii) adding, modifying or deleting
regulatory genes, (iii) altering promoter, terminator and/or
regulatory sequences, (iv) increasing copy number of genes
encoding enzymes catalyzing bottleneck reactions, and (v)
removing competing unnecessary pathways (Strohl, 2001).
It is now over 60 years since the first superior penicillin-
producing mutant, Penicillium chrysogenum X-1612, was
isolated afer X-ray mutagenesis. This heralded the beginning
of a long and successful relationship between mutational
genetics and industrial microbiology (Hersbach et al., 1984).
The improvement of penicillin production by conventional
strain improvement resulted both from enhanced gene
expression and from gene amplification (Barredo et al.,
1989; Smith et al., 1989). Increased levels of mRNA corre-
sponding to the three enzymes of penicillin G biosynthesis
were found in high-penicillin producing strains of P. chry-
sogenum as compared to wild-type strains (Smith et al.,
1990). High-producing strains contained an amplified re-
gion; a 106-kb region amplified five to six times as tandem
repeats was detected in a high-producing strain, whereas
wild-type P. chrysogenum and Fleming’s original strain of
P. notatum contained only a single copy (Fierro et al., 1995).
Strain improvement has been the main factor involved in
the achievement of impressive titers of industrial metabolites.
The production titer of tetracycline as far back as 1979 was
reported to be over 20 g L�1 (Podojil et al., 1984), mainly due
to strain improvement. Later, titers of 30–35 g L�1 were
reached for chlortetracycline and tetracycline. The production
titer of penicillin is 70 g L�1 and that of cephalosporin C over
30 g L�1 (Elander, 2003). The production titer of tylosin has
been reported to be over 15 g L�1 (Chen et al., 2004) and that
of salinomycin is 60 g L�1 (Liu, 1982).
Morphological and pigment mutants
Although almost nothing is known about the mechanisms
causing higher production in superior random or morpholo-
gical mutants, it is likely that many of these mutations involve
regulatory genes, especially as regulatory mutants obtained in
basic genetic studies are sometimes found to be altered in
colonial morphology. Thus, morphological mutants have been
very important in strain improvement. These include mutants
affected in mycelia formation, which produce colonies with a
modified appearance or a new color. Color changes have also
been important for pigment producers (Table 1).
Auxotrophic mutants
Very early in the development of the concepts of regulation,
geneticists realized that the end product of a biosynthetic
pathway to a primary metabolite excercises strict control
over the amount of an intermediate accumulated by an
auxotrophic mutant of that pathway. Only at a growth-
limiting concentration of the end product would a large
accumulation of the substrate of the deficient enzyme occur.
This principle of decreasing the concentration of an inhibi-
tory or repressive end product to bypass feedback inhibition
or repression was best accomplished by the use of auxo-
trophic mutants. Indeed, auxotrophic mutation has been a
major factor in the industrial production of primary pro-
ducts such as amino acids and nucleotides (Table 1). The
production of secondary products such as antibiotics is also
markedly affected by auxotrophic mutation, even when
auxotrophs are grown in nutritionally complete and even
complex media. Although the change in product formation
is usually in the negative direction, higher-producing auxo-
trophs are obtained from producers of antibiotics.
When several primary metabolites are produced by a
single branched pathway, mutation in one branch of the
pathway often leads to overproduction of the product of the
FEMS Microbiol Rev 30 (2006) 187–214c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
188 J. L. Adrio & A. L. Demain
Table 1. Mutations leading to increased product formation
Mutation type Organism Mutant characteristics
Overproduced
compound Reference
1. Morphological and/
or pigment change
1.1. Salmonella
typhimurium
Wrinkled colonies Histidine Roth & Ames (1966)
1.2. Streptomyces
coeruleorubidus
‘Bald colonies’; then white colonies Daunorobicin Blumauerova et al. (1978)
1.3. Streptomyces
glomeratus
Reddish-orange colonies with no
aerial mycelia
Beromycins Blumaerova et al. (1980, 1980)
1.4. Phaffia
rhodozyma
Pink colonies on agar containing
b-ionone or diphenylamine
Astaxanthin Lewis et al. (1990);
Chumpolkulwong et al. (1997)
1.5. Actinoplanes
teichomyceticus
Pink instead of brown mycelia Teichoplanins Lee et al. (2003)
2. Auxotrophic 2.1. Brevibactetrium
ammoniagenes
Guanine auxotrophy 50-Inosinic acid (IMP)
and hypoxanthine
Teshiba & Furuya (1983)
2.2. Bacillus
licheniformis
Leucine auxotrophy Bacitracin Haavik & Froyshov (1982)
2.3. Streptomyces
lipmanii
Leucine auxotrophy Cephamycin C and
penicillin N
Godfrey (1973)
3. Reversion of
auxotrophy
3.1. Streptomyces
fradiae
Non-auxotrophic for aspartate Tylosin Lee & Lee (1995)
4. Reversion of
non-production
4.1. Streptomyces
viridifaciens
Producing ability Chlortetracycline Dulaney & Dulaney (1967)
4.2. Streptomyces
goldiniensis
Producing ability Aurodox Unowsky & Hoppe (1978)
5. Antimetabolite
resistance
5.1. Candida boidinii Resistance to ethionine Methionine Tani et al. (1988)
5.2. Streptomyces
clavuligerus
Resistance to thialysine Cephamycins Mendelovitz
& Aharonowitz (1983)
5.3. Streptomyces
pilosus
Resistance to thialysine Desferrioxamine Smith (1987)
5.4. Streptomyces
cinnamonensis
Resistance to 2-ketobutyrate in
presence of valine or isoleucine
Monensins A & B Pospisil et al. (1999)
5.5. Actinoplanes
teichomyceticus
Resistance to valine hydroxamate Teichoplanins Wang et al. (1996);
Jin et al. (2002a)
5.6. Candida flareri Resistance to iron, to tubercidin,
to 2-DOG
Riboflavin Stahmann et al. (2000)
5.7. Ashbya gossypii Resistance to itaconic acid and
aminomethylphosphinic acid
Riboflavin Stahmann et al. (2000)
5.8. Penicillium
chrysogenum
Resistance to phenylacetic acid
(precursor)
Penicillin G Barrios-Gonzalez et al. (1993)
5.9. Amycolatopsis
mediterranei
Sequential resistance to tryptophan
(feedback inhibitor), p-
hydroxybenzoate, and propionate
(precursor)
Rifamycin B Jin et al. (2002)
6. Product resistance 6.1 Streptomyces
goldiniensis
Resistance to aurodox Aurodox Unowsky & Hoppe (1978)
6.2. Nocardia
uniformis
Resistance to nocardicin Nocardicin Elander & Aoki (1982)
6.3. Streptomyces
kitasatoensis
Resistance to leucomycin Leucomycin Higashide (1984)
6.4. Streptomyces
rimosus
Resistance to oxytetracycline Oxytetracycline Gravius et al. (1994)
7. Antibiotic resistance 7.1 Streptomyces
coelicolor and
Streptomyces lividans
Resistance to streptomycin,
gentamicin, paromomycin,
rifamycin and combinations
Actinorhodin Hosoya et al. (1998); Hesketh
& Ochi (1997); Okamoto et al.
(2003); Okamoto-Hosoya et al.
2000; Hu & Ochi (2001)
FEMS Microbiol Rev 30 (2006) 187–214 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
189Genetic improvement of processes yielding microbial products
other branch. Examples include the overproduction of
phenylalanine by tyrosine auxotrophs and vice versa, and
the overproduction of lysine by auxotrophs requiring threo-
nine and methionine. In the case of branched pathways
leading to a primary metabolite and a secondary metabolite,
auxotrophic mutants requiring the primary metabolite
sometimes overproduce the secondary metabolite (Table 1).
Reversion of an auxotroph to prototrophy sometimes
leads to new prototrophs possessing higher enzyme activity
than present in the original ‘grandparent’ prototroph. Such
increased enzyme activity was probably the result of a
structural gene mutation producing a more active enzyme
or an enzyme less subject to feedback inhibition (Table 1).
Revertants of non-producing mutants
A high proportion of such mutants has been found to
produce increased amounts of antibiotics (Table 1).
Antimetabolite-resistant mutants
Basic studies on regulation have shown that it is possible to
select regulatory mutants, which overproduce the end
products of primary pathways, using toxic metabolite ana-
logues. Such antimetabolite-resistant mutants often possess
enzymes that are insensitive to feedback inhibition, or
enzyme-forming systems resistant to feedback repression.
The selection of mutants resistant to toxic analogues of
primary metabolites has been widely employed by industrial
microbiologists (Table 1).
A variation of the antimetabolite selection techniques is
possible when a precursor is toxic to the producing organ-
ism. The principle here is that the mutant most capable of
detoxifying the precursor by incorporating it into the
antibiotic will be the best grower in the presence of the
precursor (Table 1). When the secondary metabolite
Table 1. Continued.
Mutation type Organism Mutant characteristics
Overproduced
compound Reference
8. Reversal of carbon
source repression
8.1. Saccharomyces
cerevisiae
Resistance to 2-deoxyglucose Cheese whey
hydrolysis
Bailey et al. (1982)
8.2. Schwanniomyces
castelli
Resistance to 2-deoxyglucose Isomaltase, amylase McCann & Barnett (1984);
Sills et al. (1984)
8.3. Pichia
polymorpha
Resistance to 2-deoxyglucose Inulinase Bajon et al. (1984)
8.4. Penicillium
chrysogenum
Resistance to 2-deoxyglucose Penicillin G Chang et al. (1980)
8.5 Aspergillus niger Rapid growth on high sucrose Citric acid Schreferl-Kunar et al. (1989)
8.6. Aspergillus niger Resistance to 2-deoxyglucose Citric acid Kirimura et al. (1992)
9. Reversal of
phosphate inhibition
9.1. Streptomyces
aureofaciens
Small colonies on phosphate-limiting
agar
Tetracycline Colombo et al. (1981)
9.2. Streptomyces
griseus
Production in excess-phosphate
medium
Candicidin Martin et al. (1979)
10. Increased
production on agar
10.1. Acremonium
chrysogenum
Increased clear zone around colony Cephalosporin C Elander (1969)
10.2. Streptomyces
viridifaciens
Increased clear zone around colony Chlortetracycline Dulaney & Dulaney (1967)
10.3. Bacillus subtilis Increased clear zone around colony Mycobacillin Bannerjee & Bose (1964)
10.4. Aspergillus
nidulans
Increased clear zone around colony Penicillin Ditchburn et al. (1974)
10.5. Aspergillus niger Increased clear zone around colony Citric acid Das & Roy (1981)
10.6. Rhyzopus oryzae Increased clear zone around colony Lactate Longacre et al. (1997)
10.7. Streptomyces
kasugaensis
Increased clear zone around plugs
of agar
Kasugamycin Ichikawa et al. (1971)
10.8. Acremoniums
chrysogenum
Increased clear zone around plugs
of agar
Cephalosporin C Chang & Elander (1979)
10.9. Streotomyces
hygroscopicus
Increased clear zone around plugs
of agar
Complex ‘165’ Gesheva (1994)
11. Change in
permeability
11.1. Brevibacterium
flavum
Inability to grow on glutamate Glutamic acid Shiio et al. (1982); Mori
& Shiio (1983)
11.2. Brevibacterium
ammoniagenes
Increased sensitivity to deoxycholate
and lysozyme
50-Inosinic acid Teshiba & Furuya (1983)
11.3. Escherichia coli Elimination of active praline uptake Proline Rancount et al. (1984)
11.4. Corynebacterium
glutamicum
Decrease in tryptophan uptake Tryptophan Ikeda & Katsumata (1995)
FEMS Microbiol Rev 30 (2006) 187–214c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
190 J. L. Adrio & A. L. Demain
produced is itself a growth inhibitor of the producing
culture, as in the case of certain antibiotics, the metabolite
can sometimes be used to select resistant mutants that are
improved producers.
Certain streptomycin resistance mutations cause in-
creased production of unrelated antibiotics. In addition to
improvement in secondary metabolite formation by muta-
tion to streptomycin resistance, resistance to gentamicin or
to paromomycin is even more effective. Furthermore,
triple mutation to resistance to streptomycin, gentamicin
and rifampicin, each of which individually increased acti-
norhodin formation, was found to be the most effective
(Table 1).
Mutants resistant to nutritional repression
Nutritional repression can also be decreased by mutation to
antimetabolite resistance. Examples of selection agents are
2-deoxyglucose (2-DOG) for enzymes and pathways con-
trolled by carbon source regulation (Table 1), methylammo-
nium for those regulated by nitrogen source repression, and
arsenate for phosphate regulation.
Mutants that use phosphate less efficiently for growth
sometimes show improved antibiotic production. Thus,
screening for small colonies on phosphate-limiting media
could be a useful strain improvement technique for phos-
phate-regulated products (Table 1).
Improved production on agar
In many cases, fermentation performance on an agar plate is
related to production in submerged liquid culture, and the
method has application as a means of detecting superior
mutants. So-called ‘zone mutants’ have proven useful for
improving several different processes (Table 1).
A widely used modification involves the production of
antibiotics by colonies on separate plugs of agar followed by
placement of these plugs on a seeded assay plate and
measurement of the resultant clear zones. The use of this
‘agar piece method’ resulted in improvement of antibiotic
production (Table 1). Agar-piece screening of antibiotic
production in the presence of inhibitory levels of phosphate
(15 mM) led to the isolation of six markedly improved and
stable Streptomyces hygroscopicus strains producing the
macrolide antifungal complex ‘165’ (Gesheva, 1994).
Permeability mutants
Product excretion in overproducing strains often occurs
when uptake and/or catabolism is impaired. Thus, genetic
lesions eliminating active uptake can be used to specifically
enhance excretion of metabolites (Table 1). It is often of
benefit to isolate mutants unable to grow on the
desired product as sole carbon or energy source. Such
mutants are often impaired in their ability to takeup the
product and they contain lower intracellular levels of the
product, thus lessening feedback regulation. In certain
improved mutants, there is an increase in sensitivity
to deoxycholate and lysozyme, indicating a change in
permeability.
Mutants showingqualitative changes in themixoffermentation products
As many organisms produce secondary metabolites as
mixtures of a chemical family or of several chemical families,
mutation has been used to eliminate undesirable products in
such fermentations. An example is that of Nakatsukasa and
Mabe (Nakatsukasa & Mabe, 1978), who found that streak-
ing out a natural single colony isolate from Streptomyces
aureofaciens (producing the polyether narasin and the
broad-spectrum antibiotic enteromycin) on galactose led to
yellow and white sectoring. The effect was specific for
galactose. Of the four colony types obtained, one produced
only narasin and two produced only enteromycin.
Streptomyces griseus ssp. cryophilus makes four R3� sulfated
and four R3� unsulfated carbapenems. The sulfated forms are
less active than the unsulfated forms. To completely eliminate
the R3 sulfated forms, sulfate transport mutants were ob-
tained. These were of two types: (i) auxotrophs for thiosulfate
or cysteine; and (ii) selenate-resistant mutants. Each type
produced completely unsulfated forms and titers were equiva-
lent to the total titer of the parent (Kitano et al., 1985).
Eight avermectins are produced by Streptomyces avermi-
tilis, of which only a small number are desirable. A non-
methylating mutant produced only four of the compounds
and a mutant that failed to make the 25-isopropyl substi-
tuent (from valine) produced a different mixture of compo-
nents. By protoplast fusion, a hybrid strain was obtained
which made only two components, B2a and B1a (Omura
et al., 1991). Stutzman-Engwall and colleagues (Stutzman-
Engwall et al., 2003) introduced random mutations by PCR
into gene aveC and obtained a mutant that produced an
avermectin B1 : B2 ratio of 2.5, much improved over the 0.6
ratio of the parent S. avermitilis strain.
Mutation was used to eliminate the undesirable polyke-
tides sulochrin and asterric acid from broths of the lovasta-
tin producer, Aspergillus terreus (Vinci et al., 1991). Mutants
have also been employed to eliminate undesirable copro-
ducts from the monensin fermentation (Pospisil et al.,
1984).
Mutantsproducingnewantibiotics
Mutant methodology has been used to produce new mole-
cules. The medically useful products demethyltetracycline
FEMS Microbiol Rev 30 (2006) 187–214 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
191Genetic improvement of processes yielding microbial products
and doxorubicin were discovered by simple mutation of
the cultures producing tetracycline and daunorubicin,
respectively. Later, the technique of ‘mutational biosynth-
esis’ (= mutasynthesis) was devised (Shier et al., 1969). In
this process, a mutant blocked in secondary metabolism is
fed analogs of the moiety whose biosynthesis is blocked. If
successful, the mutant (called an ‘idiotroph’) produces a
new antibiotic derivative (Nagaoka & Demain, 1975). The
hybramycins were the first compounds to be made this way
(Shier et al., 1969). Since then, mutational biosynthesis has
been used for the discovery of many new secondary meta-
bolites (Lemke & Demain, 1976; Daum & Lemke, 1979;
Kitamura et al., 1982). The most well-known is the com-
mercial antihelmintic agent doramectin, the production
of which employed a mutant of the avermectin producer
S. avermitilis (Cropp et al., 2000).
New anthracyclines and aglycones have been isolated
from blocked mutants of the daunorubicin and doxorubicin
producers (Cassinelli et al., 1980; McGuire et al., 1981). By
adding carminomycinone or 13-dihydrocarminomycinone
to an idiotroph of Streptomyces galilaeus (the producer of
aclacinomycin), the aglycones were glycosylated to form a
new trisaccharide anthracycline, trisarubicionol (Yoshimoto
et al., 1981).
New macrolide antibiotics have been produced from
blocked mutants of the tylosin-producer, Streptomyces fra-
diae (Kirst et al., 1983). Four new hybrid macrolide anti-
biotics were obtained by feeding erythronolide B to a
blocked mutant of the oleandomycin producer, Streptomyces
antibioticus (Spagnoli et al., 1983). A blocked-mutant of the
mycinamicin producer, Micromonospora polytrota, was fed
various rosaramicin precursors and converted them into
new rosaramicins (Lee et al., 1983).
Useofmutants to elucidatebiosyntheticpathways
A further use of mutants has been the elucidation of
metabolic pathways. This has been exploited for the bio-
synthesis of tetracyclines (McCormick, 1965), novobiocin
(Kominek, 1972), erythromycin (Martin et al., 1966;
Martin & Rosenbrook, 1967), neomycin (Pearce et al.,
1978), tylosin (Baltz et al., 1983), other aminoglycosides
(Penzikova & Levitov M, 1970; Takeda et al., 1978; Fujiwara
et al., 1980; Kase et al., 1982; Hanssen & Kirby, 1983),
rosaramicin (Vaughn et al., 1982), daunorubicin (McGuire
et al., 1981), other anthracyclines (Motamedi et al., 1986;
Yue et al., 1986), actinomycin (Troost & Katz, 1979),
carbapenems (Nozaki et al., 1984; Kojima et al., 1988),
ansamycins (Kibby et al., 1980; Ghisalba et al., 1981),
patulin (Gaucher et al., 1981) and phenazines (Byng et al.,
1979).
