Amino Acid Production by Corynebacterium

Amino Acid Production by Corynebacteriumglutamicum

Masato Ikeda and Seiki Takeno

Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

2 Amino Acid Fermentation Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

3 Overall Strategies for Strain Improvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

3.1 Terminal Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

3.2 Central Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

3.3 NADPH Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

3.4 Amino Acid Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

3.5 Respiratory Energy Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

3.6 Global Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

3.7 Stress Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

3.8 Feedstock Utilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

4 Recent Advances in Amino Acid Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

4.1 Glutamate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

4.2 Lysine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

4.3 Arginine and Citrulline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

4.4 Branched-Chain Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

4.5 Alanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

4.6 Serine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

4.7 Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

4.8 Cysteine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

5 Conclusions and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

Abstract During the half century following its discovery, the L-glutamate-producing

microorganism Corynebacterium glutamicum has played a leading role in the amino

acid fermentation industry. Due to its importance as an amino acid producer,

M. Ikeda (*) • S. Takeno

Faculty of Agriculture, Department of Bioscience and Biotechnology, Shinshu University,

8304 Minami-minoma, Nagano 399-4598, Japan

e-mail: [email protected]

H. Yukawa and M. Inui (eds.), Corynebacterium glutamicum,Microbiology Monographs 23, DOI 10.1007/978-3-642-29857-8_4,# Springer-Verlag Berlin Heidelberg 2013

107

mailto:[email protected]

C. glutamicum is also one of the best-investigated microorganisms, evidenced by the

extensive body of relevant literature and patents. In the past quarter century, various

genetic engineering tools and global analysis techniques for this bacterium have been

developed and successfully applied, giving a thorough understanding of its physiology

and permitting the development of efficient production strains. The advances enhanc-

ing the usefulness of this bacterium for amino acid production over the last decade can

be summarized in five points: (1)Metabolic engineering strategies are expanding from

the core biosynthetic pathways to include central metabolism, cofactor-regeneration

systems, uptake and export systems, energy metabolism, global regulation, and stress

responses; strain improvement is bound to thereby optimize entire cellular systems.

(2) Systems biology for this bacterium is almost capable of predicting targets to be

engineered and metabolic states that will yield maximum production; these

developments should allow rational metabolic design. (3) Rapid strides in genome

analysis have revolutionized strain improvement methodology, allowing

reengineering of more efficient producers through knowledge of the mutations that

have accumulated over years of industrial strain development. (4) The spectra of both

products and assimilable carbon sources of this bacterium have expanded, leading to

the development of, e.g., production strains of serine and methionine that could not be

produced effectively from glucose and strains that can utilize alternative feedstocks

that do not compete with human food or energy sources. (5) Recent identification of a

putative mechanosensitive channel as a possible glutamate exporter has provided

valuable insight into the glutamate production mechanism which had long been the

central question concerning the industrial biotechnology of C. glutamicum. Thischapter describes advances in the production of amino acids by C. glutamicum, withspecial focus on the technology and strategies for molecular strain improvement.

1 Introduction

Amino acids have a wide variety of characteristics in terms of nutritional value,

taste, medicinal action, and chemical properties, and thus have many potential uses,

e.g., in food additives, feed supplements, pharmaceuticals, cosmetics, polymer

materials, and agricultural chemicals. As each new use is developed, demand for

that type of amino acid grows rapidly and is followed by the development of mass

production technology for that amino acid. The annual world production of amino

acids has increased year by year (Fig. 1) and is currently estimated at more than 3.7

million metric tons (Ikeda 2003; Hermann 2003; Ajinomoto 2007). According to a

recent market research report (McWilliams 2010) and other relevant publications

(Leuchtenberger et al. 2005; Ajinomoto 2007), the global market for amino acids is

estimated to be approximately US$6.6 billion in 2009 and is growing at an annual

rate of 8–10%. Figure 2 shows the estimated global markets for amino acids of

different applications in 2009. So-called feed amino acids L-lysine, DL-methionine,

L-threonine, and L-tryptophan have the largest share of the market, generating US

$3.7 billion in 2009. The second largest share belongs to food additives, which are

comprised mainly of the flavor-enhancer monosodium glutamate and the amino

108 M. Ikeda and S. Takeno

acids L-aspartate and L-phenylalanine used as materials for the peptide sweetener

L-aspartyl L-phenylalanyl methyl ester (aspartame).

Most L-amino acids are manufactured through microbial processes, mainly

through fermentation. A pioneering study that represented the introduction of

fermentation to the industrial production of amino acids was the discovery of the

L-glutamate-producing bacterium Corynebacterium glutamicum by a research group

at Kyowa Hakko Kogyo Co. (Kinoshita et al. 1957; Udaka 1960). The success story

of the isolation of this industrially important bacterium has been described by Udaka

(2008). Within a few years after the first report of L-glutamate fermentation by

C. glutamicum, the company found that a homoserine-auxotrophic mutant of

C. glutamicum produced large amounts of L-lysine in liquid medium (Nakayama

et al. 1961), which enabled the industrial production of L-lysine by fermentation.

These successive achievements opened new avenues to the amino acid fermentation

industry. Nowadays, fermented amino acids represent highly important biotechnol-

ogy products in terms of both volume and economic value. In the world market for

fermentation products (ethanol excluded), which was estimated US$14.1 billion in

2004, the fermented amino acids constitute the second largest share (23%), after

antibiotics (35%), with an average annual growth rate of approximately 9%

(Leuchtenberger et al. 2005). C. glutamicum, which plays a principal role in the

1985

L-Glu(50%)

DL-Met(37%)

L-Lys(10%)

Others(3%)

700 L-Glu(59%)

L-Lys(18%)

DL-Met(20%)

Others(3%)

1996

1,700

2006

L-Glu(53%)

L-Lys(26%)

DL-Met(16%)

Others(2%)

L-Thr(3%)

3,700

Fig. 1 Changes in world annual production quantities of amino acids. The numbers in the squaresindicate the estimated amounts of amino acid production (1,000 metric tons)

Feed supplements(US $3.7 billion)

L-LysDL-MetL-ThrL-Trp

Food additives(US $2.1 billion)

L-GluL-PheL-Asp

Others (US $0.8 billion)

Fig. 2 Estimated 2009 global markets for amino acids segmented by applications

Amino Acid Production by Corynebacterium glutamicum 109

process of amino acid fermentation, is therefore also highly important, as

demonstrated by the increasing number of relevant research papers (Fig. 3).

Figure 3 also shows the main topics in amino acid fermentation and strain

development technology during the decades since such research began. In the early

stages, the breeding of production strains depended mostly on repeated random

mutation and selection, which resulted in many commercially potent producers

(Kinoshita and Nakayama 1978; Leuchtenberger 1996; Ikeda 2003). In 1979, a

procedure for protoplast fusion in this species was reported (Kaneko and Sakaguchi

1979); this development allowed genetic recombination in vivo (Karasawa et al.

1986). Applications of recombinant DNA technology to C. glutamicum for amino

acid production started in the 1980s when host vector systems were developed for the

microbe (Katsumata et al. 1984; Santamaria et al. 1984; Yoshihama et al. 1985;

Miwa et al. 1985). Following this, various tools for genetic engineering of thismicrobe

were exploited in the 1990s (Haynes and Britz 1990; Sch€afer et al. 1990; Schwarzerand P€uhler 1991; Ikeda and Katsumata 1998; van der Rest et al. 1999). These

molecular techniques were first applied to strain improvement for the production of

only a few amino acids such as glutamate, lysine, threonine, and the aromatic amino

acids (Ozaki et al. 1985; Katsumata et al. 1986; Ikeda and Katsumata 1992, 1999;

Katsumata and Ikeda 1993; Eggeling et al. 1998; Ikeda et al. 1994, 1999; Kimura et al.

1999). In the 2000s, research and development activities expanded to include other

amino acids such as arginine, the branched-chain amino acids, alanine, and a few

others in which high-production yields had not yet been achieved through fermenta-

tion, such as serine, methionine, and cysteine (Radmacher et al. 2002; M€ockel et al.2002;Wada et al. 2002; Peters-Wendisch et al. 2005; Ikeda et al. 2009b; Holatko et al.

2009; Jojima et al. 2010).

’50 ’60 ’70 ’80 ’90 ’00

Discovery of C. glutamicum

Host-vector systems

Genome sequencing

Glu fermentationGenetic engineering tools

Protoplast fusion

Thr fermentationLys fermentation

Global analysis techniques

0

100

200

Ann

ual nu

mbe

r of

rese

arch

pap

ers

’10

Fig. 3 History of amino acid fermentation and strain development technology in Corynebacte-rium glutamicum, together with the annual number of research papers relevant to this microbe


Most fermented amino acid production processes currently rely on modified

C. glutamicum and Escherichia coli. Almost all amino acids can be produced

technologically by either organism, but C. glutamicum is used for the industrial

production of relatively large-scale bulk amino acids such as glutamate and

lysine as well as glutamine and arginine, while E. coli is predominantly used for

the production of threonine and the branched-chain amino acids. Although

C. glutamicum has several industrially important characteristics such as its high

growth yield even under conditions of high sugar concentration, it has one draw-

back: its optimal growth temperature is around 30 �C, which is lower than that of

E. coli. For this reason, the use of C. glutamicum may be economically disadvanta-

geous, especially in tropical regions, because of the substantial cost of the utilities

necessary to maintain the optimum fermentation temperature. In an attempt to

address this issue, Corynebacterium efficiens, a related species with an optimal

growth temperature near 40 �C, has been isolated and examined for the relevant

genetic traits and glutamate production ability at higher temperatures (Fudou et al.

2002; Nishio et al. 2003; Kimura 2005). Comparative genome sequence analysis

between C. glutamicum and C. efficiens, as well as amino acid substitution analysis,

has suggested that three types of amino acid substitutions in C. efficiens (K to R,

S to A, and S to T) are important for its thermotolerance (Nishio et al. 2003).

In general, commercially potent producers have been developed by the stepwise

accumulation of beneficial genetic and phenotypic characteristics in one back-

ground through classical mutagenesis and/or recombinant DNA technology. Such

improvements involve strains capable not only of producing amino acids at higher

yields but also of producing lower quantities of by-products, as the removal of

by-products dominates the costs of downstream processing (Ikeda 2003;

Marienhagen and Eggeling 2008). The current production yields toward sugar

(w/w %) can be estimated as follows: lysine hydrochloride, 45–55; glutamate,

45–55; glutamine, 35–45; arginine, 30–40; threonine, 40–50; isoleucine, 20–30;

valine, 30–40; alanine, 45–55; serine, 30–35; methionine, 15–20; tryptophan,

20–25; phenylalanine, 20–25; and histidine, 20–30.

During the last decade, genomic and other “omics” data have accumulated for

C. glutamicum, profoundly affecting strain development methods and providing a

global understanding of this microbe (Wittmann and Heinzle 2002; Ikeda and

Nakagawa 2003; Kalinowski et al. 2003; Strelkov et al. 2004; Yukawa et al.

2007). This work has revealed new regulatory networks and functions that had

not previously been identified in this bacterium. A novel methodology that merges

genomics with classical strain improvement has been developed and used to

rationally reconstruct classically derived production strains (Ikeda et al. 2006,

2009b). In the present chapter, the processes by which amino acids are produced

by C. glutamicum are first briefly described, and strategies for molecular strain

improvement are subsequently briefly summarized and illustrated with relevant

examples. Lastly, recent advances in amino acid production by this microbe are

provided from both basic and applied perspectives. The most common approaches

to strain improvement including conventional mutagenesis and screening have been


omitted because their descriptions can be found in many other publications

(Kinoshita and Nakayama 1978; Leuchtenberger 1996; Ikeda 2003).

2 Amino Acid Fermentation Processes

Fermentation processes typically comprise three steps: cultivation of amino acid-

producing strains, purification of amino acids from fermented broth, and wastewater

treatment. The economy of these processes depends mainly on the cost of the carbon

source, the fermentation yield, purification yield, and the productivity of the overall

process. To date, many technologies have been developed in attempts to establish

economically competitive processes.

Industrial amino acid fermentations are usually performed by means of batch or

fed-batch processes using aerated agitated tank fermentors or airlift tank fermentors

in the 50- to 500-kL size range. Although batch processes are easy to run and do not

require additional tanks for feeding nutrients, industrial processes predominantly

adopt fed-batch, mainly because fed-batch processes provide improved overall

productivity by increasing yields and reducing fermentation periods, especially

when a high or changing concentration of a certain nutrient affects the yield or

productivity of the process. By lowering the initial concentration of sugar and using

subsequent feeding, the total culture period can be shortened, especially the lag

time, and in some cases the yield can also be increased. In a process where

an auxotrophic strain is employed, the yield can be maximized by growing the

auxotrophic strain in a limited amount of the required nutrient through feeding

that nutrient at a controlled rate. An example of this is tryptophan fermentation

by a phenylalanine- and tyrosine-auxotrophic strain of C. glutamicum (Ikeda

and Katsumata 1999). Furthermore, the fed-batch techniques allow substrate

concentrations to be kept low enough to prevent oxygen limitation which causes

a decreased yield with concomitant acid by-production (Ikeda 2003). In these cases,

glucose-limited fed-batch cultures are commonly employed in industrial processes.

To improve overall productivity further, it is possible to extend fed-batch

fermentation by drawing out part of the broth one or more times during the process

and refilling it through nutrient feeding (semi-continuous fermentation), or shifting

the fermentation from batch to continuous culture, where fresh medium containing

all nutrients is fed into a fermentor at a specific rate while the same quantity of broth

with a portion of the microorganisms is continuously taken from the fermentor, thus

maintaining a constant culture volume. Continuous fermentation was investigated

with a C. glutamicum lysine-producing mutant (Hirao et al. 1989); the strain in that

study yielded stable lysine production for more than 300 h, with a maximum lysine

concentration of 105 g/L and a maximum volumetric productivity value of

5.6 g/L/h. Though this strain was able to produce 100 g/L lysine within 48 h in a

fed-batch process, its productivity did not exceed 2.1 g/L/h. This means that the

productivity seen in the continuous fermentation is more than 2.5 times higher than

that seen in the fed-batch culture. The feasibility of the continuous fermentation


process is governed chiefly by the genetic stability of a production strain as well as

by the conduciveness of the process to the maintenance of the purity of the culture

over the long term. The former problem has been dealt with in the continuous

fermentation of arginine by C. glutamicum, formally classified as Corynebacteriumacetoacidophilum (Azuma and Nakanishi 1988; Azuma et al. 1988); the original

arginine producer gave rise to mutated variants with reduced productivity during

continuous culture, but one derivative isolated from the continuous culture broth

no longer caused the appearance of such variants, resulting in stable arginine

production for more than 250 h. The derivative strain was found to be less sensitive

to arginine with respect to both growth and production than the original producer,

though the genetic element(s) responsible for the change has not yet been identified.

The production profiles of amino acid fermentation processes can be growth

dependent or growth independent. Lysine and arginine are generally produced by

C. glutamicum in a growth-dependent manner, for example, while glutamate and

glutamine are produced by C. glutamicum in a growth-independent manner.

In growth-independent processes, continuous culture may not afford a higher

productivity than batch or fed-batch processes do because cells are always kept in

the growth phase. The productivity of such fermentation is likely to be improved by

incorporating cell recycling techniques in a fed-batch or continuous fermentation

process (Ishizaki et al. 1993).

Other fundamentals of amino acid fermentation processes, such as fermentation

operations, raw materials, downstream processing, and waste liquor treatment, are

thoroughly described in other publications (Leuchtenberger 1996; Ikeda 2003;

Hermann 2003).

3 Overall Strategies for Strain Improvement

The past quarter century has seen rapid developments in strain development

technology. Metabolic engineering has repeatedly led to successful yield

improvements, especially in the field of amino acid production by C. glutamicum(Eggeling and Bott 2005; Wendisch 2007; Burkovski 2008). In this latter field, the

targets of metabolic engineering have expanded beyond the core biosynthetic

pathways leading to products of interest and now include central metabolism,

cofactor-regeneration systems, uptake and export systems, energy metabolism,

global regulation, and stress responses (Fig. 4). This means that strain development

is beginning to achieve the optimization of entire cellular systems. In addition,

the product spectrum of C. glutamicum also has been expanded, and metabolic

engineering has been applied to the production of amino acids that could not be

produced effectively from glucose, such as serine and methionine. Furthermore,

strains are being engineered with environmental concerns in mind: some utilize

alternative feedstocks, such as whey, lignocellulose-derived xylose and arabinose,

and glycerol, which do not compete with human food or energy sources. The

strategies of rational strain improvement are broadly described here with lysine


production as the predominant example, though the strategies are in principle

applicable to the production of other amino acids as well.

3.1 Terminal Pathways

An essential first step for overproducing an amino acid of interest is the elimination of

bottlenecks in the core biosynthetic pathways leading to that amino acid. Strategies

for achieving this include deletion of competing pathways and desensitization of

regulatory enzymes. In lysine production, this objective can be accomplished through

mutations that reduce expression or activity of homoserine dehydrogenase or desensi-

tize aspartokinase to feedback inhibition by lysine (Shiio and Miyajima 1969;

Sano and Shiio 1971; Kase and Nakayama 1974; Pfefferle et al. 2003). These two

modifications, when combined, appear to be synergistic for production (Ohnishi et al.

2002).C. glutamicummutants with either or bothmodifications generally show lysine

production yields of 10–30% fromglucose (Sano and Shiio 1971;Kase andNakayama

1974; Ohnishi et al. 2002). A more sophisticated strategy has been applied to the

pathway engineering of a classically derived tryptophan-producing C. glutamicumstrain, resulting in remarkable gains in titer (grams of product per liter), yield (grams of

product per grams of sugar), and productivity (grams of product per liter per hour)

(Katsumata and Ikeda 1993; Ikeda et al. 1994). This significant improvement involves

Energy metabolism

Glucose

H2OH+

O2

NADPH

ADP ATP

NADP+

Amino acid

NADPH

NADP+

ATP

ADP

Amino acid

Stress responsesAlternativecarbon sources

Feedstock Utilization

Uptake & Export

Signal

H+NADHNAD+

Centralmetabolism

Cofactorregeneration

Globalregulation

Terminal pathways

H+

Fig. 4 Targets of metabolic engineering for amino acid production demonstrated inC. glutamicum


not only systematic genetic modifications that efficiently channel carbon toward

tryptophan via plasmid-mediated amplification of eight genes in all, but also the

construction of a plasmid stabilization system based on the presence of the serine-

biosynthetic gene on the plasmid and that gene’s absence from the chromosome.