Genetic recombination
In contrast to the extensive use of mutation in industry, genetic
recombination was not much used at first, despite early claims
of success (Jarai, 1961; Mindlin, 1969), mainly due to the
absence or the extremely low frequency of genetic recombina-
tion in industrial microorganisms (in streptomycetes, it was
usually 10�6 or even less). Other problems were evident with
the b-lactam-producing fungi. Although Aspergillus exhibited
sexual and parasexual reproduction, the most commercially
interesting genera, Cephalosporium and Penicillium, were the
most difficult to work with as they only reproduced para-
sexually, which rarely resulted in recombination.
Recombination was erroneously looked upon as an alter-
native to mutation instead of a method that would comple-
ment mutagenesis programs. The most balanced and
efficient strain development strategy would not emphasize
one to the exclusion of the other; it would contain both
mutagenesis-screening and recombination-screening com-
ponents. In such a program, strains at different stages of a
mutational line, or from lines developed from different
ancestors, would be recombined. Such strains would no
doubt differ in many genes and by crossing them, genotypes
could be generated which would never occur as strictly
mutational descendants of either parent. Recombination
was also of importance in the mapping of production genes.
Studies on the genetic maps of overproducing organisms
such as actinomycetes are relatively recent. The model for
such investigations was the genetic map of Streptomyces
coelicolor (Kieser et al., 1992), which was found to be very
similar to those of other Streptomyces species, such as
S. bikiniensis, S. olivaceous, S. glaucescens and S. rimosus.
Protoplast fusion
As mentioned above, genetic recombination was virtually
ignored in industry, mainly due to the low frequency of
recombination. However, use of protoplast fusion changed
the situation markedly. After 1980, there was a heightened
interest in the application of genetic recombination to the
production of important microbial products such as anti-
biotics. Today, frequencies of recombination have increased to
even greater than 10�1 in some cases (Ryu et al., 1983), and
strain improvement programs routinely include protoplast
fusion between different mutant lines. The power of recom-
bination was demonstrated by Yoneda (Yoneda, 1980), who
recombined individual mutations, each of which increased a-
amylase production by two- to seven-fold in Bacillus subtilis.
A strain constructed by genetic transformation, which con-
tained all five mutations, produced 250-fold more a-amylase.
Recombination is especially useful when combined with
conventional mutation programs to solve the problem of
‘sickly’ organisms produced as a result of accumulated
genetic damage over a series of mutagenized generations.
FEMS Microbiol Rev 30 (2006) 187–214c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
192 J. L. Adrio & A. L. Demain
For example, a cross via protoplast fusion was carried out
with strains of Cephalosporium acremonium from a commer-
cial strain improvement program. A low-titer, rapidly-grow-
ing, spore-forming strain which required methionine to
optimally produce cephalosporin C was crossed with a
high-titer, slow-growing, asporogenous strain which could
use the less expensive inorganic sulfate. The progeny in-
cluded a recombinant which grew rapidly, sporulated, pro-
duced cephalosporin C from sulfate and made 40% more
antibiotic than the high-titer parent (Hamlyn & Ball, 1979).
Protoplast fusion was used to modify the characteristics
of an improved penicillin-producing strain of P. chrysogen-
um which showed poor sporulation and poor seed growth.
Backcrossing with a low-producing (12 g L�1) strain yielded
a high-producing (18 g L�1) strain with better sporulation
and better growth in seed medium (Lein, 1986). Interspe-
cific protoplast fusion between the osmotolerant Saccharo-
myces mellis and the highly fermentative S. cerevisiae yielded
stable hybrids fermenting high concentrations of glucose
(49% w w�1) (Legmann & Margalith, 1983).
Another application of protoplast fusion is the recombi-
nation of improved producers from a single mutagenesis
treatment. By recombination, one could combine the yield-
increase mutations and obtain an even more superior
producer before carrying out further mutagenesis. Two
improved cephamycin-C producing strains from Nocardia
were fused and among the recombinants were two cultures
that produced 10–15% more antibiotic than the best parent
(Wesseling & Lago, 1981). Genetic recombination allows the
discovery of new antibiotics by fusing producers of different
or even the same antibiotics. A recombinant obtained from
two different rifamycin-producing strains of Nocardia med-
iterranei produced two new rifamycins (16,17-dihydrorifa-
mycin S and 16,17-dihydro-17-hydroxy-rifamycin S) (Tra-
xler et al., 1982). However, according to Hopwood
(Hopwood, 1983), these examples may reflect the different
expression of genes from parent A in the cytoplasm of parent
B, rather than the formation of hybrid antibiotics. Interspe-
cific protoplast fusion between S. griseus and five other
species (Streptomyces cyaneus, Streptomyces exfoliatus, Strep-
tomyces griseoruber, Streptomyces purpureus and Streptomyces
rochei) yielded recombinants of which 60% produced no
antibiotics and 24% produced antibiotics different from the
parent strains (Okanishi et al., 1996). New antibiotics can
also be created by changing the order of the genes of an
individual pathway in its native host (Hershberger, 1996).
A new antibiotic, indolizomycin, was produced by proto-
plast fusion between non-antibiotic producing mutants of
Streptomyces griseus and Streptomyces tenjimariensis (Gomi
et al., 1984). Osmotolerance of food yeasts such as
Saccharomyces cerevisiae and S. diastaticus was increased by
protoplast fusion with osmotolerant yeasts. Other traits
transferred between yeasts by protoplast fusion include
flocculation (Panchal et al., 1982), lactose utilization (Far-
ahnak et al., 1986), the killer character (Bortol et al., 1986;
Farris et al., 1992), cellobiose fermentation (Pina et al.,
1986) and methionine overproduction (Brigidi et al., 1988).
Plasmids, transposons, cosmidsand phage
Plasmid DNA has been detected in virtually all antibiotic-
producing species of Streptomyces. Some are sex plasmids
and constitute an essential part of the sexual recombination
process and others contain either structural genes or genes
somehow influencing the expression of the chromosomal
structural genes of antibiotic biosynthesis.
Most plasmids have no apparent effect on metabolite
production and only very few antibiotic biosynthesis pro-
cesses are encoded by plasmid-borne genes. However, the
production of methylenomycin A is encoded by genes
present on plasmid SCP1 in Streptomyces coelicolor. When
the plasmid was transferred to other streptomycetes, the
recipients produced the antibiotic. For many years, plasmid
SCP1 was never observed or isolated as a circular DNA
molecule, because it was a giant linear plasmid. It was
initially difficult to separate such giant linear plasmids from
chromosomal DNA but this was later accomplished by
pulsed field gel electrophoresis or orthogonal field alteration
gel electrophoresis (OFAGE) (Kinashi & Shimaji, 1987).
Some products of unicellular bacteria are plasmid-en-
coded. These include aerobactin, a hydroxamate siderophore
and virulence factor produced by Escherichia coli (McDougall
& Neilands, 1984) and other Gram-negative bacteria (Enter-
obacter aerogenes, Enterobacter cloacae, Vibrio mimicus, and
species of Klebsiella, Salmonella and Shigella). Aerobactin is
synthesized by a plasmid-borne five-gene cluster, which is
negatively regulated by iron (Roberts et al., 1986); it can also
be produced via chromosomal genes (Moon et al., 2004). It
also appears that siderophore production by Arizona hinsha-
wii is plasmid-encoded. A microcin, an antimetabolite of
methionine, which is produced by E. coli and acts as a
competitive inhibitor of homoserine-O-transuccinylase, is
encoded by a plasmid that occurs at 20 copies per genome
equivalent (Perez-Diaz & Clowes, 1980). The gene coding for
the parasporal crystal body (d-endotoxin) of Bacillus thur-
ingiensis is plasmid-borne (Whiteley & Schnepf, 1986; De
Maagd et al., 2003) in most species but is on the chromo-
some in a few other species.
Instability in Streptomyces is brought about by environ-
mentally stimulated macrolesions, e.g. deletions, transposi-
tions, rearrangements and DNA amplification. They occur
spontaneously or are induced by environmental stresses
such as intercalating dyes, protoplast formation and regen-
eration, and interspecific protoplast fusion. Streptomycetes
are the only prokaryotes known to be subject to spontaneous
DNA amplification, sometimes amounting to several
FEMS Microbiol Rev 30 (2006) 187–214 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
193Genetic improvement of processes yielding microbial products
hundred tandem copies, accounting for over 10% of total
DNA, in the absence of selection. Amplification seems to be
coupled to DNA deletion and may involve insertion se-
quence (IS)-like elements (Baltz, 1986). Ethidium bromide
cures plasmids in streptomycetes but also increases the
frequency of deletion mutations, especially in areas of the
chromosome that are already unstable (Crameri et al., 1986).
Transposable elements, DNA sequences encoding a trans-
posase enzyme (Berg & Berg, 1983) that move from one
replicon to another without host recombination functions
or extensive homology with the site of integration, have
been extremely useful for the following reasons: (i) they
usually provide stable, nonreverting mutants; (ii) they can
be used to determine the order of genes in an operon; (iii) it
is easy to select for mutants because transposons contain
antibiotic- or mercury-resistance markers; (iv) they provide
portable regions of homology for chromosomal mobiliza-
tion; (v) they provide markers for non-selectable genes and
allow the cloning of such genes which can then be used as
hybridization probes to fish out the wild-type gene from a
genomic library; and (vi) they often have unique restriction
sites, and thus are good markers for isolating defined
deletion derivatives or locating the precise position of a gene
by heteroduplex mapping.
In the daptomycin producer Streptomyces roseosporus,
some Tn 5099 transposition mutants produced 57–66%
more daptamycin than the parent, whereas others produced
less or the same (McHenney & Baltz, 1996; Baltz et al., 1997).
Transposition increased the rate-limiting step of tylosin
biosynthesis in Streptomyces fradiae, i.e. the conversion of
macrocin to tylosin. Transposing a second copy of tylF into a
neutral site on the S. fradiae chromosome increased its gene
product, macrocin O-methyltransferase, and tylosin produc-
tion, while decreasing the concentration of the final inter-
mediate (macrocin). Tylosin production was increased by up
to 60% and the total macrolide titer was unchanged (Solen-
berg et al., 1996). Transposon mutagenesis eliminated the
production of the toxic oligomycin by the avermectin-
producing Streptomyces avermitilis (Ikeda et al., 1993).
Cloning a 34-kb fragment from Streptomyces rimosus via a
cosmid into Streptomyces lividans and Streptomyces albus
resulted in oxytetracycline production by the recipients (Bin-
nie et al., 1989). Contrary to earlier reports, all the oxytetracy-
cline genes were clustered together on the S. rimosus
chromosomal map (Butler et al., 1989).
Improvementofmicrobial processesbygeneticengineering
Primarymetabolites
New processes for the production of amino acids and
vitamins have been developed by recombinant DNA tech-
nology. Escherichia coli strains were constructed with plas-
mids bearing amino acid biosynthetic operons. Plasmid
transformation was accomplished in Corynebacterium, Bre-
vibacterium and Serratia and, as a result, recombinant DNA
technology has been used routinely to improve such com-
mercial amino acid-producing strains (Sahm et al., 2000).
A recombinant strain of E. coli (made by mutating to
isoleucine auxotrophy, cloning in extra copies of the thrABC
operon, inactivating the threonine-degrading gene tdh, and
mutating to resistance to high concentrations of L-threonine
and L-homoserine) produced 80 g L�1L-threonine in 1.5
days at a yield of 50% (Eggeling & Sahm, 1999). Cloning
extra copies of threonine export genes into E. coli led to
increased threonine production (Kruse et al., 2002).
The introduction of the proline 4-hydroxylase gene from
Dactylosporangium sp. into a recombinant strain of E. coli
producing L-proline at 1.2 g L�1 lead to a new strain produ-
cing 25 g L� 1 of hydroxyproline (trans-4-hydroxy-L-pro-
line) (Shibasaki et al., 2000). When proline was added,
hydroxyproline reached 41 g L�1, with a yield of 87% from
proline.
An engineered strain of Corynebacterium glutamicum
producing 50 g L�1 of L-tryptophan was further modified
by cloning in additional copies of its own transketolase gene
to increase the level of the erythrose-4-phosphate precursor
of aromatic biosynthesis (Ikeda & Katsumata, 1999). A low
copy number plasmid increased production to 58 g L�1,
whereas a high copy number plasmid decreased production.
L-Valine production by mutant strain VAL1 of C. gluta-
micum amounted to 105 g L�1 (Radmacher et al., 2002;
Lange et al., 2003). The mutant was constructed by over-
expressing biosynthetic enzymes via a plasmid, eliminating
ilvA encoding threonine dehydratase, and deleting two genes
encoding enzymes of pantothenate biosynthesis. The culture
was grown with limitation of isoleucine and pantothenate.
By introduction of feedback-resistant threonine dehydra-
tases and additional copies of genes encoding branched
amino and biosynthetic enzymes, lysine- or threonine-
producing strains were converted into L-isoleucine produ-
cers with titers up to 10 g L�1 (Morbach et al., 1996;
Guillouet et al., 1999; Hashiguchi et al., 1999). Amplifica-
tion of the wild-type threonine dehydratase gene ilvA in a
threonine-producing strain of Corynebacterium lactofermen-
tum led to 15 g L�1 of isoleucine overproduction (Colon
et al., 1995).
Biotin has been made traditionally by chemical synthesis
but recombinant microbes have approached a competitive
economic position. The cloning of a biotin operon
(bioABFCD) on a multicopy plasmid allowed E. coli to
produce 10 000 times more biotin than did the wild-type
strain (Levy-Schil et al., 1993). Sequential mutation of
Serratia marcescens to resistance to the biotin antimetabolite
acidomycin (= actithiazic acid) led to mutant strain SB412,
FEMS Microbiol Rev 30 (2006) 187–214c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
194 J. L. Adrio & A. L. Demain
which produced 20 mg L�1 biotin (Sakurai et al., 1994).
Further improvements were made by mutating selected
strains to ethionine-resistance (strain ET2, 25 mg L�1), then
mutating ET2 to S-2-aminoethylcysteine resistance (strain
ETA23, 33 mg L�1) and finally cloning in the resistant bio
operon (Sakurai et al., 1994) yielding a strain able to
produce 500 mg L�1 in fed-batch fermentor culture along
with 600 mg L�1 of biotin vitamers. Later advances led to
production by recombinant S. marcescens of 600 mg L�1 of
biotin (Masuda et al., 1995).
A process for riboflavin production in Corynebacterium
ammoniagenes (previously Brevibacterium ammoniagenes)
was developed by cloning and overexpressing the organism’s
own riboflavin biosynthesis genes (Koizumi et al., 2000) and
its own promoter sequences. The resulting culture produced
15.3 g L�1 riboflavin in 3 days. Genetic engineering of a
Bacillus subtilis strain already containing purine analog-
resistance mutations led to the improved production of
riboflavin (Perkins & Pero, 1993). An industrial strain of B.
subtilis was produced by inserting multiple copies of the rib
operon at two different sites in the chromosome, making
purine analog-resistance mutations to increase guanosine
triphosphate (GTP; a precursor) production and a ribofla-
vin analog (roseflavin)-resistance mutation in ribC that
deregulated the entire pathway (Perkins et al., 1999).
Vitamin C (ascorbic acid) has traditionally been made in
a five-step predominantlychemical process by first convert-
ing glucose to 2-keto-L-gulonic acid (2-KGA) with a yield of
50% and then converting the 2-KGA by acid or base to
ascorbic acid. A novel process for vitamin C synthesis
involved the use of a genetically engineered Erwinia herbico-
la strain containing a gene from Corynebacterium sp. The
engineered organism converted glucose into 1 g L�1 of 2-
KGA (Anderson et al., 1985; Pramik, 1986). A better process
was devised independently, which converted 40 g L�1 glu-
cose into 20 g L�1 2-KGA (Grindley et al., 1988). This
process involved cloning and expressing the gene encoding
2,5-diketo-D-gluconate reductase from Corynebacterium sp.
into Erwinia citreus. Another process uses a recombinant
strain of Gluconobacter oxydans containing genes encoding
L-sorbose dehydrogenase and L-sorbosone dehydrogenase
from G. oxydans T-100. The new strain was an improved
producer of 2-KGA (Saito et al., 1997). Further mutation to
suppress the L-idonate pathway and to improve the promo-
ter led to the production of 130 g L�1 of 2-KGA from
150 g L�1 sorbitol.
Carotenoids were overproduced by introducing carote-
noid gene clusters from Erwinia uredovora into E. coli and
overexpressing E. coli deoxyxylulose phosphate synthase, the
key enzyme of the non-mevalonate isoprenoid biosynthetic
pathway (Matthews & Wurtzel, 2000). Lycopene accumu-
lated to 1.3 mg g�1 dry cell weight and zeaxanthin to
0.6 mg g�1.
Cloning of aldehyde dehydrogenase of Acetobacter poly-
oxogenes on a plasmid vector into Acetobacter aceti ssp.
xylinum increased the rate of acetic acid production by over
100% (1.8 g L�1 h� 1 to 4 g L�1 h�1) and titer by 40%
(68 g L�1 to 97 g L�1) (Fukaya et al., 1989).
Genetic engineering of the inosine monophosphate
(IMP) dehydrogenase gene in a B. subtilis strain producing
7 g L�1 of the desirable guanosine and 19 g L�1 of the
undesirable inosine changed production to 20 g L�1 guano-
sine and 5 g L�1 inosine (Miyagawa et al., 1986).
A recombinant E. coli strain was constructed that pro-
duced optically active pure D-lactic acid from glucose at
virtually the theoretical maximum yield, e.g. two molecules
from one molecule of glucose (Zhou et al., 2003). The
organism was engineered by eliminating genes of competing
pathways encoding fumarate reductase, alcohol/aldehyde
dehydrogenase and pyruvate formate lyase and by a muta-
tion in the acetate kinase gene.
New technologies that have proven to be very useful for
increasing production of primary metabolites include gen-
ome-based strain reconstruction, metabolic engineering,
and whole genome shuffling (see section on Novel genetic
technologies).
Secondarymetabolites
The application of recombinant DNA technology to the
production of secondary metabolites has been of great
interest (Baltz & Hosted, 1996; Diez et al., 1997). The tools
of the recombinant geneticist for increasing the titers of
secondary metabolites have included: (i) transposition mu-
tagenesis, (ii) targeted deletions and duplications by genetic
engineering and (iii) genetic recombination by protoplast
fusion (Baltz, 2003). Recent additions to these techniques
include genomics, transcriptome analysis, proteomics, me-
tabolic engineering, and whole genome shuffling (see sec-
tion on Novel gene technologies).
One of the first indications that rDNA technology could
be applied to antibiotics and other secondary metabolites
was that it could be carried out in streptomycetes (Thomp-
son et al., 1982). Plasmids were constructed from plasmid
SLP 1.2 of Streptomyces lividans and plasmid SCP2� from
Streptomyces coelicolor. In mating of plasmid-negative
S. lividans, ‘pocks’ (circular zones of sporulation inhibition
associated with plasmid transfer in the lawn of streptomy-
cete growth arising from a regenerated protoplast popula-
tion) were seen. This was due to looping out of a piece of S.
coelicolor DNA, which became a series of small S. lividans
plasmids (SLP 1.1 to 1.6) that were good cloning vehicles.