3.2 Central Metabolism

Once the core pathway is optimized, further incremental gains can be attained by

increasing precursor supply. This task usually begins with pathway analysis, which

includes laying out all the possible routes from glucose to a desired amino acid,

calculating the theoretical yield of the amino acid from glucose for each route, and

determining the most efficient route including optimal flux distributions at key

branch points. For calculation of the theoretical yield, the energy and redox

balances should be included to balance ATP and the reducing power.

Within the central metabolism, the junction between glycolysis and the tricar-

boxylic acid (TCA) cycle is particularly important for directing metabolic fluxes to

desired biosynthetic pathways, because the junction in C. glutamicum consists of

several enzyme reactions carrying fluxes that connect phosphoenolpyruvate with

oxaloacetate or pyruvate, pyruvate with oxaloacetate, and pyruvate with malate.

Several strategies have proved beneficial in this regard. For example, increased

carbon flux from pyruvate to oxaloacetate by overexpression of the pyruvate

carboxylase gene or by deletion of the phosphoenolpyruvate carboxykinase gene

resulted in significantly increased production of lysine (Petersen et al. 2001; Peters-

Wendisch et al. 2001; Riedel et al. 2001). Increasing the availability of pyruvate by

decreasing or abolishing pyruvate dehydrogenase activity can also improve lysine

production (Shiio et al. 1984; Blombach et al. 2007b). On the other hand, decreased

activity of the TCA cycle enzyme isocitrate dehydrogenase was shown to improve

lysine production, probably by means of a flux shift from the TCA cycle toward

anaplerotic carboxylation (Becker et al. 2009).

Unlike lysine production, glutamate production induced by biotin limitation

depends solely on the phosphoenolpyruvate carboxylase-catalyzed anaplerotic reac-

tion (Sato et al. 2008). In this case, increased carbon flux from phosphoenolpyruvate

to oxaloacetate by overexpression of the phosphoenolpyruvate carboxylase gene

or by deletion of pyruvate kinase gene effectively improves glutamate production

(Sato et al. 2008; Sawada et al. 2010).

Replacement of a phosphoenolpyruvate-dependent sugar phosphotransferase

system (PTS) by a PTS-independent sugar uptake system can be an alternative

strategy for increasing the availability of phosphoenolpyruvate, as has been

demonstrated in aromatic production by E. coli (Flores et al. 1996). InC. glutamicum,the PTS had long been the only known system to uptake glucose, but very recently,

potential glucose uptake systems that function as alternatives to the PTS have been

identified in this microbe (Ikeda et al. 2010, 2011). These include the iolT1 and iolT2gene products, both known as myo-inositol transporters (Krings et al. 2006).


Expression of the iolT1-specific glucose uptake bypass instead of the native PTS

resulted in approximately 20% increased lysine production (Ikeda et al. 2011).

3.3 NADPH Regeneration

Amino acid biosynthesis from sugar typically uses cofactor NADPH as the reducing

power. NADPH supply is therefore an important consideration along with pathway

engineering to direct carbon toward a desired amino acid. Availability of NADPH

is crucial especially for the production of certain amino acids which require large

quantities of NADPH for their biosynthesis, such as lysine, methionine, and arginine.

InC. glutamicum, NADPH supply has been augmented by engineering the redirection

of carbon from glycolysis into the pentose phosphate pathway through methods

such as disruption of the phosphoglucose isomerase gene (Marx et al. 2003),

overexpression of the fructose 1,6-bisphosphatase gene (Becker et al. 2005) or the

glucose 6-phosphate dehydrogenase gene (Becker et al. 2007), and introduction of a

mutant allele of the 6-phosphogluconate dehydrogenase gene encoding an enzyme

that is less sensitive to feedback inhibition (Ohnishi et al. 2005).

The importance of the pentose phosphate pathway for lysine production is

particularly obvious when fructose is used as a carbon source: only 14.4% of

carbon is channeled through the pentose phosphate pathway on fructose, in contrast

to 62.3% when glucose is used (Kiefer et al. 2004). A reason for the lower flux

channeling through the pentose phosphate pathway could be the entry point of

fructose into glycolysis: fructose mostly enters glycolysis at the level of fructose

1,6-bisphosphate which requires gluconeogenetic fructose 1,6-bisphosphatase

activity in order to direct its carbon into the pentose phosphate pathway.

This problem also has some relevance for sucrose, because the fructose unit of

sucrose follows the same metabolic fate as free fructose. To solve this problem,

direct phosphorylation of intracellular fructose produced by sucrose hydrolysis has

been attempted through the heterologous expression of the fructokinase gene from

Clostridium acetobutylicum (Moon et al. 2005). This is expected to shift the

entry point of fructose from fructose 1,6-bisphosphate to its upstream fructose

6-phosphate and thus increase the flux through the pentose phosphate pathway.

In terms of carbon yield, however, supplying carbon through the pentose phos-

phate pathway is less advantageous than supplying it via the glycolytic pathway

because the former pathway inevitably involves the release of 1 mol of carbon

dioxide (CO2) accompanied by the oxidation of 1 mol of hexose. To solve this

dilemma, an attempt was recently made to engineer a functional glycolytic pathway

in C. glutamicum supplying NADPH through a new route. In this study, endogenous

NAD-dependent glyceraldehyde 3-phosphate dehydrogenase of C. glutamicum was

replaced with nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate

dehydrogenase (GapN) of Streptococcus mutans, which catalyzes the irreversible

oxidation of glyceraldehyde 3-phosphate to 3-phosphoglycerate and the cor-

responding reduction of NADP+ to NADPH; the result was a C. glutamicum strain


with an NADPH-generating glycolytic pathway. A lysine producer derived from the

engineered strain produced considerably more lysine than the reference strain,

exceeding the reference strain’s production levels by ~70% on glucose, ~120% on

fructose, and ~100% on sucrose (Takeno et al. 2010). As an alternative to this

method, expression of the membrane-bound transhydrogenase genes from E. coli inC. glutamicum provided an alternate source of NADPH (Kabus et al. 2007a, b).

3.4 Amino Acid Transport

For certain amino acids, the biosynthetic pathways are subject to multiple

regulations at several steps. In such cases, it is not easy to completely remove all

regulatory controls existing in the pathways, but this difficulty can largely be

overcome by reducing the intracellular pool of the amino acid to a level at which

feedback control does not operate. Since the intracellular pool of an amino acid is

assumed to depend on the uptake rate of the amino acid accumulated extracellularly

as well as the efflux rate, preventing amino acid re-uptake would serve this purpose.

The impact of such transport engineering on amino acid production was first shown

for tryptophan production by C. glutamicum (Ikeda and Katsumata 1994, 1995).

A modification leading to a decreased rate of tryptophan uptake in a tryptophan-

producing mutant increased production, while plasmid-mediated amplification of

the transporter gene drastically decreased production.

Sometimes the export step is critical for achieving efficient amino acid produc-

tion in C. glutamicum (Morbach et al. 1996; Burkovski and Kr€amer 2002). In such

cases, the intrinsic capacity of this bacterium for excreting a desired amino acid

becomes the barrier to improving productivity. Recently, the discovery of active

export systems for several kinds of amino acids has made it possible to compensate

for the limited capacity of amino acid efflux through recombinant DNA technology.

The exporters so far identified for C. glutamicum are LysE, which exports the basic

amino acids lysine and arginine (Vrljic et al. 1996); ThrE, which exports threonine

and serine (Simic et al. 2001); and BrnFE, which exports the branched chain amino

acids and methionine (Kennerknecht et al. 2002; Tr€otschel et al. 2005).

Overexpression of the lysE gene resulted in a fivefold increase in the excretion

rate for lysine compared to the rate of the control strain (Vrljic et al. 1996). More

recently, the NCgl1221 gene product, a mechanosensitive channel homolog, was

identified as a possible glutamic acid exporter (Nakamura et al. 2007).

The functions of such exporters also can be transferred to heterologous bacterial

species. For example, a mutant allele of the C. glutamicum lysE gene has been

successfully used to improve lysine production in the methylotroph Methylophilusmethylotrophus (Gunji and Yasueda 2006). Likewise, the limited capacity of

C. glutamicum for threonine production was improved not only by overexpression

of the endogenous thrE gene (Simic et al. 2002) but also by heterologous expression

of an E. coli threonine exporter (Diesveld et al. 2008).


3.5 Respiratory Energy Efficiency

Like the redox balance, the energy balance is critical for efficient amino acid

production. For this reason, improving the efficiency of ATP synthesis is another

strategy for increasing amino acid production. In C. glutamicum, two terminal

oxidases are positioned in a branched respiratory chain (Bott and Niebisch 2003).

One branch is composed of the cytochrome bc1-aa3 supercomplex, which has a

threefold higher bioenergetic efficiency than the other cytochrome bd branch.

Disruption of the inefficient cytochrome bd branch caused increased lysine produc-

tion with no marked effect on growth or glucose consumption (Kabus et al. 2007a, b).

Recently, it has been shown that C. glutamicum can grow anaerobically by

means of nitrate respiration (Nishimura et al. 2007; Takeno et al. 2007). In the

presence of nitrate, lysine and arginine production occurred anaerobically, though

at a very low level, indicating the potential of this bacterium for anaerobic amino

acid production (Takeno et al. 2007).

3.6 Global Regulation

The determination of the whole genome sequence of C. glutamicum and the

development of global analysis techniques such as DNA microarray have permitted

the identification of a variety of global regulators (Burkovski 2008). These include

GlxR (Kim et al. 2004), SugR (Engels et al. 2008), RamA (Cramer et al. 2006), and

RamB (Gerstmeir et al. 2004) controlling carbon metabolism, AmtR (Beckers et al.

2005) controlling nitrogen metabolism, PhoR (Schaaf and Bott 2007) controlling

phosphorus metabolism, McbR (Rey et al. 2003, 2005) and SsuR (Koch et al. 2005)

controlling sulfur metabolism, DtxR (Wennerhold and Bott 2006) controlling iron

homeostasis, and FarR (H€anbler et al. 2007) and LtbR (Brune et al. 2007)

controlling amino acid metabolism. The existence of these regulators indicates

that amino acid biosynthesis in C. glutamicum is directly or indirectly subject to

both pathway specific and global regulation (Brockmann-Gretza and Kalinowski

2006; Kr€omer et al. 2008). Thus, global regulation is also important in strain

improvement. Another interesting finding is that the global induction of amino

acid biosynthesis genes occurs in a classically derived industrial lysine-producing

strain of C. glutamicum (Hayashi et al. 2006b): the lysC gene, encoding the key

enzyme aspartokinase, was up-regulated several fold in this strain, though a repres-

sion mechanism for lysine biosynthesis is not known in C. glutamicum. Althoughthe genetic elements responsible for these changes have not yet been identified,

it has been demonstrated that the introduction of a mutant allele of the leuC gene

into a defined lysine producer triggered a stringent-like global response and thereby

led to a significant increase in lysine production (Hayashi et al. 2006a).

Engineering of global regulation has also been demonstrated to successfully

improve valine production by C. glutamicum. A pyruvate dehydrogenase-deficient

valine producer exhibited reduced glucose metabolism and a concomitant

nonproduction phenotype in the presence of acetate which was required for its


growth. This drawback has been overcome by inactivating the global regulator SugR

which is responsible for acetate-mediated repression of the PTS (Blombach et al.

2009).

3.7 Stress Responses

Considerable heterogeneity exists within large-scale fermentors, especially with

respect to fed sugar, pH, and oxygen, due to the decrease in mixing efficiency

associated with the increase in the scale of an operation (Einsele 1978; Buckland

and Lilly 1993). Therefore, in large-scale industrial fermentations, the ambient

conditions vary considerably depending on the location of cells within the fermen-

tor. Thus, a cell’s tolerance to various stresses, referred to as cell robustness, is one

of the important characteristics which should be retained in a production strain.

Cells are assumed to possess a variety of mechanisms that allow them to adapt to

stressful conditions. A common stressor is external variation in pH, against which

each cell must maintain the internal pH of its cytoplasm. Although the mechanisms

of pH homeostasis in C. glutamicum are poorly understood, recent studies have

shown critical involvement of a putative transporter of the cation diffusion facilita-

tor family and a potential potassium channel (CglK) in alkaline and acidic pH

homeostasis, respectively (Takeno et al. 2008; Follmann et al. 2009a). Very

recently, transcriptome and proteome studies have revealed that C. glutamicumcan exhibit a variety of stress responses when subjected to acidic or alkaline pH

conditions (Follmann et al. 2009b).

Similarly,C. glutamicum is likely to possess somemeans of adaptation to conditions

of limited oxygen. Recently, the relevant genes have been isolated from the C.glutamicum genome by genetic complementation of mutants that have lost the ability

to grow under conditions of low oxygen (Ikeda et al. 2009a). Putative functions

encoded by these genes include SigD, Ferredoxin, Siderophore, and Cytidylate kinase;

there is also a set of membrane proteins which have not yet been assigned functions

even tentatively. Some of these genes have been demonstrated to show cross-

complementation of different mutants under oxygen limitation, suggesting the utility

of the genes for improving growth and production in industrial fermentation.

Examples of other stress response proteins of C. glutamicum include SigH

(Kim et al. 2005a) and WhcE (Kim et al. 2005b), which are involved in heat and

oxidative stress responses; and BetP (Peter et al. 1996), EctP (Peter et al. 1998),

ProP (Peter et al. 1998), LcoP (Steger et al. 2004), MtrB (M€oker et al. 2007), andMtrA (M€oker et al. 2007), all of which are involved in osmotic stress response.

These are potential targets for engineering in the future.

3.8 Feedstock Utilization

The main feedstocks for industrial amino acid fermentation by C. glutamicum are

sugars from agricultural crops, such as cane molasses, beet molasses, and starch


hydrolysates (glucose) from corn and cassava, but it is becoming increasingly

necessary to engineer the use of alternative raw materials, especially those that do

not compete with human food or energy sources. Wild-type C. glutamicum cannot

utilize lactose, galactose, starch, glycerol, xylose, or arabinose for growth, but

strains that do utilize these carbon sources have recently been engineered.

For example, heterologous expression of both lacYZ from Lactobacillus delbrueckiisubsp. bulgaricus and galMKTE from Lactococcus lactis subsp. cremoris in a

lysine-producing strain of C. glutamicum has resulted in a strain that is able to

produce lysine at up to 2 g/L when fed whey, which contains lactose and galactose

(Barrett et al. 2004). Another lysine-producing C. glutamicum strain has been

engineered to express the a-amylase gene from Streptomyces griseus, which allowsit to utilize soluble starch for lysine production, albeit at an efficiency lower

than that obtained using glucose (Seibold et al. 2006). More efficient lysine

production from soluble starch by C. glutamicum has been achieved by displaying

the a-amylase from Streptococcus bovis on the cell surface. As the anchor protein,

PgsA from Bacillus subtilis was fused to the N terminus of the a-amylase. A lysine

producer displaying this fusion protein on its cell surface produced 6 g/L of lysine

with a conversion yield of 18.9% on starch; this titer and yield are higher than those

obtained in glucose medium (Tateno et al. 2007). A similar cell surface display of

a-amylase has also enabled C. glutamicum to produce glutamate from starch (Yao

et al. 2009).

Glycerol, the main by-product of biodiesel production, is also a potential carbon

source for biotechnological processes. C. glutamicum has been engineered to

express the E. coli glycerol utilization genes glpF, glpK, and glpD so that it can

grow on glycerol. This allowed the production of glutamate and lysine from

glycerol with yields of 11% and 19%, respectively (Rittmann et al. 2008).

The use of lignocellulose as a feedstock is limited in part by a poor catabolism of

the xylose component. A xylose-utilizing C. glutamicum strain has been

constructed that expresses the xylA and xylB genes from E. coli on a high-copy

plasmid. It is interesting that the E. coli xylB gene contributed to improved growth

performance on xylose despite the existence of a functional xylB gene in the

C. glutamicum wild-type genome (Kawaguchi et al. 2006). Similarly, heterologous

expression of the E. coli arabinose-utilizing pathway in C. glutamicum resulted in a

strain that is able to grow on arabinose, another component of lignocellulose

(Kawaguchi et al. 2008).

4 Recent Advances in Amino Acid Production

Recently, various genetic engineering tools and global analysis techniques for

C. glutamicum as well as high-throughput genomic analysis technologies have

been successfully applied and have contributed both to the understanding of the

molecular mechanisms underlying high-level production and to the development of

more efficient production strains of this microbe. For example, DNA arrays have


been used to find engineering targets expected to result in improved valine produc-

tion (Lange et al. 2003) and to identify mutations that confer traits conducive to

high-level production in lysine-producing strains through random mutagenesis and

screening programs (Hayashi et al. 2006a; Sindelar and Wendisch 2007). Here,

recent advances in amino acid production by C. glutamicum are highlighted, with a

focus on the amino acids whose production methods have been significantly

advanced in the 2000s. These include glutamate, lysine, arginine (citrulline), the

branched-chain amino acids, alanine, serine, and the sulfur-containing amino acids

methionine and cysteine. Production technology of other industrially important

amino acids such as threonine and the aromatic amino acids has been omitted

because it is discussed in other publications (Ikeda 2003; Willis et al. 2005;

Sprenger 2007; Rieping and Hermann 2007; Dong et al. 2011).