The genetic engineering of actinomycetes was limited for
a number of years by restriction barriers hindering DNA
introduction and by the inhibition of secondary metabolism
by self-replicating plasmid-cloning vectors (Baltz & Hosted,
FEMS Microbiol Rev 30 (2006) 187–214 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
195Genetic improvement of processes yielding microbial products
1996), but these problems were mainly overcome. Early
reviews on cloning and expressing antibiotic production
genes in Streptomyces were by Martin and Gil (Martin & Gil,
1984) and Liras (Liras, 1988).
An interesting possibility was the transfer of operons
from one streptomycete to another in the hope that the
structural genes might be better able to express themselves
in another species. Clustering facilitated the transfer of an
entire pathway in a single manipulation. Studies revealed
that many antibiotic biosynthesis genes were arranged in
clusters including undecylprodigiosin, actinorhodin, chlor-
amphenicol, rifamycin, cephamycin, erythromycin, tetracy-
clines and tylosin among others. Thus, the entire
undecylprodigiosin pathway (‘red’ pathway) of S. coelicolor
was transferred on a 37-kb fragment into Streptomyces
parvulus and the antibiotic was produced (Coco et al.,
1991). Similarly, the entire cephamycin C pathway was
cloned and expressed from a cephamycin-producing strain
of Streptomyces cattleya. When the 29-kb DNA fragment was
cloned into the non-b-lactam producer, S. lividans, one
transformant (out of 30 000) made cephamycin (Chen
et al., 1988). When the fragment was introduced into
another cephamycin producer, Streptomyces lactamgens, a
two- to three-fold improvement was obtained.
In fungi making penicillin G, the three structural genes
(ACVS, cyclase and penicillin acyltransferase) are clustered
on a single chromosome of Penicillium chrysogenum (Smith
et al., 1990) and of Aspergillus nidulans (MacCabe et al.,
1990). In these fungi, the genes of the cluster are separately
transcribed. By contrast, fungal genes coding for cephalos-
porin biosynthesis are distributed among different chromo-
somes. The deacetylcephalosporin C acetyltransferase gene
from Cephalosporium acremonium (cefG) is closely linked to
the expandase (cefEF) gene (Gutierrez et al., 1992; Matsuda
et al., 1992) and both are on chromosome II, whereas the
early genes of the pathway (pcbAB, pcbC) are located on
chromosome VI.
b-Lactam antibiotics
Cloning has been very important in understanding the
biosynthesis of b-lactam antibiotics (Demain & Elander,
1999), its genetics and improving the production processes.
Early common pathway
All producers of penicillins and cephalosporins, including
cephamycins, use the same two enzymes to start the
biosynthetic process. The steps involve the condensation of
L-a-aminoadipic acid, L-cysteine and L-valine to form the
tripeptide, d-(a-aminoadipyl)-L-cysteinyl-D-valine (ACV)
by ACV synthetase (ACVS), encoded by gene pcbAB (also
known as acvA in A. nidulans). This is followed by cycliza-
tion of ACV into isopenicillin N (IPN) by IPN synthase
(cyclase; encoded by pcbB). The cloning of the gene encod-
ing ACVS from P. chrysogenum (Diez et al., 1990), C.
acremonium (Gutierrez et al., 1991) and Nocardia lactam-
durans (Castro et al., 1988) contributed greatly to the
elucidation of the biosynthetic pathway. Overexpression of
acvA in A. nidulans, by replacing the normal promoter with
the ethanol dehydrogenase promoter (Kennedy & Turner,
1996), increased penicillin production up to 30-fold. The
cyclase genes from different microorganisms were all cloned
(Aharonowitz et al., 1992; Martin et al., 1997) and provided
pure enzyme for structural studies. Cloning multiple copies
of cyclase into C. acremonium yielded an improved cepha-
losporin C-producing strain (Skatrud et al., 1987).
The hydrophobic branch
Producers of penicillin use a single step branch involving
penicillin acyltransferase acting on IPN. Its gene penDE (also
known as iat, aat and acyA in A. nidulans) was cloned from
P. chrysogenum into C. acremonium, which led to the
production of penicillin G (in the presence of exogenous
phenylacetic acid) along with cephalosporin C (Gutierrez
et al., 1991). Without cloning, C. acremonium cannot
produce penicillin G.
The hydrophilic branch
All producers of cephalosporins and cephamycins employ a
series of enzymes leading from IPN. First, IPN is epimerized
to penicillin N by IPN epimerase (encoded by cefD). The
next steps include ring expansion of penicillin N by deace-
toxycephalosporin C (DAOC) synthase (expandase, en-
coded by cefE) and hydroxylation by DAOC 30-hydroxylase
(encoded by cefF) to deacetylcephalosporin C (DAC).
Although expandase and hydroxylase are separate enzymes
encoded by separate genes in bacteria, these two activities
are found on the same protein in fungi, which is encoded by
one gene cefEF. At the DAC stage, the overall pathway again
splits into two branches. In C. acremonium, DAC is acety-
lated to cephalosporin C by DAC acetyltransferase encoded
by cefG. This step is the terminal reaction in cephalosporin-
producing fungi. By contrast, actinomycetes carbamoylate
DAC using three enzymes, encoded by cmcH, cmcI and cmcJ
genes to yield cephamycin C (Brewer et al., 1980).
When an industrial production strain of C. acremonium
394-4 was transformed with a plasmid containing the pcbC
and the cefEF gene from an early strain of the C. acremonium
mutant line, a transformant producing 50% more cephalos-
porin C than the production strain, as well as less penicillin
N, was obtained. Production in pilot plant (150 L) fermen-
tors was further improved by 15% (Skatrud et al., 1989).
One copy of the cefEF had been integrated into chromosome
III, whereas the native gene is on chromosome II.
FEMS Microbiol Rev 30 (2006) 187–214c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
196 J. L. Adrio & A. L. Demain
Transformation of P. chrysogenum with the Streptomyces
lipmanii cefD and Streptomyces clavuligerus cefE genes al-
lowed the production of the intermediate DAOC (Cantwell
et al., 1992) at titers of 2.5 g L�1. DAOC is a valuable
intermediate in the commercial production of semi-syn-
thetic cephalosporins. Also, cloning of cefE from S. clavuli-
gerus or cefEF and cefG (see next paragraph) from C.
acremonium into P. chrysogenum grown with adipic acid as
side-chain precursor (Crawford et al., 1995) resulted in
formation of adipyl-6-aminopenicillanic acid (adipyl-6-
APA) and adipyl-7-aminodeoxycephalosporanic acid (adi-
pyl-7-ADCA) in the case of cefE and adipyl-6APA, adipyl-
7ADCA, adipyl-7-DAC and adipyl-7-aminocephalosporanic
acid (7-ACA) in the case of cefEF and cefG.
Disruption and one-step replacement of the cefEF gene
of an industrial cephalosporin C production strain of A.
chrysogenum yielded strains accumulating up to 20 g L� 1 of
penicillin N. Cloning and expression of the cefE gene from S.
clavuligerus into those high-producing strains yielded re-
combinant strains producing high titers of DAOC (Velasco
et al., 2000). Production levels were nearly equivalent (80%)
to the total b-lactams biosynthesized by the parental strain.
Weak acetyltransferase promoter activity appears to be
the cause of DAC accumulation in broths of C. acremonium.
Cloning of cefG increased its copy number and cefG mRNA,
tripled acetyltransferase activity, and increased cephalospor-
in C titers in a dose-dependent manner (Matsuda et al.,
1992; Mathison et al., 1993). Cloning of the gene with its
own promoter had no effect on the low level of DAC
acetyltransferase normally observed in C. acremonium (Gu-
tierrez et al., 1997). However, the use of foreign promoters
(the gpd promoter from A. nidulans, the bla promoter from
A. niger or the pbcC promoter from P. chrysogenum) had a
major effect on the level of cefG transcripts, DAC acetyl-
transferase protein level and activity, and antibiotic produc-
tion; cephalosporin C production rose by two- to three-fold.
Of the cephalosporins produced, the undesirable DAC
decreased from 80% of the total to 30–39%, whereas
cephalosporin C increased by a similar amount.
Transformation of early strain P. chrysogenum Wis54-
1255 with individual genes, pairs of genes, and all three
genes of the penicillin pathway showed that the major
increases occurred when all three genes were overexpressed
(Theilgaard et al., 2001). The best transformant contained
three extra copies of pcbAB, one extra copy of pcbC and two
extra copies of penDE and produced 299% of control shake
flask production and 276% of control productivity in
continuous culture.
Microbial enzymes
Genes encoding many microbial enzymes have been cloned
and the enzymes expressed at levels hundreds of times
higher than those naturally produced. Recombinant DNA
technology has been used (Falch, 1991): (i) to produce in
industrial organisms enzymes obtained from microbes that
are difficult to grow or handle genetically; (ii) to increase
enzyme productivity by use of multiple gene copies, strong
promoters, and efficient signal sequences; (iii) to produce in
a safe host useful enzymes obtained from a pathogenic or
toxin-producing microorganism; and (iv) to improve the
stability, activity or specificity of an enzyme by protein
engineering. The industrial enzyme business adopted rDNA
methods to increase production levels and to produce
enzymes from industrially-unknown microorganisms in
industrial organisms such as species of Aspergillus and
Trichoderma, as well as Kluyveromyces lactis, S. cerevisiae,
Yarrowia lipolytica and Bacillus licheniformis. Virtually all
laundry detergents contain genetically-engineered enzymes
and much cheese is made with genetically-engineered en-
zymes. Indeed, over 60% of the enzymes used in the
detergent, food and starch processing industries are recom-
binant products (Cowan, 1996).
Scientists at Novo Nordisk isolated a very desirable lipase
for use in detergents from a species of Humicola. For produc-
tion purposes, the gene was cloned into Aspergillus oryzae,
where it produced 1000-fold more enzyme (Carlsen, 1990)
and is now a commercial product. Such lipases are used for
laundry cleaning, interesterification of lipids, and esterification
of glucosides producing glycolipids which have applications as
biodegradable non-ionic surfactants for detergents, skin care
products, contact lens cleaners and as food emulsifiers.
The a-amylase gene from Bacillus amyloliquefaciens was
cloned using multicopy plasmid pUB110 in B. subtilis
(Palva, 1982). Production was 2500-fold that in wild-type
B. subtilis and five-fold that of the B. amyloliquefaciens
donor. An exoglucanase from the cellulolytic Cellulomonas
fimi was overproduced after cloning in E. coli to a level of
over 20% of cell protein (O’Neill et al., 1986). The endo-b-
glucanase components of the cellulase complexes from
Thermomonospora and Clostridium thermocellum were
cloned in E. coli as was the cellobiohydrolase I gene of
Trichoderma reesei (Shoemaker et al., 1983; Teeri et al.,
1983). Pichia pastoris, a methanol-utilizing yeast, was en-
gineered to produce S. cerevisiae invertase and to excrete it
into the medium at 100 mg L�1 (Van Brunt, 1986). Interest-
ingly, in S. cerevisiae, the invertase is periplasmic. Self-
cloning of the xylanase gene in S. lividans resulted in six-
fold overproduction of the enzyme (Mondou et al., 1986).
Many enzymes are made by filamentous organisms,
which are slow-growing and difficult to handle in fermen-
tors. The transfer of these genes to rapidly-growing uni-
cellular bacteria means that rapid growth and more
reproducible production can be achieved. Other advantages
are more rapid nutrient uptake due to a greater surface/
volume ratio, better oxygen transfer, better mixing and thus
FEMS Microbiol Rev 30 (2006) 187–214 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
197Genetic improvement of processes yielding microbial products
more reliable control of pO2, pCO2 and pH, and a better
organism for mutagenesis.
Aspartase production was increased by 30-fold by cloning
in E. coli (Komatsubara et al., 1986). Captopril esterase of
Pseudomonas putida, used in preparing the chiral captopril
sidechain, was cloned in E. coli with a 38-fold increase in
activity (Elander, 1995). A 1000-fold increase in phytase
production was achieved in A. niger using recombinant
technology (Van Hartinsveldt et al., 1993). Cloning of the
benzylpenicillin acylase gene of E. coli on multicopy (50)
plasmids resulted in a 45-fold increase as compared to
uninduced wild-type production. Interestingly, the cloned
enzyme is constitutive (Mayer et al., 1980). Cloning addi-
tional penicillin V amidase genes into wild-type Fusarium
oxysporium increased enzyme titer by 130-fold (Komatsu-
bara et al., 1986).
The properties of many enzymes have been altered by
genetic means. ‘Brute force’ mutagenesis and random
screening of microorganisms over the years led to changes
in pH optimum, thermostability, feedback inhibition, car-
bon source inhibition, substrate specificity, Vmax, Km and Ki.
This information was later exploited by the more rational
techniques of protein engineering. Single changes in amino
acid sequences have yielded similar types of changes in a
large variety of enzymes. Today, it is no longer necessary to
settle for the natural properties of an enzyme; these can be
altered to suit the needs of the investigator or the process.
For example, a protease from Bacillus stearothermophilus
was increased in heat tolerance from 86 1C to 100 1C, being
made resistant to boiling. The enzyme was developed by
site-directed mutagenesis (Van den Burg et al., 1998). Only
eight amino acids had to be modified. Temperature stability
at 100 1C was increased 340-fold and activity at lower
temperature was not decreased. All eight mutations were
far from the enzyme’s active site. Washing powders have
been improved in activity and low temperature operation by
the application of recombinant DNA technology and site-
directed mutagenesis to proteases and lipases (Falch, 1991;
Wackett, 1997).
Polymers, fuels, foodsandbeverages
Microbially-produced xanthan gum is not only an accepta-
ble food-thickener but is one of the most promising agents
for enhanced oil recovery in the petroleum industry. Re-
combinant DNA manipulation of Xanthomonas campestris
increased titers of xanthan by two-fold and increased
pyruvate content by over 45% (Bigelas, 1989; Tseng et al.,
1992). The yield was 0.6 g g�1 of sucrose utilized (Letisse
et al., 2001). Ten to twenty thousand tons of xanthan are
produced annually for use in the oil, pharmaceutical,
cosmetic, paper, paint and textile industries (Becker et al.,
1998).
Escherichia coli was converted into a good ethanol produ-
cer (4.3%, v v�1) using recombinant DNA technology
(Ingram et al., 1987). Alcohol dehydrogenase II and pyr-
uvate decarboxylase genes from Zymomonas mobilis were
inserted in E. coli and became the dominant system for NAD
regeneration. Ethanol represented over 95% of the fermen-
tation products in the genetically-engineered strain. By
cloning and expressing the same two genes into Klebsiella
oxytoca, the recombinant was able to convert crystalline
cellulose to ethanol in high yield when fungal cellulase was
added (Doran & Ingram, 1993). The maximum theoretical
yield was 81–86% and titers as high as 47 g L�1 of ethanol
were produced from 100 g L�1 of cellulose.
Cloning of its ace (acetone) operon gene adc (encoding
acetoacetate decarboxylase), ctfA and ctfB (two genes encod-
ing coenzyme A transferase) on a plasmid containing the adc
promoter into Clostridium acetobutylicum resulted in a 95%
increase in production of acetone, a 37% increase in butanol,
a 90% increase in ethanol, a 50% increase in solvent yield
from glucose and a 22-fold lower production of undesirable
acids (Mermelstein et al., 1993). The introduction of the
acetone operon from C. acetobutylicum into E. coli led to
high acetone production by the latter (Bermejo et al., 1998).
Beer wort contains barley b-glucans which reduce the
filtrability of beer and lead to precipitates and haze in the
final product. The gene coding for endoglucanase was
transferred from T. reesei to brewer’s yeast and the engi-
neered yeast strain efficiently hydrolyzed the b-glucans
(Penttila et al., 1987). Similiar technology created starch-
utilizing S. cerevisiae strains and wine yeast strains produ-
cing lower acidity and enhanced flavor. Brewing yeasts were
modified using recombinant DNA technology so that they
could produce A. niger amyloglucosidase and break down
unfermentable dextrins for light beer production (Van
Brunt, 1986; Hammond, 1988). The glucoamylase gene
from Aspergillus awamori was cloned and expressed stably
in polyploid distiller’s yeast. A high level of glucoamylase
was secreted. Almost all (95%) of the carbohydrates in the
25% starch substrate were utilized and high levels of ethanol
were produced. The engineered strain outperformed S.
diastaticus (Cole et al., 1988).
Brewing yeasts were engineered to produce acetolactate
decarboxylase from Enterobacter aerogenes or A. aceti. This
enzyme eliminated diacetyl and the requirement for the
three- to five-week flavor maturation period which normally
follows a one-week fermentation stage (Sone et al., 1988).
The resulting beer suffered no loss of quality or flavor
(Holzman, 1994).
Bioconversions
Recombinant DNA techniques have been useful in develop-
ing new bioconversions and improving old ones. Using a
FEMS Microbiol Rev 30 (2006) 187–214c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
198 J. L. Adrio & A. L. Demain
plasmid containing tryptophan synthase plus induction
with 3-indole acrylate, recombinant E. coli was able to
produce 180 g L�1 of L-tryptophan from indole plus
L-serine in 8 h (Yukawa et al., 1988). Whereas S. cerevisiae
normally produces 2 g L�1 of malic acid from fumaric acid,
a recombinant strain containing a cloned fumarase gene was
able to produce 125 g L�1 with a yield of almost 90%
(Neufeld et al., 1991).
An oxidative bioconversion of saturated and unsaturated
linear aliphatic 12–22 carbon substrates to their terminal
dicarboxylic acids was developed by gene disruption and
gene amplification (Picataggio et al., 1992). Product con-
centrations reached 200 g L�1 and problematic side-reac-
tions such as unsaturation, hydroxylation and chain-
shortening did not occur.
3-0-Acetyl-40 0-0-isovaleryltylosin (AIV) is useful in veter-
inary medicine against tylosin-resistant Staphylococcus aur-
eus. It is made by first producing tylosin with Streptomyces
fradiae and then using Streptomyces thermotolerans (produ-
cer of carbomycin) to bioconvert tylosin into AIV. A new
direct fermentation organism was constructed by trans-
forming S. fradiae with S. thermotolerans plasmids contain-
ing acyl transferase genes (Arisawa et al., 1996).
Recombinant Candida pasteurianum can carry out the
conversion of glycerol to 1,3-propanediol (Luers et al.,
1997). A more economical process involving conversion of
the less expensive glucose to 1,3-propanediol has been
achieved with a recombinant E. coli strain (Nakamura &
Whited, 2003). The project is a collaborative effort by
Genencor International and DuPont (Potera, 1997). The
recombinant strain contains two metabolic pathways, one
for conversion of glucose to glycerol and the other for
conversion of glycerol to 1,3-propanediol (Tong et al.,
1991; Laffend et al., 1996). The 1,3-propanediol (also known
as trimethylene glycol or 3G) is used as the building block to
produce a new biodegradable polyester (3G1).