4.1 Glutamate

Since the discovery of C. glutamicum as a producer of the food flavoring

monosodium glutamate, commercial production of glutamate has been conducted

exclusively by this microbe. In 2006, the global demand for monosodium glutamate

amounted to almost two million metric tons (Fig. 1), and the market is expected to

continue its gradual expansion at an average annual rate of 3–4% (Ajinomoto

2007). Glutamate production by C. glutamicum is induced by biotin limitation or

by treatment with certain fatty acid ester surfactants or with b-lactam antibiotics

such as penicillin. Although the induction treatment is the core technology involved

in industrial glutamate production processes, the molecular basis of the induction of

glutamate secretion was long unknown. In recent years, however, a valuable insight

into the secretion mechanism has been gained in the form of the identification of the

NCgl1221 gene product as a possible glutamate exporter (Nakamura et al. 2007).

An intriguing finding is that only a specific point mutation in the NCgl1221 gene

resulted in glutamate secretion without any induction treatments. It has also been

shown that amplification of the wild-type NCgl1221 gene increases glutamate

secretion while its disruption substantially abolishes secretion accompanied by an

increase in the intracellular glutamate pool under the induction conditions men-

tioned earlier. The gene in question encodes the YggB protein which has been

described as a putative mechanosensitive channel (Nottebrock et al. 2003). Based

on the possible function as a mechanosensitive channel, the following mechanism

has been proposed: the induction conditions, such as biotin limitation and penicillin

treatment, alter membrane tension by inhibiting lipid or peptidoglycan synthesis.

This triggers conformational changes in the NCgl1221 gene product, which in turn

enables the protein to export glutamate (Fig. 5). To confirm this model, one must

determine how energy is supplied into the glutamate secretion process because

carrier-mediated glutamate secretion by C. glutamicum was shown to be energy

dependent (Gutmann et al. 1992).

The observation mentioned earlier raises the question of how the new model

is congruent with the accepted notion that a decrease in the activity of the


2-oxoglutarate dehydrogenase complex (ODHC) is crucial for glutamate production

(Shingu and Terui 1971; Kawahara et al. 1997; Kimura 2003; Asakura et al. 2007;

Kim et al. 2009a, b). Although the new model seems to explain the basics of the

mechanism underlying the induction of glutamate secretion, it is probably not

sufficient to explain the entire process of glutamate production by C. glutamicum.Recently, a possible connection at a molecular level has been uncovered between

ODHC activity and glutamate production (Fig. 5). A novel 15 kDa protein OdhI was

identified as a regulator of ODHC (Niebisch et al. 2006). The unphosphorylated

form of OdhI binds to the OdhA protein, one of the subunits of ODHC, and inhibits

the ODHC activity. This inhibition can be prevented by the PknG-catalyzed

phosphorylation of OdhI. A phospho-serine/threonine protein phosphatase

responsible for dephosphorylation of OdhI has also been identified (Niebisch et al.

2006). Interestingly, disruption of the odhI gene was shown to abolish glutamate

production even under the induction conditions (Schultz et al. 2007), suggesting a

close relationship between the regulator protein and the reduction of the ODHC

activity that occurs during glutamate production. It is also worth noting that

proteome analyses have revealed a significant increase in the OdhI protein upon

penicillin treatment, which has become a conventional industrial method to trigger

glutamate production (Kim et al. 2009a, b). These findings have confirmed the

existence of a connection between ODHC activity and glutamate production, but it

should be noted that an ODHC-activity-reducing metabolic change alone is not

sufficient to induce glutamate production (Kim et al. 2009a, b).

These results taken all together, the evidence to date suggests a link between

the induction treatments, such as biotin limitation and penicillin treatment, and

Glucose

Glutamate

Phosphoenol -pyruvate

Pyruvate

Acetyl-CoA

Biotin

Fatty acid

Oxaloacetate

OxoglutarateYggB

?

AccBCAccBCDtsRDtsR

PknGPknG

OdhIOdhI

ODHCODHC

Fig. 5 Possible mechanism triggering glutamate overproduction under the induction conditions in

C. glutamicum. In this model, proteins AccBC and DtsR form the biotin-dependent acetyl-CoA

carboxylase complex required for fatty acid biosynthesis; this biotin–enzyme complex is thought

to be the primary target of biotin limitation and surfactant addition


glutamate production. In our proposed mechanism, the induction treatments

enhance the synthesis of the regulator protein OdhI in its unphosphorylated form

and thereby inhibit ODHC activity. This causes a metabolic shift at the branch point

of 2-oxoglutarate, which channels carbon toward glutamate. Intracellularly

accumulated glutamate is then secreted into the medium via the NCgl1221 gene

product, a possible glutamate exporter, which has been activated in response to

altered membrane tension (Fig. 5). Questions for the future include why and how

the OdhI protein is overexpressed in response to the induction treatment and what

conditions are required for the phosphorylation and dephosphorylation of OdhI.

Continuous efforts have been made not only to understand glutamate production

but also to improve the process. In addition to the general approaches, in which

metabolic fluxes are directed into glutamate (Kimura 2003; Sato et al. 2008;

Sawada et al. 2010), an innovative metabolic design allowing an increased

maximum theoretical yield has recently been reported (Chinen et al. 2007).

Glutamate biosynthesis from glucose in C. glutamicum is inevitably associated

with the release of CO2 in the pyruvate dehydrogenase reaction, but the creation

of a novel metabolic route by installing the phosphoketolase pathway of

Bifidobacterium animalis allowed the CO2-releasing pyruvate dehydrogenase

reaction to be bypassed, and thereby led to increased glutamate production

coupled with the suppression of CO2 emission. On the other hand, expression

of the Vitreoscilla hemoglobin gene vgb under a tac promoter in a wild-type

C. glutamicum strain has been shown to increase glutamate production in both

shake-flask and fermentor cultivations (Liu et al. 2008), probably due to the

enhancement of respiration by the hemoglobin (Webster 1987; Kallio et al. 1994;

Zhang et al. 2007a, b).

4.2 Lysine

Lysine, one of the essential amino acids for animals, has a significant commercial

value as a feed additive to promote the growth of animals including swine and

poultry, and thus is the second-ranking amino acid after glutamate in terms of

worldwide annual production. The scale of the lysine market in 2006 has been

estimated at approximately 960,000 metric tons (Fig. 1), and the market is expected

to grow continuously at an annual rate of 8–10% (Ajinomoto 2007). Because of the

growing market for lysine, exhaustive studies have been undertaken in an attempt to

engineer the metabolism of C. glutamicum for lysine production. These studies

have resulted in several effective strategies for rational strain improvement, includ-

ing engineering of terminal pathways, central metabolism, cofactor-regeneration

systems, export systems, energy metabolism, and global regulation. Typical

examples of these have been discussed earlier.

Recently, a genome-scale model of the C. glutamicum metabolic network has

been constructed, based on the annotated genome, available literature, and various

“omic” data (Kjeldsen and Nielsen 2009). The constructed metabolic model


consists of 446 reactions and 411 metabolites; the predicted metabolic fluxes during

lysine production and growth under various conditions are highly consistent with

experimental values. The ability to predict the metabolic state associated with

maximum production yield can be used to guide strain engineering. This strategy

has been proven through the rational design of high lysine-producing strains of

C. glutamicum (Kr€omer et al. 2004; Becker et al. 2005; Wendisch et al. 2006).

In addition to such metabolic engineering approaches, a genome-based reverse

engineering approach has been employed to create a more robust and efficient

lysine producer (Ohnishi et al. 2002, 2003; Ikeda and Nakagawa 2003; Ikeda et al.

2006). Reverse engineering is so called because it traces backwards to an existing

classical producer. First, the genome sequence of an industrial lysine producer was

compared with a corresponding wild-type sequence to identify the mutational

differences. The mutations were then sequentially introduced by allelic replace-

ment into the wild-type genome (Ikeda et al. 2005). Mutations in the relevant

terminal pathways were introduced first, followed by those in central metabolism,

and finally those in genes involved in global regulation. Each of the strains thus

constructed was evaluated to determine the contribution of each mutation to

production. When the mutation was beneficial, the resulting strain was used as

the parent to which further mutations were introduced for evaluation. This iterative

cycle has led to a minimally mutated strain having only useful mutations. It should

be noted that the particular host strain used at the beginning of the process can have

a significant impact on the ultimate outcome. When reverse engineering was

applied to lysine production, lysC311, a key mutation that confers high-level lysine

production on wild-type C. glutamicumwas used to screen various wild-type strains

of C. glutamicum to identify the best background with which to begin the process

(Ohnishi and Ikeda 2006).

Among the useful mutations identified through the reverse engineering process

were two (hom59 and lysC311) that are located in the terminal pathway to lysine

(Ohnishi et al. 2002), three (pyc458, gnd361, and mqo224) involved in central

metabolism (Ohnishi et al. 2002, 2005; Mitsuhashi et al. 2006), and one (leuC456)causing global induction of the amino acid-biosynthetic genes and thereby further

increasing production (Hayashi et al. 2006a). The subsequent assembly of these six

useful mutations into the industrially robust wild-type strain chosen as the best

background was shown to substantially improve producer performance, resulting in

a final titer of 100 g/L after only 30 h of 5-L jar fermentor cultivation at a

suboptimal temperature of 40 �C (Fig. 6, Ikeda et al. 2006).

Most recently, the cumulative body of knowledge on lysine production was

combined with metabolic flux profiling and modeling technologies and systemized

to predict a combination of genetic modifications that would lead to the theoretically

best flux scenario for optimum lysine production (Becker et al. 2011). Ultimately,

the process has identified 12 steps of modifications to a wild-type genome leading to

the lysine hyper-producer, LYH-12 (Fig. 7), which can achieve a final titer of 120 g/L

with a conversion yield of 55% on glucose after 30 h of 5-L jar fermentor cultivation

at 30�C. Among the twelve modifications were six (introduction of the lysC311 and

hom59 mutations, duplication of the ddh and lysA genes, and overexpression of the


lysC and dapB genes under a strong promoter) that cause increased flux through

the lysine biosynthetic pathway, three (introduction of the pyc458 mutation,

overexpression of the pyc gene under a strong promoter, and deletion of the pckgene) that cause increased flux toward oxaloacetate through anaplerotic carboxyla-

tion, two (overexpression of the fbp gene and the zwf-opcA-tkt-tal operon under strongpromoters) that cause increased flux through the pentose phosphate pathway for

NADPH supply, and one (replacement of the start codon ATG by the rare GTG in

the icd gene) that causes reduced flux through the TCA cycle and thereby increases the

availability of oxaloacetate.

4.3 Arginine and Citrulline

Arginine, a semi-essential amino acid, has lately attracted considerable attention for

being a precursor to nitric oxide (NO), a key component of endothelial-derived

relaxing factor (Appleton 2002). Citrulline, a precursor of arginine biosynthesis, is

also important for human health since it is a source of endogenous arginine in the

body (Curis et al. 2007). As the economic values of these amino acids have

lysC

hom

pyc gnd mqo

lysC

AK -1

AHD-2 AHP-3 APG -4 AGM -5Wild

HD-1

leuC

AGL -6

Wild AK -1AHD-2

AHP-3APG -4

AGM -5HD-1

50

100

0

AGL -6

Lys

ine

(g/L

)

40ºC

Fig. 6 Schematic diagram of the creation of the defined lysine producers and their lysine

production capabilities at 40 �C in 5-L jar fermentor cultivation


increased, considerable attention has recently been given to the development of

more efficient production strains, as well as to the analysis of arginine metabolism

in microbes (Utagawa 2004; Glansdorff and Xu 2007; Lee et al. 2010). The latest

research in the field of production technology involves the reengineering of an

arginine and citrulline-producing strain of C. glutamicum; in this experiment,

positive mutations derived from three different lines of classical producers were

systematically assembled into a single wild-type background (Fig. 8) (Ikeda et al.

2009b). The procedure and impact of this advanced approach are summarized

as follows:

1. The first step was to identify the basic mutation(s) causing arginine and citrulline

overproduction in wild-type C. glutamicum. For this purpose, three independentlyderived industrial arginine and citrulline producer strains were sequenced and

compared to their wild-type ancestors. This identified a variety of mutations

potentially associated with arginine biosynthesis. Among these, five specific

mutations (argB26, argB31, argR123, argG92up, and argG45) located within

arg operons were examined in a wild-type background for their relevance to

arginine and citrulline production. argB26 and DargR (argR123-derived deletion

mutation) were found to be the basic mutations.

2. The second step was to screen for the wild-type background giving the best

performance. The two basic mutations, argB26 and DargR, were introduced into

Glucose

Phosphoenolpyruvate

Pyruvate

Oxaloacetate

Oxoglutarate

Glucose 6-P

Fructose 6-P

Fructose1,6-BP

Glyceraldehyde 3-P

Ribulose 5-P

Xylulose 5-P Ribose 5-P

Erythrose 5-P Sedoheptulose 7-P

Aspartate

Aspartyl-P

Aspartatesemialdehyde

Threonine

Lysine

Diaminopimelate

Piperideinedicarboxylate

Lysine

Isocitrate

zwf pgl

tkttkt

tal

icd

pyc

lysC

dapB

ddh

lysA

fbp

hom

pck

Overexpression

Attenuation

opcA

Fig. 7 Schematic diagram of genetic modifications to a wild-type genome leading to the lysine

hyper-producer C. glutamicum LYS-12


six different C. glutamicum wild-type strains to generate isogenic mutants,

which were then screened for their ability to produce arginine and citrulline

under suboptimal temperature conditions (38�C). This revealed that strain

ATCC 13032 has the highest potential for production at elevated temperatures.

By combining the two basic mutations in the best host, a robust producer was

obtained, but its production was still only one-third of that of the best classically

derived strain.

3. The third step was to identify what was limiting production in the new strain.

Transcriptome analyses revealed that the arg operon in the classically derived

strain was much more highly expressed than it was in the new strain. This

brought up the possibility that one of the steps in the arginine-biosynthetic

pathway was rate limiting. Replacement of the endogenous argB with the

heterologous E. coli argB, which is natively insensitive to arginine inhibition,

increased production threefold, revealing that a prime target for engineering was

the properties of the argB product, the key regulatory enzyme for arginine

biosynthesis in C. glutamicum.4. The final step was to engineer the argB product, N-acetyl-L-glutamate kinase, so

that it would not be feedback inhibited by arginine. To this end, in addition to

argB26, the argB31mutation was introduced into the new strain, causing a more

complete deregulation of the enzyme and resulting in dramatically increased

production. This reconstructed strain, designated strain RBid (Fig. 8), displayed

StrainI-30

?

?

?

StrainA-27

StrainD-77

argB26

(A26V)

argR123

(A123V) DargR

argB31

(M31V)

New strain RBid

Wild strain

Sequencing & identifying

useful mutations

Assembling

useful mutations

Fig. 8 Reengineering method for the creation of a robust and efficient producer of arginine and

citrulline (strain RBid) using useful genetic traits identified in three different lines of classical

producers (strains I-30, A-27, and D-77). Useful mutations relevant to production are indicated

(stars), together with unnecessary mutations (multiplication symbols)


significantly higher productivity of arginine and citrulline even at the suboptimal

temperature of 38 �C. The enhanced performance of the new strain is obvious

from the differences in fermentation kinetics between strain RBid and the best

classical producer, strain A-27 (Fig. 9).

4.4 Branched-Chain Amino Acids

The branched-chain amino acids, valine, leucine, and isoleucine, are all essential for

human and animal nutrition, and all have increasing uses in various fields including

pharmaceuticals, cosmetics, agricultural chemicals, dietary supplements, and feed

additives. Currently, their most popular use is as a supplement for athletes to promote

strength; this use is based on the nutraceutical effect of these amino acids on skeletal

muscles (Shimomura et al. 2006). The intermediates for these amino acids can also be

used for the production of biofuels (Atsumi et al. 2008). InC. glutamicum, all three ofthese amino acids share common uptake and export systems (Ebbighausen et al.

1989; Kennerknecht et al. 2002), as well as common substrates and enzymes for their

biosynthesis, and thus are closely related in their metabolic fate.

In the last decade, rational metabolic engineering has been applied to the produc-

tion of the branched-chain amino acids by C. glutamicum many times, with a special

emphasis on valine production (Patek 2007; Park and Lee 2010). The strategies used

to improve production of valine include (1) eliminating bottlenecks in the terminal

pathway, either by conferring isoleucine auxotrophy which allows the attenuation

control of the ilvBNC operon to be circumvented (Radmacher et al. 2002), by

deregulating the key regulatory enzyme acetohydroxyacid synthase (Elisakova et al.

0 20 40 60

Gro

wth

(O

D66

0)

Arg

inin

e/C

itru

lline

(m

M)

Time (h)

40

80

100

0

500

400

300

200

0

60

20

80

Reengineered strain

RBid

Classical producer

A-27

Fig. 9 Fermentation kinetics of the newly developed strain RBid at 38�C in 5-L jar fermentor

cultivation. For comparison, the profiles of best classical producer A-27, which was cultured under

its optimal 30 �C conditions, are shown as controls. Open circles arginine and citrulline of strain

RBid, closed circles growth of strain RBid, open squares arginine and citrulline of strain A-27,

closed squares growth of strain A-27


2005), or by overexpressing the gene set responsible for valine biosynthesis

(Radmacher et al. 2002; Blombach et al. 2007a; Bartek et al. 2010a, b); (2) increasing

the availability of precursor pyruvate, either by blocking pantothenate synthesis

(Radmacher et al. 2002; Bartek et al. 2008) or by inactivating pyruvate dehydroge-

nase, pyruvate carboxylase, and pyruvate:quinine oxidoreductase (Blombach et al.

2007a, 2008, 2009); and (3) increasing NADPH supply by inactivating phos-

phoglucose isomerase (Blombach et al. 2008; Bartek et al. 2010a, b). These

modifications have mostly been achieved through plasmid-mediated amplification

and/or deletion of the targeted genes, possibly leading to perturbations of the natural

homeostatic mechanisms of the cell. To alleviate such side effects on cell physiology,

the desired metabolic engineering has been achieved through purposeful mutagenesis

of promoters of the chromosomal genes involved in the valine biosynthesis pathway

and in competing pathways (Holatko et al. 2009). The resulting plasmid-free valine

producer was auxotrophic to pantothenate and bradytrophic to isoleucine, carried

a feedback-resistant acetohydroxy acid synthase, and expressed the genes ilvD and

ilvE from strong mutant promoters. This new type strain with all mutations

constructed within the chromosome has been shown to produce 136 mM valine

from 4% glucose after 48 h of flask cultivation.