Novel genetic technologies
A new genomic technique called ‘genome-based strain
reconstruction’ allows one to construct a strain superior to
the production strain because it only contains mutations
crucial to hyperproduction, but not other unknown muta-
tions which accumulate by brute-force mutagenesis and
screening (Ohnishi et al., 2002). This approach was used to
improve the lysine production rate of Corynebacterium
glutamicum by comparing high producing strain B-6 devel-
oped by Hirao and coworkers (Hirao et al., 1989) (produc-
tion rate slightly less than 2 g L�1 h�1) and a wild-type
strain. Comparison of 16 genes from strain B-6, encoding
enzymes of the pathway from glucose to lysine, revealed
mutations in five of the genes. Introduction of three of these
mutations into the wild-type created a new strain which
produced 80 g L�1 in 27 h, at a rate of 3 g L�1 h�1, the
highest rate ever reported for a lysine fermentation.
‘Metabolic engineering’ is the directed improvement of
product formation or cellular properties through the mod-
ification of specific biochemical reactions or introduction of
new ones using recombinant DNA technology (Stephano-
poulos, 1999; Nielsen, 2001). Its essence is the combination
of analytical methods to quantify fluxes and the control of
fluxes with molecular biological techniques to implement
suggested genetic modifications. Flux is the focal point of
metabolic engineering. Different means of analyzing flux
are: (i) kinetic based models; (ii) control theories; (iii) tracer
experiments; (iv) magnetization transfer; (v) metabolite
balancing; (vi) enzyme analysis and (vii) genetic analysis
(Eggeling et al., 1996). Metabolic control analysis revealed
that the overall flux through a metabolic pathway depends
on several steps, not just a single rate-limiting reaction
(Kacser & Acerenza, 1993).
Metabolic engineering has been applied to antibiotic
production (Khetan & Hu, 1999, 1999; Thykaer & Nielsen,
2003). The increases in metabolic flux were carried out by
enhancing enzymatic activity, manipulating regulatory
genes, enhancing antibiotic resistance and heterologous
expression of novel genes. Table 2 summarizes several
examples of progress on the production of those secondary
metabolites.
The production of amino acids shows many examples of
this approach. A useful review of metabolic engineering in
C. glutamicum, especially in relation to L-lysine production,
was published by Sahm and colleagues (Sahm et al., 2000).
Metabolic flux studies of wild-type C. glutamicum and four
improved lysine-producing mutants available from the
ATCC showed that yield increased from 1.2% to 24.9%
relative to the glucose flux. Other recent examples are on
overproduction of aromatic amino acids and derivatives
(Bongaerts et al., 2001), L-lysine (Wittmann & Heinzle,
2002) and glutamate (Kimura, 2003).
There are many other successful applications of metabolic
engineering for products such as 1,3-propanediol (Naka-
mura & Whited, 2003), carotenoids (Rohlin et al., 2001;
Visser et al., 2003; Wang & Keasling, 2003), organic acids
(Kramer et al., 2003), ethanol (Nissen et al., 2000), vitamins
(Zamboni et al., 2003; Sybesma et al., 2004) and complex
polyketides in bacteria (Pfeifer et al., 2001; Khosla &
Keasling, 2003).
During the last few years, an expanded view of the cell has
been possible due to impressive advances in all the ‘omics’
techniques (genomics, proteomics, metabolomics, etc.) and
high-throughput technologies for measuring different
classes of key intracellular molecules. ‘Systems biology’ has
recently emerged as a term to describe an approach that
considers genome-scale and cell-wide measurements in
elucidating processes and mechanisms (Stephanopoulos
FEMS Microbiol Rev 30 (2006) 187–214 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
199Genetic improvement of processes yielding microbial products
et al., 2004). Progress in strain development will depend, not
only on all the technologies mentioned above, but also on
the development of mathematical methods that facilitate the
elucidation of mechanisms and identification of genetic
targets for modification.
Integrating transcriptional and metabolite profiles from
21 strains of A. terreus producing different levels of lovasta-
tin and another 19 strains with altered (1)-geodin levels led
to an improvement in lovastatin production of over 50%
(Askenazi et al., 2003). The approach, named ‘association
analysis’, was used to reduce the complexity of profiling data
sets to identify those genes in which expression was most
tightly linked to metabolite production. Such an application
is suitable to all industrially useful organisms for which
genome data are limited.
A genome-wide transcript expression analysis called
‘massive parallel signature sequencing’ (Brenner et al.,
2000) was used successfully to discover new targets for
further improvement of riboflavin production by the fungus
A. gossypii (Karos et al., 2004). The authors identified 53
genes of known function, some of which could clearly be
related to riboflavin production. This approach also allowed
the finding of sites within the genome with high transcrip-
tional activity during riboflavin biosynthesis that are suita-
ble integration loci for the target genes found.
Gene expression analysis of wild-type and improved
production strains of Saccharopolyspora erythraea and S.
fradiae using microarrays of S. coelicolor revealed that
regulation of antibiotic biosynthetic enzymes as well as
enzymes involved in precursor metabolism were altered in
Table 2. Metabolic engineering of antibiotics
Target Result Reference
1.Manipulation of
structural genes
1.1. Amplifying an entire pathway 2.3-fold increase in cephamycin C Chen et al. (1988)1.2. Amplifying a segment of a pathway 7-fold increase in daunorubicin Otten et al. (1990)
30% increase in tetracenomycin C Decker et al. (1994)
3- to 4-fold increase in spinosyn Madduri et al. (2001)
1.3. Enhancing resistance 7- fold increase in neomycin Crameri & Davies (1986)
2. Manipulation of
regulatory genes
2.1. Amplifying positive regulatory genes
Pathway specific regulators 5-fold increase in spiramycin Geistlich et al. (1992)
1.6-fold increase in mithramycin Lombo et al. (1999)
Global regulators Increase in actinorhodin and
undecylprodigiosin
Voegtli et al. (1994)
2.2. Disrupting negative regulatory genes 1.5-3.5 fold increase in avermectin Hwang et al. (2003); Lee et al. (2000)
Pathway specific regulators increase in methylenomycin; in mito-
mycin C; 7- to 10-fold in lovastatin
Chater & Bruton (1985); Mao et al.
(1999); Kennedy et al. (1999)
Global regulators increase in actinorhodin and
undecylprodigiosin
Brian et al. (1996)
3. Engineering of
well-known pathways
3.1. Kinetic analysis 2- to 5-fold increase in cephamycin C Malmberg et al. (1995)
3.2. Increasing expression of rate-limiting
enzymes
30-fold increase in penicillin increase in
tylosin
Kennedy & Turner (1996)
Cox et al. (1987); Fishman et al.
(1987)
3.3. Eliminating accumulation and
excretion of intermediate
Elimination of excretion of penicillin N;
15% increase in cephalosporin C
Skatrud (1992)
3.4. Deleting gene leading to a side
product
Elimination of oligomycin production Ikeda et al. (1993)
3.5. Biosynthesizing compounds
previously made semisynthetically
Production of 7-
aminodeacetoxycephalosporanic acid
in Acremonium chrysogenum
Velasco et al. (2000)
3.6. Biosynthesizing new compounds Production of adipyl-7-ADCA, adipyl-7-
ACA in P. chrysogenum
Crawford et al. (1995)
3.7. Increasing oxygen availability 60% increase in erythromycin production Brunker et al. (1998); Minas et al.
(1998)
3.8. Enhancing precursor uptake 4-fold increase in deoxyerythronolide B
and 8, 8a-deoxyoleoandolide
Lombo et al. (2001)
FEMS Microbiol Rev 30 (2006) 187–214c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
200 J. L. Adrio & A. L. Demain
those mutated strains (Lum et al., 2004). The S. erythraea
overproducer expressed the entire erythromycin gene cluster
for several more days than the wild-type. It seemed that the
eryA gene and protein expression differences observed for
the overproducer could account for over 50% of the total
erythromycin titer increase. The S. fradiae mutant expressed
the tylosin biosynthetic genes in a similar way to the wild-
type strain; however, two genes, aco (encoding acyl-CoA
dehydrogenase) and icmA (encoding isobutyryl-CoA mu-
tase), were expressed more highly than in the wild-type
strain. The induction of these two genes could increase the
flux of metabolites from fatty acids to tylosin precursors in
the overproducer.
These recent technologies and mathematical approaches
will all contribute to the generation and characterization of
microorganisms able to synthesize large quantities of com-
mercially important metabolites. The ongoing sequencing
projects involving hundreds of genomes, the availability of
sequences corresponding to model organisms, new DNA
microarray and proteomics tools, as well as the new techni-
ques for mutagenesis and recombination described above
will accelerate strain improvement programs. The develop-
ment and combined application of these technologies will
help to develop what was already succinctly described
several years ago as ‘Inverse netabolic engineering’ (Bailey
et al., 1996), which means to identify, construct or calculate
a desired phenotype, identify the molecular basis of that
desirable property, and incorporate that phenotype into
another strain or other species by genetic and environmental
manipulations.
‘Directed evolution’ (= applied molecular evolu-
tion = directed molecular evolution) is a rapid and inexpen-
sive way of finding variants of existing enzymes that work
better than naturally occurring enzymes under specific
conditions (Kuchner & Arnold, 1997; Skandalis et al., 1997;
Arnold, 1998). The process involves evolutionary design
methods using random mutagenesis, gene recombination
and high-throughput screening (Arnold, 2001). Diversity is
initially created by in vitro mutagenesis of the parent gene
using repeated cycles of mutagenic polymerase chain reac-
tion (error-prone PCR) (Leung et al., 1989), repeated
oligonucleotide-directed mutagenesis (Reidhaar-Olson
et al., 1991), mutator strains (Bornscheuer et al., 1998) or
chemical agents (Taguchi et al., 1998). A key limitation of
these strategies is that they introduce random ‘noise’ muta-
tions into the gene at every cycle and hence improvements
are limited to small steps. This strategy has been used
successfully in different applications (Zhao et al., 2002).
‘Molecular breeding techniques’ (DNA shuffling, Mole-
cular BreedingTM) come closer to mimicking natural
recombination by allowing in vitro homologous
recombination (Ness et al., 2000). These techniques not
only recombine DNA fragments but also introduce point
mutations at a very low controlled rate (Stemmer, 1994;
Zhao & Arnold, 1997). Unlike site-directed mutagenesis,
this method of pooling and recombining parts of similar
genes from different species or strains has yielded remark-
able improvements in enzymes in a very short amount of
time (Patten et al., 1997). A step forward in this technique
was breeding a population with high genetic variability as a
starting point to generate diversity (DNA Family Shuffling).
This approach led to a 240- to 540-fold improvement in
cephalosporinase activity when four cephalosporinase genes
were shuffled as a starting point (Crameri et al., 1998).
When each of these genes was shuffled independently, only
eight-fold improvements were obtained. Innovations that
expand the formats for generating diversity by recombina-
tion include formats similar to DNA shuffling and others
with few or no requirements for parental gene homology
(Kurtzman et al., 2001; Lutz et al., 2001).
Random redesign techniques are currently being used to
generate enzymes with improved properties such as: activity
and stability at different pH values and temperatures (Ness
et al., 1999), increased or modified enantioselectivity (Jaeger
& Reetz, 2000), altered substrate specificity (Suenaga et al.,
2001), stability in organic solvents (Song & Rhee, 2001),
novel substrate specificity and activity (Raillard et al., 2001),
increased biological activity of protein pharmaceuticals and
biological molecules (Patten et al., 1997; Kurtzman et al.,
2001) as well as novel vaccines (Marshall, 2002; Locher et al.,
2004). Proteins from directed evolution work were already
on the market by 2000 (Tobin et al., 2000). These were green
fluorescent protein of Clontech (Crameri et al., 1996) and
Novo Nordisk’s LipoPrimes lipase.
‘Whole genome shuffling (WGS)’ is a novel technique for
strain improvement combining the advantage of multi-
parental crossing allowed by DNA shuffling with the recom-
bination of entire genomes. This method was applied
successfully to improved tylosin production in S. fradiae
(Zhang et al., 2002). Historically, 20 cycles of classical strain
improvement at Eli Lilly and Co. carried out over 20 years
employing about one million assays improved production
six-fold. In contrast, two rounds of WGS with seven early
strains each were sufficient to achieve similar results in one
year and involved only 24 000 assays. Such recursive
genomic recombination has also been used to improve the
acid-tolerance of a commercial lactic acid-producing Lacto-
bacillus sp. (Patnaik et al., 2002).
‘Combinatorial biosynthesis’ is being used for the dis-
covery of new and modified drugs (Hutchinson, 1998;
Reeves, 2003). In this technique, recombinant DNA techni-
ques are utilized to introduce genes coding for antibiotic
synthases into producers of other antibiotics or into non-
producing strains to obtain modified or hybrid antibiotics.
The first demonstration of this technology involved gene
transfer from a streptomycete strain producing the
FEMS Microbiol Rev 30 (2006) 187–214 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
201Genetic improvement of processes yielding microbial products
isochromanequinone antibiotic actinorhodin into strains
producing granaticin, dihydrogranaticin and mederomycin
(which are also isochromanequinones). This led to the
discovery of two new antibiotic derivatives, mederrhodin A
and dihydrogranatirhodin. Since this breakthrough paper by
Hopwood and coworkers (Hopwood et al., 1985), many
hybrid antibiotics have been produced by recombinant DNA
technology.
Hundreds of new polyketides have been made by combi-
natorial biosynthesis (Rodriguez & McDaniel, 2001; Dona-
dio & Sosio, 2003; Kantola et al., 2003). Manipulations
include: (i) deletion of one of the domains of a particular
module; (ii) addition of a copy of the thioesterase domain to
the end of an earlier module resulting in a shortened
polyketide; (iii) replacement of an AT domain of a polyke-
tide synthase (PKS) with an AT domain from another PKS,
resulting in addition of a methyl group at a particular site or
removal of a methyl group; (iv) addition of a reductive
domain(s) to a particular module, thus changing a keto
group to a double bond or to a methylene group; (v) use of
synthetic diketides delivered as N-acetylcysteamine thioe-
sters to load onto the active site of the ketosynthase (KS) in
module 2 and to be taken all the way to a novel final
product; (vi) replacement of the loading module of one
PKS with the loading module of another PKS, thus changing
the starter unit from propionate to acetate, for example; and
(vii) replacement of the hydroxylase or glycosylase enzymes
from one pathway to another, thus modifying the ring
structure with respect to OH groups and/or sugars (Staun-
ton, 1998).
As mentioned above, there are many examples of new
polyketides been made by combining polyketide biosyn-
thetic genes from different producers (McAlpine et al., 1987;
Epp et al., 1989; Donadio et al., 1991, 1993; Weber et al.,
1991; Hara & Hutchinson, 1992; Decker & Hutchinson,
1993; Hopwood, 1993; Katz & Donadio, 1993; Khosla et al.,
1993; McDaniel et al., 1993a, b, 1999; Hutchinson & Fujii,
1995; Kao et al., 1995; Tsoi & Khosla, 1995; Pacey et al., 1998;
Wohlert et al., 1998; Xue et al., 1999; Pfeifer & Khosla, 2001).
Some of these novel polyketides contain sugars at normally
unglycosylated positions (Trefzer et al., 2002) or as new
sugar moieties (Zhao et al., 1999; Mendez & Salas, 2001).
New anthracyclines (Bartel et al., 1990; Strohl et al., 1991;
Hwang et al., 1995; Niemi & Mantsala, 1995; Kim et al.,
1996; Ylihonko et al., 1996) and peptide antibiotics (Sta-
chelhaus et al., 1995) have also been made by combinatorial
biosynthesis.
Concluding remarks
Microorganisms produce many compounds of industrial
interest. These may be very large materials such as proteins,
nucleic acids, carbohydrate polymers, or even cells, or they
can be smaller molecules that can be essential for vegetative
growth or inessential, i.e. primary and secondary metabo-
lites, respectively. The power of the microbial culture in the
competitive world of commercial synthesis can be appre-
ciated by the fact that even simple molecules are made by
fermentation rather than by chemical synthesis. Most nat-
ural products are so complex that they probably will never
be made commercially by chemical synthesis. Strains iso-
lated from nature produce only tiny amounts of product.
This is because they need these secondary metabolites for
their own competitive benefit, and they do not overproduce
these metabolites. Regulatory mechanisms have evolved in
microorganisms which enable a strain to avoid excessive
production of its metabolites, thus, strain improvement
programs are required for commercial application. The goal
is to isolate cultures exhibiting desired phenotypes. Most
commonly, the ability of a strain to improve titer is what is
desired, although the other traits may also be improved on.
The tremendous increases in fermentation productivity and
the resulting decreases in costs have come about mainly by
using mutagenesis. In recent years, recombinant DNA
technology has also been applied. The promise of the future
is via extensive use of new genetic techniques such as: (i)
metabolic engineering accomplishing quantification and
control of metabolic fluxes and including inverse metabolic
engineering and transcript expression analyses such as
association analysis and massive parallel signature sequen-
cing; (ii) directed evolution; (iii) molecular breeding in-
cluding DNA shuffling and whole genome shuffling; and
(iv) combinatorial biosynthesis. These efforts will facilitate
not only the isolation of improved strains but also the
elucidation and identification of new genetic targets to be
used in strain improvement programs.
Acknowledgements
The authors thank the following colleagues for supplying
information: Richard H. Baltz, Graham S. Byng, Richard P.
Elander, David A. Hopwood, Daslav Hranueli, Krishna
Madduri, Jaraslav Spizek, William R. Strohl and J. Mark
Weber.
References
Aharonowitz Y, Cohen G & Martin JF (1992) Penicillin and
cephalosporin biosynthetic genes: structure, organization,
regulation and evolution. Ann Rev Microbiol 46: 461–495.
Anderson S, Marks CB, Lazarus R, Miller J, Stafford K, Seymour J,
Light D, Rastetter W & Estell D (1985) Production of 2-keto-
L-gulonate: an intermediate in L-ascorbate synthesis by a
genetically modified Erwinia herbicola. Science 230: 44–149.
FEMS Microbiol Rev 30 (2006) 187–214c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
202 J. L. Adrio & A. L. Demain
Arisawa A, Kawamura N, Narita T, Kojima I, Okamura K,
Tsunekawa H, Yoshioka T & Okamoto R (1996) Direct
fermentative production of acyltylosins by genetically-
engineered strains of Streptomyces fradiae. J Antibiot 49:
349–354.
Arnold FH (1998) Design by directed evolution. Acc Chem Res 31:
125–131.
Arnold FH (2001) Combinatorial and computational challenges
for biocatalyst design. Nature 409: 253–257.
Askenazi M, Driggers EM, Holtzman DA, et al. (2003) Integrating
transcriptional and metabolite profiles to direct the
engineering of lovastatin-producing fungal strains. Nature
Biotechnol 21: 150–156.