An H+-ATPase defect has also been shown to be effective for accelerating sugar

metabolism and increasing valine production in C. glutamicum (Wada et al. 2008).

Proteomic analysis has revealed various metabolic responses to this defect, including

upregulated expression of 6-phosphofructokinase and pyruvate kinase in the glycolytic

pathway (Li et al. 2007). These increases may contribute to the enhanced glycolysis

observed in the mutant, and thus to the increased availability of precursor pyruvate.

For the production of the branched-chain amino acids which are mainly used for

pharmaceutical purposes and are therefore required to have the highest degree of

purity, it is desirable to minimize by-production of other amino acids to a level at

which supplementary purification of the desired amino acid is not necessary.

By-production of alanine occurs during valine production, but the enzymes

involved in alanine formation from pyruvate by C. glutamicum had not been

identified until recently. Double knockout mutants of the alanine aminotransferase

gene (alaT, NCgl2747) and the alanine-valine transaminase gene (avtA, NCgl2510)were shown to be auxotrophic for alanine, revealing that the two gene products are

the only aminotransferases involved in alanine biosynthesis by C. glutamicum(Marienhagen and Eggeling 2008). Deletion of the alaT gene, which is primarily

responsible for alanine formation, in a valine producer actually reduces the extra-

cellular alanine accumulation, thereby facilitating cost-effective downstream

processing (Marienhagen and Eggeling 2008).

4.5 Alanine

Alanine, a nonessential amino acid, is used mainly as a sweetener in dairy products,

a natural moisture balancer in cosmetics, and an ingredient in infusion solutions and

pharmaceutical products. Unlike most amino acids, which are currently produced


by means of fermentative processes, alanine, the simplest optically active amino

acid, is produced commercially through the enzymatic decarboxylation of aspartate

(Shibatani et al. 1979) which is synthesized from fumarate and ammonia with

immobilized cells expressing aspartase. However, since the initial substrate, fuma-

rate, is produced primarily from petroleum, a fermentation process that uses

renewable feedstocks to produce alanine has attracted increasing attention.

Several alanine-producing microorganisms have long been known, including

C. glutamicum and E. coli (Kitai 1972), but alanine production at a useful level was

first demonstrated in a natural isolate, Arthrobacter oxydans HAP-1 (Hashimoto and

Katsumata 1993, 1998). It has since been determined that alanine hyperproduction

results from the combination of the presence of the glucose-nonrepressive, NADH-

dependent alanine dehydrogenase and decreases in the activities of pyruvate dehydro-

genase and NADH oxydase in the stationary phase, which allows balanced coupling of

redox potential between glycolysis and the dehydrogenase pathway (Hashimoto and

Katsumata 1999). Aside from the successful example utilizing the intrinsic

characteristics of A. oxydans, metabolic engineering has enabled several other

microorganisms such as Zymomonas mobilis, Lactococcus lactis, andE. coli to producehigh levels of alanine from sugar (Uhlenbusch et al. 1991; Hols et al. 1999; Smith et al.

2006; Zhang et al. 2007a, b). The most common strategies for alanine production

involve the heterologous expression of NADH-dependent alanine dehydrogenase in

hosts, some of which already possess defects in alanine racemase and/or the pathways

that compete with alanine biosynthesis. C. glutamicum has also been demonstrated to

be a useful host for alanine production (Katsumata and Hashimoto 1996). As in many

other microorganisms, biosynthesis of alanine in C. glutamicum depends on the

transamination reaction from pyruvate and glutamate. Expression of alanine dehydro-

genase from A. oxydans in an alanine racemase-deficient C. glutamicum strain has

resulted in hyperproduction of L-alanine isomer under oxygen limitation (Fig. 10).

The process of alanine production by C. glutamicum is economically significant,

but has the drawback of relatively low productivity due to decreased glucose

metabolism under oxygen-limited conditions. Very recently, it has been shown by

a bioprocess using growth-arrested packed cells that homologous overexpression of

the glyceraldehyde 3-phosphate dehydrogenase gene in a C. glutamicum strain

expressing alanine dehydrogenase from Lysinibacillus sphaericus and simulta-

neously deficient in the genes associated with by-production of organic acids can

dramatically improve glucose metabolism and thereby also alanine productivity

under conditions of oxygen deprivation (Fig. 10, Jojima et al. 2010). Further inacti-

vation of alanine racemase has led to the production of L-alanine isomer with a chiral

purity greater than 99.5%. This bioprocess has achieved a final titer of 98 g/L of L-

alanine with a conversion yield of 83% on glucose after 32 h.

4.6 Serine

Serine is likewise a nonessential amino acid, but one that is important in metabo-

lism because it participates in the biosynthesis of many important metabolites such


as purines, pyrimidines, cysteine, and tryptophan. Although the amount of serine

produced each year worldwide is relatively small compared with the amounts of the

other amino acids mentioned earlier, serine is of significant commercial value as an

ingredient in pharmaceuticals such as infusion solutions, a natural moisturizing

material in cosmetics, and a feed additive.

Serine is among the few amino acids for which high-production yields were not

achieved by fermentation directly from sugar until recently. Classical mutagenesis

and screening for a strain producing serine from glucose had not resulted in

a practical production method. Recently, however, an attempt at systematic

metabolic engineering resulted in a C. glutamicum strain that produces considerable

amounts of serine, with help in the form of interventions at several points in the

complicated metabolism of the amino acid (Fig. 11, Peters-Wendisch et al. 2005).

Initial overexpression of serine biosynthesis genes (serAD197, serC, and serB)coding for deregulated enzymes did not lead to significant serine accumulation,

nor did the further deletion of the serine dehydratase gene (sdaA) that catalyzesserine degradation to pyruvate. Only by reducing the glyA-encoded serine

hydroxymethyltransferase (SHMT) activity was considerable serine accumulation

ultimately achieved. Since SHMT, being the unique route to the C1 supply, is

essential for growth of C. glutamicum, reducing the activity of this enzyme by

exactly the right amount was key. This was initially accomplished by replacing the

glyA promoter with the tac promoter, which reduced glyA expression in the absence

of isopropyl-thio-b-D-galactopyranoside. As it turned out, this method of glyAcontrol was unstable: since mutations in lacIq restored expression. As an alternate

method of glyA expression control, a more reliable physiological method was

developed in which the strain is made auxotrophic for folate by deleting the

Glucose

NADH

NAD+

Hypoxic conditions

L-Alanine L-AlaninePyruvateLactate

Glutamate Oxoglutarate

D-Alanine

NH4+

AlaATAlaAT

GAPDHGAPDH

AlaDHAlaDHLDHLDH

ALRALR

Fig. 10 Outline of metabolic engineering for L-alanine production inC. glutamicum. AlaDH alanine

dehydrogenase from A. oxydans or L. sphaericus, ALR alanine racemase, AlaAT alanine aminotrans-

ferase, GAPDH glyceraldehydes 3-phosphate dehydrogenase, LDH lactate dehydrogenase


pabABC genes so that SHMT activity can be controlled by the availability of

5,6,7,8-tetrahydrofolate (THF). This resulted in an accumulation of 345 mM of

serine in a 20-L controlled fed-batch culture (Stolz et al. 2007).

4.7 Methionine

Methionine, another essential amino acid for animals, has a great deal of commer-

cial value as a feed additive. For this purpose, methionine is produced exclusively

by chemical synthesis in D, L-forms, as this amino acid is considered to have a

similar effect on animal nutrition in both L- and D, L-forms. Nowadays, however,

there is an increasing interest in the development of environmentally friendly

fermentation methods using renewable feedstocks to produce methionine.

In the hope of discovering a method for the rational construction of a methionine

producer, methionine biosynthesis and its regulation are being studied in

C. glutamicum. Recently, C. glutamicum was shown to possess both transsulfuration

and direct sulfhydrylation pathways, in contrast to most microorganisms, including

E. coli, which utilize only one of these two pathways (Lee and Hwang 2003; Hwanget al. 2007). Metabolic engineering to redirect carbon from the lysine pathway into

the methionine pathway led to a C. glutamicum strain that produced 2.9 g/L of

methionine, together with 23.8 g/L of lysine (Park et al. 2007). Overexpression of

the homologous metX and metY genes in another lysine-producing C. glutamicumstrain was reported in a patent byM€ockel et al. (2002) to result in a final titer of 16 g/Lof methionine. Two regulatory genes in C. glutamicum have been identified as being

relevant to methionine biosynthesis: mcbR (cg3253) and NCgl2640. Inactivation of

Glucose

Serine3-Phosphoglycerate

Pyruvate

GlycineMethylene-

THF

THF

Plasmid

Serine

Folate

DsdaADsdaA

serACBserACB

SHMTSHMT

DpabABCDpabABC

Fig. 11 Schematic diagram of genetically engineered serine-producing C. glutamicum strain


either in wild-type C. glutamicum results in increased methionine production

(Mampel et al. 2005; Rey et al. 2003, 2005).