Bailey RM, Benitez T & Woodward A (1982) Saccharomyces
cerevisiae mutants resistant to catabolite repression: use in
cheese whey hydrolysate fermentation. Appl Environ Microbiol
44: 631–639.
Bailey JE, Sburlati A, Hatzimanikatis V, Lee K, Renner WA & Tsai
PS (1996) Inverse metabolic engineering: a strategy for
directed genetic engineering of useful phenotypes. Biotechnol
Bioeng 52: 109–121.
Bajon AM, Guiraud JP & Galzy P (1984) Isolation of an inulinase
derepressed mutant of Pichia polymorpha for the production
of fructose. Biotechnol Bioeng 26: 128–133.
Baltz RH (1986) Mutagenesis in Streptomyces. Manual of
Industrial Microbiology and Biotechnology (Demain AL &
Solomon NA, eds), pp. 184–190. American Society for
Microbiology, Washington, DC.
Baltz RH (1995) Gene expression in recombinant Streptomyces.
Gene Expression in Recombinant Microorganisms (Smith A, ed),
pp. 309–381. Marcel Dekker, New York.
Baltz RH (1998) Genetic manipulation of antibiotic producing
Streptomyces. Trends Microbiol 6: 76–83.
Baltz RH (1999) Mutagenesis. Encyclopedia of Bioprocessing
Technology: Fermentation, Biocatalysis, and Separation
(Flickinger MC & Drew SW, eds), pp. 1819–1822. Wiley, New
York.
Baltz RH (2003) Genetic engineering solutions for natural
products in actinomycetes. Handbook of Industrial Cell
Culture: Mammalian, Microbial, and Plant Cells (Vinci VA
& Parekh SR, eds), pp. 137–170. Humana Press, Totowa, NJ.
Baltz RH & Hosted TJ (1996) Molecular genetic methods for
improving secondary-metabolite production in
actinomycetes. Trends Biotechnol 14: 245–250.
Baltz RH, McHenney MA, Cantwell CA, Queener SW &
Solenberg PJ (1997) Applications of transposition mutagenesis
in antibiotic producing streptomyces. Ant v Leeuwenhoek 71:
179–187.
Baltz RH, Seno ET, Stonesifer J & Wild GM (1983) Biosynthesis
of the macrolide antibiotic tylosin: a preferred pathway from
tylactone to tylosin. J Antibiot 36: 131–141.
Bannerjee AB & Bose SK (1964) A rapid method for isolating
mutants of Bacillus subtilis producing increased or decreased
amounts of the antibiotic, mycobacillin. J Appl Bacteriol 27:
93–95.
Barredo JL, Diez B, Alvarez E & Martin JF (1989) Large
amplification of a 35-kb DNA fragment carrying two penicillin
biosynthetic genes in high penicillin producing strains of
Penicillium. chrysogenum Curr Genet 16: 453–459.
Barrios-Gonzalez J, Montenegro E & Martin JF (1993) Penicillin
production by mutants resistant to phenylacetic acid. J Ferm
Bioeng 76: 455–458.
Bartel PL, Zhu CB, Lampel JS, Dosch DC, Connors NC, Strohl
WR, Beale JM Jr. & Floss HG (1990) Biosynthesis of
anthraquinones by interspecies cloning of actinorhodin
biosynthesis genes in streptomycetes; clarification of
actinorhodin gene functions. J Bacteriol 172: 4816–4826.
Becker A, Katzen F, Puehler A & Ielpi L (1998) Xanthan gum
biosynthesis and application: a biochemical/genetic
perspective. Appl Microbiol Biotechnol 50: 145–152.
Berg DE & Berg CM (1983) The prokaryotic transposable
element Tn5. Bio/Technology 1: 417–435.
Bermejo LL, Welker NE & Papoutsakis ET (1998) Expression of
Clostridium acetobutylicum ATCC 824 genes in Escherichia coli
for acetone production and acetate detoxification. Appl
Environ Microbiol 64: 1079–1085.
Bigelas R (1989) Industrial products of biotechnology:
application of gene technology. Biotechnology, Vol. 7b
(Jacobson GK & Jolly SO, eds), pp. 230–259. VCH, Weinheim.
Binnie C, Warren M & Butler MJ (1989) Cloning and
heterologous expression in Streptomyces lividans of
Streptomyces rimosus genes involved in oxytetracycline
biosynthesis. J Bacteriol 171: 887–895.
Blumaerova M, Podojil M, Gauze GF, Maksikmova TS, Panos J &
Vanek Z (1980) Effect of cultivation conditions on the activity
of the beromycin producer Streptomyces glomeratus 3980 and
its spontaneous variants. Folia Microbiol 25: 213–218.
Blumaerova M, Podojil M, Gauze GF, Maksikmova TS, Panos J &
Vanek Z (1980) Spontaneous variability of Streptomyces
glomeratus, a producer of anthracycline antibiotics
beromycins. Folia Microbiol 25: 207–212.
Blumauerova M, Pokorny V, Stastna J, Hostalek Z & Vanek Z
(1978) Developmental mutants of Streptomyces
coeruleorubidis, a producer of anthracyclines: isolation and
preliminary characterization. Folia Microbiol 23: 177–182.
Bongaerts J, Kramer M, Muller U, Raeven L & Wubbolts M (2001)
Metabolic engineering for microbial production of aromatic
amino acids and derived compounds. Metab Eng 3: 289–300.
Bornscheuer UT, Altenbuchner J & Meyer HH (1998) Directed
evolution of an esterase for the stereoselective resolution of a
key intermediate in the synthesis of epothilones. Biotechnol
Bioeng 58: 554–559.
Bortol A, Nudel C, Fraile E, de Torres R, Giulietti A, Spencer JFT
& Spencer D (1986) Isolation of yeast with killer activity and
its breeding with an industrial baking strain by protoplast
fusion. Appl Microbiol Biotechnol 24: 414–416.
Brenner S, Johnson M, Bridgham J, et al. (2000) Gene expression
analysis by massively parallel signature sequencing (MPSS) on
microbead arrays. Nature Biotechnol 18: 630–634.
FEMS Microbiol Rev 30 (2006) 187–214 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
203Genetic improvement of processes yielding microbial products
Brewer SJ, Taylor PM & Turner MK (1980) An adenosine
triphosphate-dependent carbamoylphosphate-3-
hydroxymethylcephem O-carbamoyltransferase from
Streptomyces clavuligerus. Biochem J 185: 555–564.
Brian P, Riggle PJ, Santos RA & Champness WC (1996) Global
negative regulation of Streptomyces coelicolor antibiotic
synthesis mediated by an absA-encoded putative transduction
system. J Bacteriol 178: 3221–3231.
Brigidi P, Matteuzzi D & Fava F (1988) Use of protoplast fusion to
introduce methionine overproduction into Saccharomyces
cerevisiae. Appl Microbiol Biotechnol 28: 268–271.
Brunker P, Minas W, Kallio PT & Bailey JE (1998) Genetic
engineering of an industrial strain of Saccharopolyspora
erythrea for stable expression of the Vitreoscilla hemoglobin
gene (vhb). Microbiology 144: 2441–2448.
van Brunt J (1986) Fungi: the perfect hosts? Bio/Technology 4:
1057–1062.
van den Burg B, Vriend G, Veltman OR, Venema G & Eijsink
VGH (1998) Engineering an enzyme to resist boiling. Proc Natl
Acad Sci USA 95: 2056–2060.
Butler MJ, Friend EJ, Hunter IS, Kaczmarek FS, Sugden DA &
Warren M (1989) Molecular cloning of resistance genes and
architecture of a linked gene cluster involved in biosynthesis of
oxytetracycline by Streptomyces rimosus. Molec Gen Genet 215:
231–238.
Byng GS, Eustice DC & Jensen RA (1979) Biosynthesis of
phenazine pigments in mutant and wild-type cultures of
Pseudomonas aeruginosa. J Bacteriol 138: 846–852.
Cantwell C, Beckmann R, Whiteman P, Queener SW & Abraham
EP (1992) Isolation of deacetoxycephalosporin C from
fermentation broths of Penicillium chrysogenum
transformants: construction of a new fungal biosynthetic
pathway. Proc R Soc Lond (Biol) 248: 283–289.
Carlsen S (1990) Molecular biotechnology in the research and
production of recombinant enzymes. Industrial Use of
Enzymes: Technical and Economic Barriers (Wolnak B & Scher
M, eds), pp. 52–69. Bernard Wolnak and Associates, Chicago.
Cassinelli G, Di Matteo F, Forenza S, Ripamonti MC, Rivola G,
Arcamone F, Di Marco A, Cassaza AM, Soranzo C & Pratesi G
(1980) New anthracycline glycosides from Micromonospora. II.
Isolation, characterization and biological properties. J Antibiot
33: 1468–1473.
Castro JM, Liras P, Laiz L, Cortes J & Martin JF (1988)
Purification and characterization of the isopenicillin N
synthase of Streptomyces lactandurans. J Gen Microbiol 134:
133–141.
Chang LT & Elander RP (1979) Rational selection for improved
cephalosporin C productivity in strains of Acremonium
chrysogenum. Devel Indust Microbiol 20: 367–379.
Chang LT, McGrory EL & Elander RP (1980) Penicillin
production by glucose-derepressed mutants of Penicillium
chrysogenum. J Indust Microbiol 6: 165–169.
Chater KF & Bruton CJ (1985) Resistance, regulatory and
production genes for the antibiotic methylenomycin are
clustered. EMBO J 4: 1893–1897.
Chen CW, Lin HF, Kuo CL, Tsai HL & Tsai JFY (1988) Cloning
and expression of a DNA sequence conferring cephamycin C
production. Bio/Technology 6: 122–1224.
Chen W-Q, Yu ZN & Zheng Y-H (2004) Expression of Vitreoscilla
hemoglobin gene in Streptomyces fradiae and its effect on cell
growth and synthesis of tylosin. Chin J Antibiot 29: 516–520.
Chumpolkulwong N, Kakizono T, Nagai S & Nishio N (1997)
Increased astaxanthin production by Phaffia rhodozyma
mutants isolated as resistant to diphenylamine. J Ferm Bioeng
83: 429–434.
Coco EA, Narva KE & Feitelson JS (1991) New classes of
Streptomyces coelicolor A3(2) mutants blocked in
undecylprodigiosin (Red) biosynthesis. Mol Gen Genet 227:
28–32.
Cole GE, McCabe PC, Inlow D, Gelfand DH, Ben-Bassat A &
Innis MA (1988) Stable expression of Aspergillus awamori
glucoamylase in distiller’s yeast. Bio/Technology 6: 417–421.
Colombo AL, Crespi-Pevellino N, Grein A, Minghetti A & Spalla
CJ (1981) Metabolic and genetic aspects of the relationship
between growth and tetracycline production in Streptomyces
aureofaciens. Biotechnol Lett 3: 71–76.
Colon GE, Nguyen TT, Jetten MSM, Sinskey AJ &
Stephanopoulos G (1995) Production of isoleucine by
overexpression of ilvA in a Corynebacterium lactofermentum
threonine producer. Appl Microbiol Biotechnol 43: 482–488.
Cowan D (1996) Industrial enzyme technology. Trends Biotechnol
14: 177–178.
Cox KL, Fishman SE, Larson JL, Stanzak R, Reynolds PA, Yeh W-
K, Van Frank RM, Birmingham VA, Hershberger CL & Seno
ET (1987) Cloning and characterization of genes involved in
tylosin biosynthesis. Genetics of Industrial Microorganisms,
Part B (Alacevic M, Hranueli D & Toman Z, eds), pp. 337–346.
Pliva, Zagreb.
Crameri R & Davies JE (1986) Increased production of
aminoglycosides associated with amplified antibiotic
resistance genes. J Antibiot 39: 128–135.
Crameri R, Davies JE & Huetter R (1986) Plasmid curing and
generation of mutations induced with ethidium bromide in
streptomycetes. J Gen Microbiol 132: 819–824.
Crameri A, Raillard SA, Bermudez E & Stemmer WP (1998) DNA
shuffling of a family of genes from diverse species accelerates
directed evolution. Nature 391: 288–291.
Crameri A, Whitehorn A & Stemmer WPC (1996) Improved
green fluorescent protein by molecular evolution using DNA
shuffling. Nature Biotechnol 14: 315–319.
Crawford L, Stepan AM, Mcada PC, Rambosek JA, Conder MJ,
Vinci VA & Reeves CD (1995) Production of cephalosporin
intermediates by feeding adipic acid to recombinant
Penicillium chrysogenum strains expressing ring expansion
activity. Bio/Technology 13: 58–62.
Cropp TA, Wilson DJ & Reynolds KA (2000) Identification of a
cyclohexylcarbonyl CoA biosynthetic gene cluster and
application in the production of doramectin. Nature
Biotechnol 18: 980–983.
FEMS Microbiol Rev 30 (2006) 187–214c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
204 J. L. Adrio & A. L. Demain
Das A & Roy P (1981) Rapid strain selection for citric acid
production. Adv Biotechnol 1: 51–55.
Daum SJ & Lemke JR (1979) Mutational biosynthesis of new
antibiotics. Ann Rev Microbiol 33: 241–265.
De Maagd RA, Bravo A, Berry C, Crickmore N & Schnepf HE
(2003) Structure, diversity, and evolution of protein toxins
from spore-forming entemopathogenic bacteria. Annu Rev
Genet 37: 409–433.
Decker H & Hutchinson CR (1993) Transcriptional analysis of
the Streptomyces glaucescens tetracenomycin biosynthesis gene
cluster. J Bacteriol 175: 3887–3892.
Decker H, Summers RG & Hutchinson CR (1994)
Overproduction of the acyl carrier protein component of
a type II polyketide synthase stimulates production of
tetracenomycin biosynthetic intermediates in Streptomyces
glaucescens. J Antibiot 47: 54–63.
Demain AL & Elander RP (1999) The b-lactam antibiotics: past,
present, and future. Ant v Leeuwenhoek 75: 5–19.
Diez B, Gutierrez S, Barredo JL, van Solinger P, van der Voort
LHM & Martin JF (1990) The cluster of penicillin biosynthetic
genes. Identification and characterization of the pcbAB gene
encoding the a-aminoadipyl-cysteinyl-valine synthetase and
linkage to the pcbC and pcbDE genes. J Biol Chem 265:
16358–16365.
Diez B, Mellado E, Rodriguez R, Fouces R & Barredo JL (1997)
Recombinant microorganisms for industrial production of
antibiotics. Biotech Bioeng 55: 216–226.
Ditchburn P, Giddings B & MacDonald KD (1974) Rapid
screening for the isolation of mutants of Aspergillus nidulans
with increased penicillin yields. J Appl Bacteriol 37: 515–523.
Donadio S, McAlpine JB, Sheldon PA, Jackson MA & Katz L
(1993) An erythromycin analog produced by reprogramming
of polyketide synthesis. Proc Natl Acad Sci USA 90: 7119–7123.
Donadio S & Sosio M (2003) Strategies for combinatorial
biosynthesis with modular polyketide synthases. Comb Chem
High Throughput Screen 6: 489–500.
Donadio S, Staver MJ, McAlpine JB, Swanson SJ & Katz L (1991)
Modular organization of genes required for complex
polyketide biosynthesis. Science 252: 675–679.
Doran JB & Ingram LO (1993) Fermentation of crystalline
cellulose to ethanol by Klebsiella oxytoca containing
chromosomally integrated Zymomonas mobilis genes.
Biotechnol Prog 9: 533–538.
Dulaney EL & Dulaney DD (1967) Mutant populations of
Streptomyces viridifaciens. Trans N Y Acad Sci 29: 782–799.
Eggeling L & Sahm H (1999) Amino acid production: principles
of metabolic engineering. Metabolic Engineering (Lee SY &
Papoutsakis ET, eds), pp. 153–176. Marcel Dekker, New York.
Eggeling L, Sahm H & de Graaf AA (1996) Quantifying and
directing metabolic flux: application to amino acid
overproduction. Adv Biochem Eng Biotechnol 54: 1–30.
Elander RP (1995) Genetic engineering applications in the
development of selected industrial enzymes and therapeutic
proteins. Microbes for Better Living (Sankaran R & Manja KS,
eds), pp. 619–628. Defense Food Research Laboratory, Mysore,
India.
Elander RP (2003) Industrial production of b-lactam antibiotics.
Appl Microbiol Biotechnol 61: 385–392.
Elander RP (1969) Applications of microbial genetics to
industrial fermentations. Fermentation Adavances (Perlman D,
ed), pp. 89–114. Academic Press, New York.
Elander RP & Aoki H (1982) b-Lactam producing
microorganisms–their biology and fermentation behavior.
Chemistry and Biology of b-Lactam Antibiotics, Vol. 3 (Morin
RB & Gorman M, eds), pp. 83–153. Academic Press, New York.
Epp JK, Huber MLB, Turner JR, Goodson T & Schoner BE (1989)
Production of hybrid macrolide antibiotic in Streptomyces
ambofaciens and Streptomyces lividans by introduction of a
cloned carbomycin biosynthetic gene from Streptomyces
thermotolerans. Gene 85: 293–301.
Falch E (1991) Industrial enzymes–developments in production
and application. Biotech Adv 9: 643–658.
Farahnak F, Seki T, Ryu DDY & Ogrydziak D (1986) Construction
of lactose-assimilating and high-ethanol-producing yeasts by
protoplast fusion. Appl Environ Microbiol 51: 362–367.
Farris GA, Fatichenti F, Bifulco L, Berardi E, Deiana P & Satta T
(1992) A genetically improved wine yeast. Biotechnol Lett 14:
219–222.
Fierro F, Barredo JL, Diez B, Gutierrez S, Fernandez FJ & Martin
JF (1995) The penicillin gene cluster is amplified in tandem
repeats linked by conserved hexanucleotide sequences. Proc
Natl Acad Sci USA 92: 6200–6204.
Fishman SE, Cox K, Larson JL, Reynolds PA, Seno ET, Yeh WK,
Van Frank R & Hershberger CL (1987) Cloning genes for the
biosynthesis of a macrolide antibiotic. Proc Natl Acad Sci USA
84: 8248–8252.
Fujiwara T, Takahashi Y, Matsumoto K & Kondo E (1980)
Isolation of an intermediate of 2-deoxystreptamine
biosynthesis from a mutant of Bacillus circulans. J Antibiot 33:
824–829.
Fukaya M, Tayama K, Tamaki T, Tagami H, Okumura H,
Kawamura Y & Beppu T (1989) Cloning of the membrane-
bound aldehyde dehydrogenase gene of Acetobacter
polyoxogenes and improvement of acetic acid production by
use of the cloned gene. Appl Environ Microbiol 55: 171–176.
Gaucher GM, Lam KS, Grootwassink JWD, Neway J & Deo YM
(1981) The initiation and longevity of patulin biosynthesis.
Devel Indust Microbiol 22: 219–232.
Geistlich M, Losick R, Turner JR & Rao RN (1992)
Characterization of a novel regulatory gene governing the
expression of a polyketide synthase gene in Streptomyces
ambofaciens. Mol Microbiol 6: 2019–2029.
Gesheva V (1994) Isolation of spontaneous Streptomyces
hygroscopicus 111-81 phosphate-deregulated mutants
hyperproducing its antibiotic complex. Biotechnol Lett 16:
443–448.
Ghisalba O, Fuhrer H, Richter W & Moss S (1981) A genetic
approach to the biosynthesis of the rifamycin-chromophore in
Nocardia mediterranei. III. Isolation and identification of an
FEMS Microbiol Rev 30 (2006) 187–214 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
205Genetic improvement of processes yielding microbial products
early ansamycin-precursor containing the seven-carbon amino
starter-unit and three initial acetate/propionate-units of the
ansa chain. J Antibiot 34: 58–63.
Godfrey O (1973) Isolation of regulatory mutants of the aspartic
and pyruvic acid families and their effect on antibiotic
production in Streptomyces lipmanii. Antimicrob Agents
Chemother 4: 73–79.
Gomi S, Ikeda D, Nakamura H, Naganawa H, Yamashita F, Hotta
K, Kondo S, Okami Y, Umezawa H & Iitaka Y (1984) Isolation
and structure of a new antibiotic, indolizomycin, produced by
a strain SK2-52 obtained by interspecies fusion treatment.
J Antibiot 37: 1491–1494.
Gravius B, Glocker D, Pigac J, Pandza K, Hranueli D & Cullum J
(1994) The 387 kb linear plasmid pPZG101 of Streptomyces
rimosus and its interactions with the chromosome.
Microbiology 140: 2271–2277.
Grindley JF, Payton MA, van de Pol H & Hardy KG (1988)
Conversion of glucose to 2-keto-L-gulonate, an intermediate
in L-ascorbate synthesis by a recombinant strain of Erwinia
citreus. Appl Environ Microbiol 54: 1770–1775.
Guillouet S, Rodal AA, An G-H, Lessard PA & Sinskey AJ (1999)
Expression of the Escherichia coli catabolic threonine
dehydratase in Corynebacterium glutamicum and its effect on
isoleucine production. Appl Environ Microbiol 65: 3100–3107.
Gutierrez S, Diez B, Alvarez E, Barredo JL & Martin JF (1991)
Expression of the penDE gene of Penicillium chrysogenum
encoding isopenicillin N acyltransferase in Cephalosporium
acremonium: production of benzyl penicillin by the
transformants. Mol Gen Genet 225: 56–64.
Gutierrez S, Velasco J, Fernandez FJ & Martin JF (1992) The cefG
gene of Cephalosporium acremonium is linked to the cefEF gene
and encodes a deacetylcephalosporin C acetyltransferase
closely related to homoserine O-acetyltransferase. J Bacteriol
174: 3056–3064.
Gutierrez S, Velasco J, Marcos AT, Fernandez FJ, Fierro F, Barredo
JL, Diez B & Martin JF (1997) Expression of the cefG gene is
limiting for cephalosporin biosynthesis in Acremonium
chrysogenum. Appl Microbiol Biotechnol 48: 606–614.
Haavik HI & Froyshov O (1982) On the role of L-leucine in the
control of bacitracin formation by Bacillus licheniformis.
Peptide Antibiotics: Biosynthesis and Functions (Kleinkauf H &
von Doehren H, eds), pp. 155–159. Walter de Gruyter, Berlin.
Hamlyn PF & Ball C (1979) Recombination studies with
Cephalosporium acremonium. Genetics of Industrial
Microorganisms (Sebek OK & Laskin AI, eds), pp. 185–191.
American Society for Microbiology, Washington, DC.
Hammond JRM (1988) Brewery fermentation in the future.
J Appl Bacteriol 65: 169–177.
Hanssen R & Kirby R (1983) The induction by N-methyl-N0-
nitro-nitrosoguanidine of multiple closely linked mutations in
Streptomyces bikiniensis ISP5235 affecting streptomycin
resistance and streptomycin biosynthesis. FEMS Microbiol Lett
17: 317–320.
Hara O & Hutchinson CR (1992) A macrolide 3-O-
acyltransferase gene from the midecamycin-producing species
Streptomyces mycarofaciens. J Bacteriol 174: 5141–5144.
van Hartinsveldt W, van Zeijl CM, Harteeld GM, et al. (1993)
Cloning, characterization and overexpression of the phytase-
encoding gene (phyA) of Aspergillus niger. Gene 127: 87–94.
Hashiguchi K, Takesada H, Suzuki E & Matsui H (1999)
Construction of an L-isoleucine overproducing strain of
Escherichia coli K-12. Biosci Biotechnol Biochem 63: 672–679.
Hersbach GJM, van der Beck CP & van Dijck PWM (1984) The
penicillins: properties, biosynthesis, and fermentation.
Biotechnology of Industrial Antibiotics (Vandamme EJ, ed),
pp. 45–140. Marcel Dekker, New York.
Hershberger CL (1996) Metabolic engineering of polyketide
biosynthesis. Curr Opin Biotechnol 7: 560–562.
Hesketh A & Ochi K (1997) A novel method for improving
Streptomyces coelicolor A3(2) for production of actinorhodin
by introduction of rpsL (encoding ribosomal protein S12)
mutations conferring resistance to streptomycin. J Antibiot 50:
532–535.
Higashide E (1984) The macrolides; properties, biosynthesis, and
fermentation. Biotechnology of Industrial Antibiotics
(Vandamme EJ, ed), pp. 451–509. Marcel Dekker, New York.
Hirao T, Nakano T, Azuma T, Sugimoto M & Nakanishi T (1989)
L-Lysine production in continuous culture of an L-lysine
hyperproducing mutant of Corynebacterium glutamicum. Appl
Microbiol Biotechnol 32: 269–273.
Holzman D (1994) Engineered yeasts available but not yet used
for brewing. ASM News 60: 585.
Hopwood DA (1983) Actinomycete genetics and antibiotic
production. Biochemistry and Genetic Regulation of
Commercially Important Antibiotics (Vining LC, ed), pp. 1–23.
Addison Wesley, Reading, MA.
Hopwood DA (1993) Genetic engineering of Streptomyces to
create hybrid antibiotics. Curr Opin Biotechnol 4: 531–537.
Hopwood DA (1999) Forty years of genetics with Streptomyces:
from in vivo to in vitro to in silico. Microbiology 145:
2183–2202.
Hopwood DA, Malpartida F, Kieser HM, Ikeda H, Duncan J, Fujii
I, Rudd BAM, Floss HG & Omura S (1985) Production of
‘hybrid’ antibiotics by genetic engineering. Nature 314:
642–644.
Hosoya Y, Okamoto S, Muramatsu H & Ochi K (1998)
Acquisition of certain streptomycin resistant (str) mutations
enhances antibiotic production in bacteria. Antimicrob Agents
Chemother 42: 2041–2047.
Hu H & Ochi K (2001) Novel approach for improving the
production of antibiotic-producing strains by inducing
combined resistant mutations. Appl Environ Microbiol 67:
1885–1892.
Hutchinson CR (1998) Combinatorial biosynthesis for new drug
discovery. Curr Opin Microbiol 1: 319–329.
Hutchinson CR & Fujii I (1995) Polyketide synthase gene
manipulation: a structure-function approach in engineering
novel antibiotics. Annual Rev Microbiol 49: 201–238.
FEMS Microbiol Rev 30 (2006) 187–214c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
206 J. L. Adrio & A. L. Demain
Hwang Y-S, Kim E-S, Biro S & Choi C-Y (2003) Cloning and
analysis of a DNA fragment stimulating avermectin
production in various Streptomyces avermitilis strains. Appl
Environ Microbiol 69: 1263–1269.
Hwang CK, Kim HS, Hong YS, Kim YH, Hong SK, Kim SJ & Lee
JJ (1995) Expresssion of Streptomyces peucetius genes for
doxorubicin resistance and aklavinone 11-hydroxylase in
Streptomyces galilaeus ATCC 31133 and production of a hybrid
aclacinomycin. Antimicrob Agents Chemother 39: 1616–1620.
Ichikawa T, Date M, Ishikura T & Ozaki A (1971) Improvement
of kasugamycin-producing strain by agar piece method and
the prototroph method. Folia Microbiol 16: 218–224.
Ikeda M & Katsumata R (1995) Tryptophan production by
transport mutants of Corynebacterium glutamicum. Biosci
Biotechnol Biochem 59: 1600–1602.
Ikeda M & Katsumata R (1999) Hyperproduction of tryptophan
by Corynebacterium glutamicum with the modified pentose
pathway. Appl Environ Microbiol 65: 2497–2502.
Ikeda H, Takada Y, Pang C-H, Tanaka H & Omura S (1993)
Transposon mutagenesis by Tn4560 and applications with
avermectin-producing Streptomyces avermitilis. J Bacteriol 175:
2077–2082.
Ingram LO, Conway T, Clark DP, Sewell GW & Preston JF (1987)
Genetic engineering of ethanol production in Escherichia coli.
Appl Environ Microbiol 53: 2420–2425.
Jaeger KE & Reetz MT (2000) Directed evolution of
enantioselective enzymes for organic chemistry. Curr Opin
Chem Biol 4: 68–73.
Jarai M (1961) Genetic recombination in Streptomyces
aureofaciens. Acta Microbiol Acad Sci Hungary 8: 73–79.
Jin ZH, Lin JP, Xu ZN & Cen PL (2002) Improvement of
industry-applied rifamycin B-producing strain, Amycolatopsis
mediterranei, by rational screening. J Gen Appl Microbiol 48:
329–334.
Jin ZH, Wang MR & Cen PL (2002a) Production of teichoplanin
by valine-analogue resistant mutant strains of Actinoplanes
teichomyceticus. Appl Microbiol Biotechnol 58: 63–66.
Kacser H & Acerenza L (1993) A universal method for achieving
increases in metabolite production. Eur J Biochem 216:
361–367.
Kantola J, Kunnari T, Mantsala P & Ylihonko K (2003) Expanding
the scope of aromatic polyketides by combinatorial
biosynthesis. Comb Chem High Throughput Screen 6: 501–512.
Kao CM, Luo GL, Katz L, Cane DE & Khosla C (1995)
Manipulation of macrolide ring size by directed mutagenesis
of a modular polyketide synthase. J Amer Chem Soc 117:
9105–9106.
Karos M, Vilarino C, Bollschweiler C & Revuelta JL (2004) A
genome-wide transcription analysis of a fungal rivoflavin
overproducer. J Biotechnol 113: 69–76.
Kase H, Odakura Y & Nakayama K (1982) Sagamycin and the
related aminoglycosides: fermentation and biosynthesis. I.
Biosynthetic studies with the blocked mutants of
Micromonospora sagamiensis. J Antibiot 35: 1–9.
Katz L & Donadio S (1993) Polyketide synthesis: prospects for
hybrid antibiotics. Annu Rev Microbiol 47: 875–912.
Keller U (1983) Highly efficient mutagenesis of Claviceps
purpurea by using protoplasts. Appl Envir Microbiol 46:
580–584.
Kennedy J, Auclair K, Kendrew SG, Park C, Vederas JC &
Hutchinson CR (1999) Modulation of polyketide synthase
activity by accessory proteins during lovastatin biosynthesis.
Science 284: 1368–1372.
Kennedy J & Turner G (1996) d-(L-a-aminoadipyl)-L-cysteinyl-
D-valine synthetase is a rate limiting enzyme for penicillin
production in Aspergillus nidulans. Mol Gen Genet 253:
189–197.
Khetan A & Hu W-S (1999) Metabolic engineering of antibiotic
biosynthetic pathways. Manual of Industrial Microbiology and
Biotechnology (Demain AL & Davies JE, eds), pp. 717–724.
ASM Press, Washington, DC.
Khetan A & Hu W-S (1999) Metabolic engineering of antibiotic
biosynthesis for process improvement. Metabolic Engineering
(Lee SY & Papoutsakis ET, eds), pp. 177–202. Marcel Dekker,
New York.
Khosla C & Keasling JD (2003) Metabolic engineering for drug
discovery and development. Nature Rev/Drug Disc 2:
1019–1025.
Khosla C, McDaniel R, Ebert-Khosla S, Torres R, Sherman DH,
Bibb MJ & Hopwood DA (1993) Genetic construction and
functional analysis of hybrid polyketide synthases containing
heterologous acyl carrier proteins. J Bacteriol 175: 2197–2204.
Kibby JJ, McDonald IA & Rickards RW (1980) 3-Amino-5-
hydroxybenzoic acid as a key intermediate in ansamycin and
maytansinoid biosynthesis. J Chem Soc, Chem Comm 768–769.
Kieser HM, Kieser T & Hopwood DA (1992) A combined genetic
and physical map of the Streptomyces coelicolor A3(2)
chromosome. J Bacteriol 174: 5496–5507.
Kim KS, Cho NY, Pai HS & Ryu DDY (1983) Mutagenesis of
Micromonospora rosaria by using protoplasts and mycelial
fragments. Appl Environ Microbiol 46: 689–693.
Kim HS, Hong YS, Kim YH, Yoo OJ & Lee JJ (1996) New
anthracycline metabolites produced by the aklavinone 11-
hydroxylase gene in Streptomyces galilaeus ATCC 31133.
J Antibiot 49: 355–360.
Kimura E (2003) Metabolic engineering of glutamate production.
Adv Biochem Eng Biotechnol 79: 37–57.
Kinashi H & Shimaji M (1987) Detection of giant linear plasmids
in antibiotic producing strains of Streptomyces by the OFAGE
technique. J Antibiot 40: 913–916.
Kirimura K, Saragbin S, Rugsaseel S & Usami S (1992) Citric acid
production by 2-deoxyglucose-resistant mutants of Aspergillus
niger. Appl Microbiol Biotechnol 36: 573–577.
Kirst HA, Wild GM, Baltz RH, Seno ET, Hamill RL, Paschal JW &
Dorman DE (1983) Elucidation of structure of novel
macrolide antibiotics produced by mutant strains of
Streptomyces fradiae. J Antibiot 36: 376–382.
Kitamura S, Kase H, Odakura Y, Iida T, Shirahata K & Nakayama
K (1982) 2-Hydroxysagamicin: a new antibiotic produced by
FEMS Microbiol Rev 30 (2006) 187–214 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
207Genetic improvement of processes yielding microbial products
mutational biosynthesis of Micromonospora sagamiensis.
J Antibiot 35: 94–97.
Kitano K, Nozaki Y & Imada A (1985) Selective accumulation of
unsulfated carbapenem antibiotics by sulfate transport-
negative mutants of Streptomyces griseus subsp. cryophilus C-
19393. Agric Biol Chem 49: 677–684.
Koizumi S, Yonetani Y, Maruyama A & Teshiba S (2000)
Production of riboflavin by metabolically engineered
Corynebacterium ammoniagenes. Appl Microbiol Biotechnol 51:
674–679.
Kojima I, Fukagawa Y, Okabe M, Ishikura T & Shibamoto N
(1988) Mutagenesis of OA-6129 carbapenem-producing
blocked mutants and the biosynthesis of carbapenems.
J Antibiot 41: 899–907.
Komatsubara S, Taniguchi T & Kisumi M (1986) Overproduction
of aspartase of Escherichia coli K-12 by molecular cloning.
J Biotechnol 3: 281–291.
Kominek LA (1972) Biosynthesis of novobiocin by Streptomyces
niveus. Antimicrob Agents Chemother 1: 123–134.
Kramer M, Bongaerts J, Bovenberg R, Kremer S, Muller U, Orf S,
Wubbolts M & Raeven L (2003) Metabolic engineering for
microbial production of shikimic acid. Metab Eng 5: 277–283.
Kruse D, Kraemer R, Eggeling L, Rieping M, Pfefferle W, Tchieu
JH, Chung YJ, Saier MH Jr & Burkorski A (2002) Influence of
threonine exporters on threonine production in Escherichia
coli. Appl Microbiol Biotechnol 59: 205–210.
Kuchner O & Arnold FH (1997) Directed evolution of enzyme
catalysis. Trends Biotechnol 15: 523–530.
Kurtzman AL, Govindarajan S, Vahle K, Jones JT, Heinrichs V &
Patten PA (2001) Advances in directed protein evolution by
recursive genetic recombination: applications to therapeutic
proteins. Curr Opin Biotechnol 12: 361–370.
Kurzatkowski W, Kurylowicz W, Solecka J & Penyige A (1986)
Improvement of Streptomyces strains by the regeneration of
protoplasts. Biological, Biochemical and Biomedical Aspects of
Actinomycetes, Part A (Szabo G, Biro S & Goodfellow M, eds),
pp. 289–292. Academiai Kiado, Budapest.
Laffend LA, Nagarajan V & Nakamura CE (1996) Bioconversion
of a fermentable carbon source to 1,3-propanediol by a single
microorganism. Patent WO 96/53.796 (E. I. DuPont de
Nemours and Genencor International).
Lange C, Rittmann D, Wendisch VF, Bott M & Sahm H (2003)
Global expression profiling and physiological characterization
of Corynebacterium glutamicum grown in the presence of L-
valine. Appl Environ Microbiol 69: 2521–2532.
Lee JY, Hwang YS, Kim SS, Kim ES & Choi CY (2000) Effect of a
global regulatory gene, afsR2, from Streptomyces lividans on
avermectin production in Streptomyces avermitilis. J Biosci
Bioeng 89: 606–608.
Lee SH & Lee KJ (1995) Threonine dehydratases in different
strains of Streptomyces fradiae. J Biotechnol 43: 95–102.
Lee J-C, Park H-R, Park D-J, Son KH, Yoon K-H, Kim Y-B & Kim
C-J (2003) Production of teicoplanin by a mutant of
Actinoplanes teicomyceticus. Biotechnol Lett 25: 537–540.
Lee BK, Puar MS, Patel M, Bartner P, Lotvin J, Munayyer H &
Waitz JA (1983) Multistep bioconversion of 20-deoxo-20-
dihydro-12, 13-deepoxy-12, 13-dehydrorosaranolide to 22-
hydroxy-23-o-mycinosyl-20-deoxo-20-dihydro-12, 13-
deepoxy-rosaramicin. J Antibiot 36: 742–743.
Legmann R & Margalith P (1983) Interspecific protoplast fusion
of Saccharomyces cerevisiae and Saccharomyces mellis. Eur J
Appl Microbiol Biotechnol 18: 320–322.
Lein J (1986) The Panlabs penicillin strain improvement
program. Overproduction of Microbial Metabolites; Strain
Improvement and Process Control Strategies (Vanek Z &
Hostalek Z, eds), pp. 105–139. Butterworth Publishers,
Boston.
Lemke JR & Demain AL (1976) Preliminary studies on
streptomutin A. Eur J Appl Microbiol 2: 91–94.
Letisse F, Chevallereau P, Simon J-L & Lindley ND (2001) Kinetic
analysis of growth and xanthan gum production with
Xanthomonas campestris on sucrose, using sequentially
consumed nitrogen sources. Appl Microbiol Biotechnol 55:
417–422.
Leung DW, Chen E & Goeddel DV (1989) A method for random
mutagenesis of a defined DNA segment using a modified
polymerase chain reaction. Technique 1: 11–15.