Very recently, an attempt at systematic metabolic engineering resulted in an

E. coli strain that produces methionine at an industrially useful level (Figge et al.

2009). The key to success here was achieving a balanced supply of three important

precursors for methionine biosynthesis: O-succinylhomoserine, cysteine, and a C1

carbon, methyl-tetrahydrofolate (CH3-THF). An imbalanced supply of these

precursors causes the formation of undesired by-products, such as homolanthionine

and isoleucine, through the involvement of certain methionine-biosynthetic

enzymes themselves. The procedure and impact of the metabolic engineering

involved in the production of this strain can be summarized as follows (Fig. 12):

1. The first step was to eliminate bottlenecks in the core pathways leading to three

precursors. For this purpose, the metF, metH, and cysE genes, as well as mutant

alleles of the metA and thrA genes that encodes enzymes less sensitive to

feedback inhibition, were overexpressed in an E. coli strain deficient in the

methionine repressor gene metJ. This engineering resulted in methionine pro-

duction at the yield of 6.7% on glucose in 50-mL batch culture.

2. The second step was to make more sulfur available for cysteine biosynthesis.

For this purpose, the cysPUWAM and cysJIH gene clusters were overexpressed,

increasing the yield up to 8.5%.

Glucose

3-Phospho-glycerate

Phosphoenol-pyruvate

Pyruvate

Serine

H2S

CysteineTHF

CH2-THF CH3-THF

Glycine

Oxaloacetate

Aspartate HomoserineO-succinyl-homoserine

γ-Cystathioine

HomocysteineMethionine

COOH

S-adenosyl-methionine

O-Acetylserine

Asp-P Asp-SA

Threonine

Formyl-THFHomo-

lanthionine

Isoleucine

metB

metC

metB

metE

metL metL

Lysine

succinate

HistidinePurines

ppc

cysPUWA

cysJIH

cysM

metF

metA*

metH

serACB cysE

glyA

thrA*thrA*

ΔpykA

ΔpykF

ΔpurU

metA

metB

metC metEmetF

ΔmetJOverexpression Disruption

gcvTHP

NADPH

metB

Metabolic pathway

Overexpression

Feedback inhibition

S2O3

S2O3

Fig. 12 Schematic diagram of genetically engineered methionine-producing E. coli strain.

Asterisked genes encode enzymes less sensitive to feedback inhibition


3. The third step was to increase the availability of phosphoenolpyruvate for

biosynthesis of the precursor O-succinylhomoserine. Deletion of two pyruvate

kinase genes, pykA and pykF, further increased the yield up to 10.2%.

4. The fourth step was to channel more carbon toward a C1 source, in this case,

methyl-tetrahydrofolate CH3-THF, via serine and glycine. Overexpression of the

relevant genes including serACB, glyA, gcvTHP, andmetF, as well as deletion ofthe purU gene, further boosted the yield to 12.9%.

The engineered E. coli strain has achieved a yield of 19.9% after 50 h in

fed-batch fermentation without the formation of any detectable undesirable by-

products. Based on this yield, the methionine titer is estimated at more than 35 g/L.

Though some progress has been made toward creating improved methionine

producers, methionine yields still remain low compared with those attained for

other amino acids. Metabolic pathway analysis has been used to evaluate the

theoretical maximum yields of methionine production on the substrates glucose,

sulfate, and ammonia in C. glutamicum and E. coli (Kr€omer et al. 2006). The

theoretical yield (mol-C methionine per mol-C glucose) of C. glutamicumwas 0.49,

while that of E. coli was somewhat higher at 0.52. This analysis also showed that

introduction of the E. coli glycine cleavage system into C. glutamicum as an

additional C1 source and replacing sulfate with thiosulfate or sulfide, thereby

avoiding the need for reduction of oxidized sulfur, would increase the theoretical

maximal methionine yields in C. glutamicum to 0.57 and 0.68, respectively.

Furthermore, when methanethiol (also known as methylmercaptan) is used as a

combined source for a C1 carbon and sulfur in C. glutamicum, the theoretical yieldwas estimated to reach its highest potential value at 0.91 (Kr€omer et al. 2006).

Most recently, the potential utilization of methanethiol and its dimeric form

dimethyldisulfide as both the C1 source and the sulfur source has been experimen-

tally verified in C. glutamicum (Bolten et al. 2010). Isotope experiments have

revealed that the S-CH3 group is entirely added to O-acetylhomoserine, directly

yielding methionine (Fig. 13). It has also been shown that the reaction is catalyzed

by MetY, creating a shortcut for methionine biosynthesis. The problem in this case

would be the toxicity of these sulfur compounds to cells. A delivery system using a

beaded macroporous polystyrene resin has been suggested to be a way of alleviating

the toxic effects (Bolten et al. 2010).

4.8 Cysteine

Cysteine, the other sulfur-containing amino acid, is a nonessential amino acid but

has a crucial function in metabolism as a precursor of sulfur-containing compounds

such as methionine, thiamine, biotin, lipoic acid, and coenzyme A. In addition to its

biological significance, cysteine is important commercially because of its various

applications in the pharmaceutical, cosmetic, food, and livestock industries. Due to

the lack of an efficient method of producing cysteine through fermentation, its


production has depended on other methods including microbial conversion from

DL-2-amino-D2thiazoline 4-carboxylic acid (Sano et al. 1977) and extraction from

natural protein-rich resources such as hair and keratin.

As with methionine, it has been difficult to engineer strains that produce high

yields of cysteine, though this amino acid is synthesized in C. glutamicum from

serine via O-acetyl-serine in only two steps (Haitani et al. 2006). Typical strategiesinclude deregulation of the key regulatory enzyme serine O-acetyltransferase,deletion of the cysteine desulfhydrase gene that catalyzes cysteine degradation to

pyruvate, and overexpression of cysteine exporters; these have been shown to be

effective for cysteine production in both E. coli and C. glutamicum, though the finaltiters were below 2 g/L (Wada et al. 2002; Wada and Takagi 2006). It has been

suggested that the combination of these strategies and the improvement of other

factors including an increased supply of the precursor serine and a decreased

reuptake of the product would lead to further improvement (Wada and Takagi

2006). Recently, ydeD and yfiK, both of which are involved in efflux of cysteine andrelated compounds, have been identified as the genes underlying the augmentation

of cysteine yield in a cysteine-producing E. coli strain (Dassler et al. 2000; Franke

et al. 2003). This suggests that cysteine efflux is the step that limits cysteine

production, and thus is an important consideration in the construction of cysteine

overproducers.

SO42-

SO42-

SO32-

Adenylyl-sulfate

H2S

NADPH

3 × NADPH

CH3-THF

THF

Homocysteine

Methionine

Aspartate

O-Acetylhomoserine

NADPH

NADPH

Cysteine

Acetate

SerineGlycine

Homoserine

Asp-P

Asp-SA Lysine

γ-Cystathionine

Methanethiol(CH3-SH)

Dimethyldisulfide(CH3-S-S-CH3)

Acetate

MetYMetY

MetYMetY

Direct sulfhydrylation pathwayTranssulfuration pathway

Fig. 13 Proposed pathway for assimilation in C. glutamicum of methanethiol and dimethyl-

disulfide into methionine in addition to two known pathways of transsulfuration and direct

sulfhydrylation


5 Conclusions and Outlook

The determination of the complete genome sequence of C. glutamicum was

obviously an important milestone in the history of amino acid fermentation. The

subsequent rapid progress in C. glutamicum genomics and so-called post-genome

technologies has opened up new avenues for the development of various global

analysis techniques, which have led to our current understanding of whole cellular

metabolism and systems in this microbe. Furthermore, these advances have dra-

matically transformed our approaches toward strain development. For example, in

silico modeling and simulation approaches are nowadays being used routinely to

help identify new targets for further engineering and strain improvement.

The power of such systems-level approaches will surely increase as modeling is

combined with the ever-accumulating “-omics” data. Meanwhile, the availability of

high-throughput DNA sequencing has made it feasible to decode the genomes of

classical industrial producers and thereby to identify important genetic traits that

distinguish them from their wild-type ancestors. As a result, the conventional style

of selecting improved strains by phenotypes, formerly the standard practice in

industry, is rapidly being replaced by the new method of reengineering strains by

assembling desirable genotypes. The reconstructed strains can be more robust, give

higher fermentation yields in less time, and resist stressful conditions better than

classical industrial producers.

In addition, these technologies can help us meet new and possibly unforeseen

challenges in the future: the amino acid industry is now beginning to consider

sustainable and environmentally friendly manufacturing systems in response to the

continuing crisis of global warming. There is a powerful drive to develop highly

efficient fermentation processes, especially for methionine and cysteine, the sulfur-

containing amino acids, using feedstocks that are renewable and that do not

compete with human food or energy sources. The development of strains that

enable the reduction in effluents and wastes generated during fermentation and

purification processes is also expected. These remain important themes for future

engineering.

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Amino Acid Production by Corynebacterium