Levy-Schil S, Debussche L, Rigault S, Soubrier F, Bacchette F,
Lagneaux D, Schleuniger J, Blanche F, Crouzet J & Mayaux JF
(1993) Biotin biosynthetic pathway in a recombinant strain of
Escherichia coli overexpressing bio genes: evidence for a
limiting step upstream from KAPA. Appl Microbiol Biotechnol
38: 755–762.
Lewis MJ, Ragot N, Berlant MC & Miranda M (1990) Selection of
astaxanthin-overproducing mutants of Phaffia rhodozyma
with b-ionone. Appl Environ Microbiol 56: 2944–2945.
Liras P (1988) Cloning of antibiotic biosynthetic genes. Use of
Recombinant DNA Techniques for Improvement of Fermentation
Organisms (Thompson JA, ed), pp. 217–253. CRC Press, Boca
Raton, FL.
Liu C-M (1982) Microbial aspects of polyether antibiotics:
activity, production and biosynthesis. Polyether antibiotics.
Naturally Occurring Acid Ionophores, Vol. 1. Biology (Westley
JW, ed), pp. 43–102. Marcel Dekker, New York.
Locher CP, Soong NW, Whalen RG & Punnonen J (2004)
Development of novel vaccines using DNA shuffling and
screening strategies. Curr Opi Mol Ther 6: 34–39.
Lombo F, Brana AF, Mendez C & Salas JA (1999) The
mithramycin gene cluster of Streptomyces argillaceus contains a
positive regulatory gene and two repeated DNA sequences that
are located at both ends of the cluster. J Bacteriol 181: 642–647.
Lombo F, Pfeifer B, Leaf T, Ou S, Kim YS, Cane DE, Licari P &
Khosla C (2001) Enhancing the atom economy of polyketide
biosynthetic processes through metabolic engineering.
Biotechnol Prog 17: 612–617.
Longacre A, Reimers JM, Gannon JE & Wright BE (1997) Flux
analysis of glucose metabolism in Rhizopus oryzae for the
purpose of increasing lactate yields. Fungal Genet Biol 21:
30–39.
FEMS Microbiol Rev 30 (2006) 187–214c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
208 J. L. Adrio & A. L. Demain
Luers F, Seyfried M, Daniel R & Gottschalk G (1997) Glycerol
conversion to 1,3 propanediol by Clostridium pasteurianum:
cloning and gene expression of the gene encoding 1,3
propanediol dehydrogenase. FEMS Microbiol Lett 154:
337–335.
Lum AM, Huang J, Hutchinson CR & Kao CM (2004) Reverse
engineering of industrial pharmaceutical-producing
actinomycete strains using DNA microarrays. Metab Eng 6:
186–196.
Lutz S, Ostermeier M, Moore GL, Maranas CD & Benkovic SP
(2001) Creating multiple-crossover DNA libraries
independent of sequence identity. Proc Natl Acad Sci USA 98:
11248–11253.
MacCabe AP, Riach MBR, Unkles SE & Kinghorn JR (1990) The
Aspergillus nidulans npeA locus consists of three contiguous
genes required for penicillin biosynthesis. EMBO J 9: 279–287.
Madduri K, Waldrom M, Matsushima P, Broughton MC,
Crawford K, Merlo DJ & Baltz RH (2001) Genes for the
biosynthesis of spinosyns: applications for yield improvement
in Saccharopolyspora spinosa. J Indust Microbiol Biotechnol 27:
399–402.
Malik VS (1979) Genetics of applied microbiology. Adv Genet 29:
37–126.
Malmberg LH, Hu W-S & Sherman DH (1995) Effects of
enhanced lysine epsilon-aminotransferase activity on
cephamycin biosynthesis in Streptomyces clavuligerus. Appl
Microbiol Biotechnol 44: 198–205.
Mao Y, Varoglu M & Sherman DH (1999) Molecular
characterization and analysis of the biosynthetic gene cluster
for the antitumor antibiotic mitomycin C from Streptomyces
lavendulae NRRL 2564. Chem Biol 6: 251–263.
Marshall SH (2002) DNA shuffling: induced molecular breeding
to produce new generation long-lasting vaccines. Biotechnol
Avd 20: 229–238.
Martin JF & Gil JA (1984) Cloning and expression of antibiotic
production genes. Bio/Technology 2: 63–72.
Martin JF, Gutierrez S & Demain AL (1997) b-lactams. Fungal
Biotechnology (Anke T, ed), pp. 91–127. Chapmam & Hall,
Weinheim.
Martin JF, Naharro G, Liras P & Villanueva JR (1979) Isolation of
mutants deregulated in phosphate control of candicidin
biosynthesis. J Antibiot 32: 600–606.
Martin JR, Perun TJ & Girolami RL (1966) Studies on the
biosynthesis of erythromycins. I. Isolation of an intermediate
glycoside, 3-alpha-L-mycarosylerythronolide B. Biochemistry
5: 2852–2856.
Martin JR & Rosenbrook WR (1967) Studies on the biosynthesis
of erythromycins. II. Isolation and structure of a biosynthetic
intermediate, 6-deoxyerythronolide B. Biochemistry 6:
435–440.
Masuda M, Takahashi K, Sakurai N, Yanagiya K, Komatsubara S
& Tosa T (1995) Further improvement of D-biotin production
by a recombinant strain of Serratia marcescens. Proc Biochem
30: 553–562.
Mathison L, Soliday C, Stepan T, Aldrich T & Rambosek J (1993)
Cloning, characterization, and use in strain improvement of
the Cephalosporium acremonium gene cefG encoding acetyl
transferase. Curr Genet 23: 33–41.
Matsuda A, Sugiura H, Matsuyama K, Matsumoto H, Ichikawa S
& Komatsu K-I (1992) Molecular cloning of acetyl coenzyme
A: deacetylcephalosporin C O-acetyltransferase cDNA from
Acremonium chrysogenum: sequence and expression of
catalytic activity in yeast. Biochem Biophys Res Commun 182:
995–1001.
Matthews PD & Wurtzel ET (2000) Metabolic engineering of
carotenoid accumulation in Escherichia coli by modulation of
the isoprenoid precursor pool with expression of
deoxyxylulose phosphate synthase. Appl Microbiol Biotechnol
53: 396–400.
Mayer H, Collins J & Wagner F (1980) Cloning of the penicillin
G-acylase gene of Escherichia coli ATCC 11105 on multicopy
plasmids. Enzyme Engineering, Vol. 5 (Weetall HH & Royer GP,
eds), pp. 61–69. Plenum, New York.
McAlpine JB, Tuan JS, Brown DP, Grebner KB, Whittern DN,
Buko A & Katz L (1987) New antibiotics from genetically
engineered actinomycetes. I. 2-Norerythromycins, isolation
and structural determinations. J Antibiot 40: 1115–1122.
McCann AK & Barnett JA (1984) Starch utilization by yeasts:
mutants resistant to carbon catabolite repression. Curr Genet
8: 525–530.
McCormick JRD (1965) Biosynthesis of the tetracyclines.
Biogenesis of Antibiotic Substances (Vanek Z & Hostalek Z, eds),
pp. 73–91. Publishing House of the Czechoslovak Academy of
Science, Prague.
McDaniel R, Ebert-Khosla S, Hopwood D & Khosla C (1993a)
Engineered biosynthesis of novel polyketides: manipulation
and analysis of an aromatic polyketide synthase with unproven
catalytic specificities. J Amer Chem Soc 115: 11671–11675.
McDaniel R, Ebert-Khosla S, Hopwood D & Khosla C (1993b)
Engineered biosynthesis of novel polyketides. Science 262:
1546–1550.
McDaniel R, Thamchaipenet A, Gustafsson C, Fu H, Betlach M,
Betlach M & Ashley G (1999) Multiple genetic modifications
of the erythromycin polyketide synthase to produce a library
of novel ‘‘unnatural’’ natural products. Proc Natl Acad Sci USA
96: 1846–1851.
McDougall S & Neilands JB (1984) Plasmid- and chromosome-
coded aerobactin synthesis in enteric bacteria: insertion
sequences flank operon in plasmid-mediated systems.
J Bacteriol 159: 300–305.
McGuire J, Thomas MC, Pandey RC, Toussaint M & White RJ
(1981) Biosynthesis of daunorubicin glycosides: analysis with
blocked mutants. Advances in Biotechnology, Vol. III,
Fermentation Products (Moo-Young M, ed), pp. 117–122.
Pergammon Press, New York.
McHenney MA & Baltz RH (1996) Gene transfer and
transposition mutagenesis in Streptomyces roseosporus:
mapping of insertions that influence daptomycin or pigment
production. Microbiology 142: 2363–2373.
FEMS Microbiol Rev 30 (2006) 187–214 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
209Genetic improvement of processes yielding microbial products
Mendelovitz S & Aharonowitz Y (1983) b-Lactam antibiotic
production by Streptomyces clavuligerus mutants impaired in
regulation of aspartokinase. J Gen Microbiol 129: 2063–2069.
Mendez C & Salas JA (2001) Altering the glycosylation pattern of
bioactive compounds. Trends Biotechnol 19: 449–456.
Mermelstein LD, Papoutsakis ET, Petersen DJ & Bennett GN
(1993) Metabolic engineering of Clostridium acetobutylicum
ATCC 824 for increased solvent production by enhancement
of acetone formation enzyme activities using a synthetic
operon. Biotechnol Bioeng 42: 1053–1060.
Minas W, Brunker P, Kallio PT & Bailey JE (1998) Improved
erythromycin production in a genetically engineered industrial
strain of Saccharopolyspora erythraea. Biotechnol Prog 1:
561–566.
Mindlin SZ (1969) Genetic recombination in the actinomycete
breeding. Genetics and Breeding of Streptomyces (Sermonti G &
Alacevic M, eds), pp. 147–159. Yugoslav Academy of Sciences
and Arts, Zagreb.
Miyagawa K, Kimura H, Nakahama K, Kikuchi M, Doi M,
Akiyama S & Nakao Y (1986) Cloning of the Bacillus subtilis
IMP dehydrogenase gene and its application to increased
production of guanosine. Bio/Technology 4: 225–228.
Mondou F, Shareck F, Morosoli R & Kleupfel D (1986) Cloning of
the xylanase gene of Streptomyces lividans. Gene 49: 323–329.
Moon YH, Tanabe T, Funahashi T, Shiuchi K, Nakao H &
Yamamoto S (2004) Identification and characterization of two
contiguous operons required for aerobactin transport and
biosynthesis in Vibrio mimicus. Microbiol Immunol 48:
389–398.
Morbach S, Sahm H & Eggeling L (1996) L-Isoleucine production
with Corynebacterium glutamicum: further flux increase and
limitation of export. Appl Environ Microbiol 62: 4345–4351.
Mori M & Shiio I (1983) Glutamate transport and production in
Brevibacterium flavum. Agric Biol Chem 47: 983–990.
Motamedi H, Wendt-Pientowski E & Hutchinson CR (1986)
Isolation of tetracenomycin C non-producing Streptomyces
glaucescens mutants. J Bacteriol 167: 575–580.
Nagaoka K & Demain AL (1975) Mutational biosynthesis of a
new antibiotic, streptomutin A, by an idiotroph of
Streptomyces griseus. J Antibiot 28: 627–635.
Nakamura CE & Whited GM (2003) Metabolic engineering for
the microbial production of 1,3-propanediol. Curr Opin
Biotechnol 14: 1–6.
Nakatsukasa WM & Mabe JA (1978) Galactose induced colonial
dissociation in Streptomyces aureofaciens. J Antibiot 31:
805–808.
Ness JE, Welch M, Giver L, Bueno M, Cherry JR, Borchert TV,
Stemmmer WP & Minshull J (1999) DNA shuffling of
subgenomic sequences of subtilisin. Nature Biotechnol 17:
893–896.
Ness JE, del Cardayre SB, Minshull J & Stemmer WP (2000)
Molecular breeding: the natural approach to protein design.
Adv Protein Chem 55: 261–292.
Neufeld RJ, Peleg Y, Rokem JS, Pines O & Goldberg I (1991) L-
Malic acid formation by immobilized Saccharomyces cerevisiae
amplified for fumarase. Enzyme Microb Technol 13: 991–996.
Nielsen J (2001) Metabolic engineering. Appl Microbiol Biotechnol
55: 263–283.
Niemi J & Mantsala P (1995) Nucleotide sequences and
expression of genes from Streptomyces purpurescens that cause
the production of new anthracyclines in Streptomyces galilaeus.
J Bacteriol 177: 2942–2945.
Nissen TL, Kielland-Brandt MC, Nielsen J & Villadsen J (2000)
Optimization of ethanol production in Saccharomyces
cerevisiae by metabolic engineering of the ammonium
assimilation. Metab Eng 2: 69–77.
Nozaki Y, Kitano K & Imada I (1984) Blocked mutants in the
biosynthesis of carbapenem antibiotics from Streptomyces
griseus subsp. cryophilus. Agric Biol Chem 48: 37–44.
Ohnishi J, Mitsuhashi S, Hayashi M, Ando S, Yokoi H, Ochiai K &
Ikeda M (2002) A novel methodology employing
Corynebacterium glutamicum genome information to generate
a new L-lysine-producing mutant. Appl Microbiol Biotechnol
58: 217–223.
Okamoto S, Lezhava A, Hosaka T, Okamoto-Hosoya Y & Ochi K
(2003) Enhanced expression of S-adenosylmethionine
synthetase causes overproduction of actinorhodin in
Streptomyces coelicolor A3(2). J Bacteriol 185: 601–609.
Okamoto-Hosoya Y, Sato T & Ochi K (2000) Resistance to
paromomycin is conferred by rpsL mutations, accompanied by
an enhanced antibiotic production in Streptomyces coelicolor
A3(2). J Antibiot 53: 1424–1427.
Okanishi M, Suzuki N & Furuta T (1996) Variety of hybrid
characters among recombinants obtained by interspecific
protoplast fusion in streptomycetes. Biosci Biotechnol Biochem
60: 1233–1238.
Omura S, Ikeda H & Tanaka H (1991) Selective production of
specific components of avermectins in Streptomyces
avermitilis. J Antibiot 44: 560–563.
Otten SL, Stutzman-Engwall J & Hutchinson CR (1990) Cloning
and expression of daunorubicin biosynthesis genes from
Streptomyces peucetius and S. peucetius subsp. caisius.
J Bacteriol 172: 3427–3434.
O’Neill GP, Kilburn DG, Warren RAJ & Miller RC Jr. (1986)
Overproduction from a cellulase gene with a high guanosine-
plus-cytosine content in Escherichia coli. Appl Environ
Microbiol 52: 737–743.
Pacey MS, Dirlam JP, Geldart RW, Leadlay PF, McArthur HA,
McCormick EL, Monday RA, O’Connell TN, Staunton J &
Winchester TJ (1998) Novel erythromycins from a
recombinant Saccharopolyspora erythraea strain NRRL
2338pIG1. I. Fermentation, isolation and biological activity.
J Antibiot 51: 1029–1034.
Palva I (1982) Molecular cloning of a-amylase gene from Bacillus
amyloliquefaciens and its expression in Bacillus subtilis. Gene
19: 81–87.
FEMS Microbiol Rev 30 (2006) 187–214c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
210 J. L. Adrio & A. L. Demain
Panchal CJ, Harbison A, Russell I & Stewart GG (1982) Ethanol
production of genetically modified strains of Saccharomyces.
Biotechnol Lett 4: 33–38.
Parekh S, Vinci VA & Strobel RJ (2000) Improvement of
microbial strains and fermentation processes. Appl Microbiol
Biotechnol 54: 287–301.
Patnaik R, Louie S, Gavrilovic V, Perry K, Stemmer WPC, Ryan
CM & del Cardayre S (2002) Genome shuffling of Lactobacillus
for improved acid tolerance. Nature Biotechnol 20: 707–712.
Patten PA, Howard RJ & Stemmer WP (1997) Applications of
DNA shuffling to pharmaceuticals and vaccines. Curr Opin
Biotechnol 8: 724–733.
Pearce CJ, Akhtar M, Barnett JEG, Mercier D, Sepulchre A-M &
Gero S (1978) Sub-unit assembly in the biosynthesis of
neomycin. The synthesis of 5-O-b-D-ribofuranosyl and 4-O-
b-D-ribofuranosyl-2,6-dideoxystreptamines. J Antibiot 31:
74–81.
Penttila ME, Suihko M-L, Lehtinen U, Nikkola M & Knowles JKC
(1987) Construction of brewer’s yeasts secreting fungal endo-
b-glucanase. Curr Genet 12: 413–420.
Penzikova GA & Levitov MM (1970) A study on transamidinase
activity with respect to streptomycin biosynthesis. Biologiya
39: 337–342.
Perez-Diaz JC & Clowes RC (1980) Physical characterization of
plasmids determining synthesis of a microcin which inhibits
methionine synthesis in Escherichia coli. J Bacteriol 141:
1015–1023.
Perkins JB & Pero J (1993) Bacillus subtilis and Other Gram
Positive Bacteria: Biochemistry, Physiology and Molecular
Genetics (Sonenshein AL, ed. in Chief), pp. 319–334. ASM
Press, Washington, DC.
Perkins JB, Sloma A, Hermann T, et al. (1999) Genetic
engineering of Bacillus subtilis for the commercial production
of riboflavin. J Indust Microbiol Biotechnol 22: 8–18.
Pfeifer BA, Admiraal SJ, Gramajo H, Cane DE & Khosla C (2001)
Biosynthesis of complex polyketides in a metabolically
engineered strain of Escherichia coli. Science 291: 1790–1792.
Pfeifer BA & Khosla C (2001) Biosynthesis of polyketides in
heterologous hosts. Microbiol Molec Biol Revs 65: 106–118.
Picataggio S, Rohver T, Deander K, Lanning D, Reynolds R,
Mielenz J & Eirich LD (1992) Metabolic engineering of
Candida tropicalis for the production of long-chain
dicarboxylic acids. Bio/Technology 10: 894–898.
Pina A, Calderon IL & Benitez T (1986) Intergeneric hybrids of
Saccharomyces cerevisiae and Zygosaccharomyces fermentati
obtained by protoplast fusion. Appl Environ Microbiol 51:
995–1003.
Podojil M, Blumauerova M, Culik K & Vanek Z (1984) The
tetracycyclines: properties, biosynthesis and fermentation.
Biotechnology of Industrial Antibiotics (Vandamme EJ, ed), pp.
259–279. Marcel Dekker, New York.
Pospisil S, Kopecky J, Prikrylova V & Spizek J (1999)
Overproduction of 2-ketoisovalerate and monensin
production by regulatory mutants of Streptomyces
cinnamonensis resistant to 2-ketobutyrate and amino acids.
FEMS Microbiol Lett 172: 197–204.
Pospisil S, Peterkova M, Krumphanzl V & Vanek Z (1984)
Regulatory mutants of Streptomyces cinnamonensis producing
monensin A. FEMS Microbiol Lett 24: 209–213.
Potera C (1997) Genencor & DuPont create ‘‘green’’ polyester.
Gen Eng News 17(11): 17.
Pramik MJ (1986) Genentech develops recombinant technique
for producing vitamin C. Gen Eng News 2(6): 9–12.
Radmacher A, Vaitsikova A, Burger U, Krumbach K, Sahm H &
Eggeling L (2002) Linking central metabolism with increased
pathway flux: L-valine accumulation by Corynebacterium
glutamicum. Appl Environ Microbiol 68: 2246–2250.
Raillard S, Krebber A, Chen Y, et al. (2001) Novel enzyme
activities and functional plasticity revealed by recombining
highly homologous enzymes. Chem Biol 8: 891–898.
Rancount DE, Stephenson JT, Vickell GA & Wood JM (1984)
Proline excretion by Escherichia coli K12. Biotechnol Bioeng 26:
74–80.
Reeves CD (2003) The enzymology of combinatorial
biosynthesis. Crit Rev Biotechnol 23: 95–147.
Reidhaar-Olson J, Bowie J, Breyer RM, Hu JC, Knight KL, Lim
WA, Mossing MC, Parsell DA, Shoemaker KR & Sauer RT
(1991) Random mutagenesis of protein sequences using
oligonucleotide cassettes. Methods Enzymol 208: 564–586.
Roberts M, Leavitt RW, Carbonetti NH, Ford S, Cooper RA
& Williams PH (1986) RNA-DNA hybridization analysis of
transcription of the plasmid ColV-K30 aerobactin gene cluster.
J Bacteriol 167: 467–472.
Rodriguez E & McDaniel R (2001) Combinatorial biosynthesis of
antimicrobials and other natural products. Curr Opin
Microbiol 4: 526–534.
Rohlin L, Oh MK & Liao JC (2001) Microbial pathway
engineering for industrial processes: evolution, combinatorial
biosynthesis and rational design. Curr Opin Microbiol 4:
350–355.
Roth JR & Ames BN (1966) Histidine regulatory mutants in
Salmonella typhimurium II. Histidine regulatory mutants
having altered histidyl-tRNA synthetase. J Mol Biol 22:
325–334.
Ryu DDY, Kim KS, Cho NY & Pai HS (1983) Genetic
recombination in Micromonospora rosaria by protoplast
fusion. Appl Environ Microbiol 45: 1854–1858.
Sahm H, Eggeling L & de Graaf AA (2000) Pathway analysis and
metabolic engineering in Corynebacterium glutamicum. Biol
Chem 381: 899–910.
Saito Y, Ishii Y, Hayashi H, et al. (1997) Cloning of genes coding
for L-sorbose and L-sorbosone dehydrogenases from
Gluconobacter oxydans and microbial production of 2-keto-
gulonate, a precursor of L-ascorbic acid, in a recombinant
G. oxydans strain. Appl Environ Microbiol 63: 454–460.
Sakurai N, Imai Y, Masuda M, Komatsubara S & Tosa T (1994)
Improvement of a d-biotin-hyperproducing recombinant
strain of Serratia marcescens. J Biotechnol 36: 63–73.
FEMS Microbiol Rev 30 (2006) 187–214 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
211Genetic improvement of processes yielding microbial products
Schreferl-Kunar G, Grotz M, Roehr M & Kubicek CP (1989)
Increased citric acid production by mutants of Aspergillus niger
with increased glycolytic capacity. FEMS Microbiol Lett 59:
297–300.
Shibasaki T, Hashimoto S, Mori H & Ozaki A (2000)
Construction of a novel hydroxyproline-producing
recombinant Escherichia coli by introducing a proline
4-hydroxylase gene. J Biosci Bioeng 90: 522–525.
Shier WT, Rinehart KL Jr & Gottlieb D (1969) Preparation of four
new antibiotics from a mutant of Streptomyces fradiae. Proc
Natl Acad Sci USA 63: 198–204.
Shiio I, Mori M & Ozaki H (1982) Amino acid aminotransferases
in an amino acid-producing bacterium, Brevibacterium
flavum. Agric Biol Chem 46: 2967–2977.
Shoemaker S, Schweickart V, Ladner M, Gelfand D, Kwok S,
Myambo K & Innis M (1983) Molecular cloning of exo-
cellobiohydrolase I derived from Trichoderma reesei strain L27.
Bio/Technology 1: 691–696.
Sills AM, Zygora PSJ & Stewart GG (1984) Characterization of
Schwanniomyces castelli mutants with increased productivity
of amylases. Appl Microbiol Biotechnol 20: 124–128.
Simpson IN & Caten CE (1979) Induced quantitative variation
for penicillin titre in clonal populations of Aspergillus nidulans.
J Gen Microbiol 110: 1–12.
Skandalis A, Encell LP & Loeb LA (1997) Creating novel enzymes
by applied molecular evolution. Chem Biol 4: 889–898.
Skatrud PL (1992) Genetic engineering of a beta-lactam
antibiotic biosynthetic pathway in filamentous fungi. Trends
Biotechnol 10: 324–329.
Skatrud PL, Fisher DL, Ingolia TD & Queener SW (1987)
Improved transformation of Cephalosporium acremonium.
Genetics of Industrial Microorganisms, Part B (Alacevic M,
Hranueli D & Toman Z, eds), pp. 111–119. Pliva, Zagreb.
Skatrud PL, Tietz AJ, Ingolia TD, Cantwell CA, Fisher DL,
Chapman JL & Queener SW (1989) Use of recombinant DNA
to improve production of cephalosporin C by Cephalosporium
acremonium. Bio/Technology 7: 477–485.
Smith A (1987) Enzyme regulation of desferrioxamine
biosynthesis: a basis for a rational approach to process
development. Fifth International Symposium on the Genetics of
Industrial Microorganisms, 1986 (Alacevic M, Hranueli D &
Toman Z, eds), pp. 513–527. Pliva, Zagreb.
Smith DJ, Bull JH, Edwards J & Turner G (1989) Amplification of
the isopenicillin N synthetase gene in a strain of Penicillium
chrysogenum producing high levels of penicillin. Mol Gen
Genet 216: 492–497.
Smith DJ, Burnham MKR, Bull JH, Hodgson JE, Ward JM,
Browne P, Brown J, Barton B, Earl AJ & Turner G (1990) b-
Lactam antibiotic biosynthesis genes have been conserved in
clusters in prokaryotes and eukaryotes. EMBO J 9: 741–747.
Solenberg PJ, Cantwell CA, Tietz AJ, McGilvray D, Queener SW &
Baltz RH (1996) Transposition mutagenesis in Streptomyces
fradiae: identification of a neutral site for the stable insertion
of DNA by transposon exchange. Gene 16: 67–72.
Sone H, Fujii T, Kondo K, Shimizu F, Tanaka J-I & Inoue T (1988)
Nucleotide sequence and expression of Enterobacter aerogenes
alpha-acetolactate decarboxylase gene in brewers’ yeast. Appl
Environ Microbiol 54: 38–42.
Song JK & Rhee JS (2001) Enhancement of stability and activity
of phospholipase A(1) in organic solvents by directed
evolution. Biochim Biophys Acta 1547: 370–378.
Spagnoli R, Cappalletti L & Toscano L (1983) Biological
conversion of erythronolide B, an intermediate of
erythromycin biogenesis, into new ‘‘hybrid’’ macrolide
antibiotics. J Antibiot 36: 365–375.
Stachelhaus T, Schneider A & Marahiel MA (1995) Rational
design of peptide antibiotics by targeted replacement of
bacterial and fungal domains. Science 269: 69–72.
Stahmann KP, Revuelta JL & Seulberger H (2000) Three
biochemical processes using Ashbya gossypii, Candida famata,
or Bacillus subtilis compete with chemical riboflavin
production. Appl Microbiol Biotechnol 53: 509–516.
Staunton J (1998) Combinatorial biosynthesis of erythromycin
and complex polyketides. Curr Opin Chem Biol 2: 339–345.
Stemmer WP (1994) Rapid evolution of a protein in vitro by
DNA shuffling. Nature 370: 389–391.
Stephanopoulos G (1999) Metabolic fluxes and metabolic
engineering. Metab Eng 1: 1–11.
Stephanopoulos G, Alper H & Moxley J (2004) Exploiting
biological complexity for strain improvement through systems
biology. Nature Biotechnol 22: 1261–1267.
Strohl WR (2001) Biochemical engineering of natural product
biosynthesis pathways. Metab Eng 3: 4–14.
Strohl WR, Bartel PL, Li Y, Connors NC & Woodman RH (1991)
Expression of polyketide biosynthesis and regulatory genes in
heterologous streptomycetes. J Indust Microbiol 7: 163–174.
Stutzman-Engwall K, Conlon S, Fedechko R, Kaczmarek F,
McArthur H, Krebber A, Chen Y, Minshull J, Raillard SA &
Gustafsson C (2003) Engineering the aveC gene to enhance the
ratio of doramectin to its CHC-B2 analogue produced in
Streptomyces avermitilis. Biotechnol Bioeng 82: 359–369.
Suenaga H, Mitsokua M, Ura Y, Watanabe T & Furukawa K
(2001) Directed evolution of biphenyl dioxygenase: emergence
of enhanced degradation capacity for benzene, toluene, and
alkylbenzenes. J Bacteriol 183: 5441–5444.
Sybesma W, Burgess C, Starrenburg M, van Sinderen D &
Hugenholtz J (2004) Multivitamin production in Lactococcus
lactis using metabolic engineering. Metab Eng 6: 109–115.
Taguchi S, Ozaki A & Momose H (1998) Engineering of a cold-
adapted protease by sequential random mutagenesis and a
screening system. Appl Environ Microbiol 64: 492–495.
Takebe H, Imai S, Ogawa H, Satoh A & Tanaka H (1989) Breeding
of bialaphos producing strains from a biochemical engineering
viewpoint. J Ferm Bioeng 67: 226–232.
Takeda K, Aihara K, Furumai T & Ito Y (1978) An approach to
the biosynthetic pathway of butirosins and the related
antibiotics. J Antibiot 31: 250–253.
FEMS Microbiol Rev 30 (2006) 187–214c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
212 J. L. Adrio & A. L. Demain
Tani Y, Lim W-J & Yang H-C (1988) Isolation of an L-
methionine-enriched mutant of a methylotrophic yeast,
Candida boidinii No. 2201. J Ferm Technol 66: 153–158.
Teeri T, Salovuori I & Knowles J (1983) The molecular cloning of
the major cellulase gene from Trichoderma reesei strain L27.
Bio/Technology 1: 696–699.
Teshiba S & Furuya A (1983) Mechanisms of 50-inosinic acid
accumulation by permeability mutants of Brevibacterium
ammoniagenes. II. Sensitivities of a series of mutants to various
drugs. Agric BiolChem 47: 1035–1041.
Theilgaard HA, van den Berg MA, Mulder CA, Bovenberg RAL &
Nielsen J (2001) Quantitative analysis of Penicillium
chrysogenum Wis54-1255 transformants overexpressing the
penicillin biosynthetic genes. Biotechnol Bioeng 72: 379–388.
Thompson CJ, Ward JM & Hopwood DH (1982) Cloning of
antibiotic resistance and nutritional genes in streptomycetes.
J Bacteriol 151: 668–677.
Thykaer J & Nielsen J (2003) Metabolic engineering of b-lactam
production. Metab Eng 5: 56–69.
Tobin MB, Gustafsson C & Huisman GW (2000) Directed
evolution: the ‘rational’ basis for ‘irrational’ design. Curr Opin
Struct Biol 10: 421–427.
Tong I-T, Liao JJ & Cameron DC (1991) 1,3-Propanediol
production by Escherichia coli expressing genes from the
Klebsiella pneumoniae dha region. Appl Environ Microbiol 57:
3541–3546.
Traxler P, Schupp T & Wehrli W (1982) 16,17-dihydrorifamycin S
and 16,17-dihydro-17-hydroxyrifamycin S, two novel
rifamycins from a recombinant strain C5/42 of Nocardia
mediterranei. J Antibiot 35: 594–601.
Trefzer A, Blanco G, Remsing L, et al. (2002) Rationally designed
glycosylated premithramycins: hybrid aromatic polyketides
using genes fom three different biosynthetic pathways. J Amer
Chem Soc 124: 6056–6062.
Troost T & Katz E (1979) Phenoxazinone biosynthesis:
accumulation of a precursor, 4-methyl-3-hydroxyanthranilic
acid, by mutants of Streptomyces parvulus. J Gen Microbiol 11:
121–132.
Tseng YH, Ting WY, Chou HC, Yang BY & Chun CC (1992)
Increase of xanthan production by cloning xps genes into wild-
type Xanthomonas campestris. Lett Appl Microbiol 14: 43–46.
Tsoi CJ & Khosla C (1995) Combinatorial biosynthesis of
unnatural natural products–the polyketide example. Chem
Biol 2: 355–362.
Unowsky J & Hoppe DC (1978) Increased production of the
antibiotic aurodox (X-5108) by aurodox-resistant mutants.
J Antibiot 31: 662–666.
Vaughn RV, Lotvin J, Puar MS, Patel M, Kershner A, Kalyanpur
MG, Marquez J & Waitz JA (1982) Isolation and
characterization of two 16-membered lactones: 20-
deoxorosaramicin and 20-deoxo-12,13-desepoxy-12,13-
dehydrorosaramicin aglycones, from a mutant strain of
Micromonospora rosaria. J Antibiot 35: 251–253.
Velasco J, Adrio JL, Moreno MA, Diez B, Soler G & Barredo JL
(2000) Environmentally safe production of 7-
aminodeacetoxycephalosporanic acid (7-ADCA) using
recombinant strains of Acremonium chrysogenum. Nature
Biotechnol 18: 857–861.
Vinci VA & Byng G (1999) Strain improvement by
nonrecombinant methods. Manual of Industrial Microbiology
and Biotechnology, 2nd edn (Demain AL & Davies JE, eds), pp.
103–113. ASM Press, Washington, DC.
Vinci VA, Hoerner TD, Coffman AD, Schimmel TG, Dabora RL,
Kirpekar AC, Ruby CL & Stieber RW (1991) Mutants of a
lovaststin-hyperproducing Aspergillus terreus deficient in the
production of sulochrin. J Indust Microbiol 8: 113–120.
Visser H, van Ooyen AJ & Verdoes JC (2003) Metabolic
engineering of the astaxanthin-biosynthetic pathway of
Xanthophyllomyces dendrorhous. FEMS Yeast Res 4: 221–231.
Voegtli M, Chang PC & Cohen SN (1994) afsR2: a previously
undetected gene encoding a 63-amino acid protein that
stimulates antibiotic production in Streptomyces lividans. Mol
Microbiol 14: 643–653.
Wackett LP (1997) Bacterial biocatalysis: stealing a page from
nature’s book. Nature Biotechnol 15: 415–416.
Wang MR, Ding H & Hu YJ (1996) The action of arginine and
valine in the biosynthesis of teicoplanin. Chin J Antibiot
21(suppl): 77–80.
Wang GY & Keasling JD (2003) Amplification of HMG-CoA
reductase production enhances carotenoid accumulation in
Neurospora crassa. Metab Eng 43: 193–201.
Weber JM, Leung JO, Swanson SJ, Idler KB & McAlpine JB (1991)
An erythromycin derivative produced by targeted gene
disruption in Saccharopolyspora erythraea. Science 252:
114–117.
Wesseling AC & Lago B (1981) Strain improvement by genetic
recombination of cephamycin producers, Nocardia
lactamdurans and Streptomyces griseus. Devel Indust Microbiol
22: 641–651.
Whiteley HR & Schnepf HE (1986) The molecular biology of
parasporal crystal body formation in Bacillus thuringiensis.
Ann Rev Microbiol 40: 549–576.
Wittmann C & Heinzle E (2002) Geneology profling through
strain improvement using metabolic network analysis-
metabolic flux geneology of several generations of lysine
producing corynebacteria. Appl Environ Microbiol 68:
5843–5859.
Wohlert S-E, Blanco G, Lombo F, et al. (1998) Novel hybrid
tetracenomycins through combinatorial biosynthesis using a
glycosyltransferase encoded by the elm-genes in cosmid 16F4
and which shows a very broad sugar substrate specificity.
J Amer Chem Soc 120: 10596–10601.
Xue Q, Hutchinson CR & Santi DV (1999) A multi-plasmid
approach to preparing large libraries of polyketides. Proc Natl
Acad Sci USA 96: 11740–11745.
Ylihonko K, Hakala J, Kunari T & Mantsala P (1996) Production
of hybrid anthracycline antibiotics by heterologous expression
of Streptomyces nogalater nogalamycin biosynthesis genes.
Microbiology 142: 1965–1972.
FEMS Microbiol Rev 30 (2006) 187–214 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
213Genetic improvement of processes yielding microbial products
Yoneda Y (1980) Increased production of extracellular enzymes
by the synergistic effect of genes introduced into Bacillus
subtilis by stepwise transformation. Appl Environ Microbiol 39:
274–276.
Yoshimoto A, Matsuzawa Y, Matsuhashi Y, Oki T, Takeuchi T &
Umezawa H (1981) Trisarubicinol, new antitumor
anthracycline antibiotic. JAntibiot 34: 1492–1494.
Yue S, Motamedi H, Wendt-Pienskowski E & Hutchinson CR
(1986) Anthracycline metabolites of tetracenomycin-
non-producing Streptomyces glaucescens. J Bacteriol 167:
581–586.
Yukawa H, Kurusu Y, Shimazu M, Yamagata H & Teresawa M
(1988) Stabilization of an Escherichia coli plasmid by a mini-F
fragment of DNA. J Indust Microbiol 2: 323–328.
Zamboni N, Mouncey N, Hohman HP & Sauer U (2003)
Reducing maintenance metabolism by metabolic engineering
of respiration improves rivoflavin production by Bacillus
subtilis. Metab Eng 5: 49–55.
Zhang Y-X, Perry K, Vinci VA, Powell K, Stemmer WPC &
del Cardayre SB (2002) Genome shuffling leads to rapid
phenotypic improvement in bacteria. Nature 415:
644–646.
Zhao L, Ahlert J, Xue Y, Thorson JS, Sherman DH & Liu H-W
(1999) Engineering a methymycin/pikromycin-calicheamicin
hybrid: construction of two new macrolides carrying a
designed sugar moiety. J Amer Chem Soc 121: 9881–9882.
Zhao H & Arnold FH (1997) Optimization of DNA shuffling
for high fidelity recombination. Nucleic Acids Res 25:
1307–1308.
Zhao H, Chockalingam K & Chen Z (2002) Directed evolution of
enzymes and pathways for industrial biocatalysis. Curr Opin
Biotechnol 13: 104–110.
Zhou S, Causey TB, Hasona A, Shanmugam KT & Ingram LO
(2003) Production of optically pure D-lactic acid in mineral
salts medium by metabolically engineered Escherichia coli
W3110. Appl Environ Microbiol 69: 399–407.
FEMS Microbiol Rev 30 (2006) 187–214c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
214 J. L. Adrio & A. L. Demain