Biocatalysis in pharmaceutical preparation and alteration

Biocatalysis in pharmaceutical preparation and alterationBarrie Wilkinson1 and Brian O Bachmann2

The term ‘synthetic biology’ is being used with increasing

frequency to describe the biocatalytic generation of small

molecules, either via stepwise biotransformation or engineered

biosynthetic pathways. The flexibility of this newly coined term

encompasses the historically separate fields of natural product

biosynthesis and metabolic engineering. This review discusses

the state of the art of these two disciplines in the context of the

discovery and development of bioactive precursors and

products.

Addresses1 Biotica technology Ltd, Chesterford Research Park, Little Chesterford,

Essex CB10 1XL, UK2 College of Arts and Science, Vanderbilt University, 7921 Stevenson

Center, Nashville, TN 37235-1822, USA

Corresponding authors: Wilkinson, Barrie

([email protected]); Bachmann, Brian O

([email protected])

Current Opinion in Chemical Biology 2006, 10:169–176

This review comes from a themed issue on

Biocatalysis and biotransformation

Edited by Ben Davis and Grace DeSantis

Available online 24th February 2006

1367-5931/$ – see front matter

# 2005 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.cbpa.2006.02.006

IntroductionEngineering-based methods for ‘improving’ natural pro-

duct structures have succeeded in generating focused

libraries of structurally related analogues anticipated to

have improved pharmaceutical properties. These altera-

tions have been achieved through modification of the

biosynthetic machinery responsible for assembling both

the initial template structures, such as from polyketide

biosynthesis, and then the elaboration of template struc-

tures, such as by oxidation, methylation and glycosyla-

tion. We report on recent ambitious efforts toward the

expression and engineering of natural and non-natural

biosynthetic pathways.

The ultimate utility of a newly discovered therapeutic

agent is a function of both its pharmaceutical properties

and the viability of its large-scale production. Advances in

the manipulation of natural product biosynthetic path-

ways, originally developed as tools for basic science and

combinatorial biosynthesis, also lend themselves toward

application in drug and/or drug precursor production

platforms. This has traditionally been the domain of

www.sciencedirect.com

metabolic engineering, which has historically provided

systems for optimized individual biocatalytic steps [1].

We discuss recent developments in pathway expression

and engineering of multiple biocatalytic steps. Pathway

engineering efforts amplify the successes of single-step

biocatalysis and rely on advances in both natural product

biosynthesis and metabolic engineering methodologies.

Recent advances in technology are redefining both dis-

ciplines, underlining significant overlapping opportu-

nities for both drug discovery and production.

In vivo biosynthetic engineering of naturalproduct core structuresBiosynthetic engineering has been utilized to produce

analogues of rapamycin. This is the only validated inhi-

bitor of mTOR (mammalian target of rapamycin), a

serine/threonine kinase and central controller of eukar-

yotic cellular processes related to growth and prolifera-

tion. The analogues were created by deleting multiple

genes responsible for the tailoring of the pre-rapamycin

macrolactone from the biosynthetic gene cluster of Strep-tomyces hygroscopicus; pre-rapamycin is the first enzyme-

free intermediate of the biosynthetic pathway [2]. As

shown in Figure 1a, there are five modifications (two

regiospecific oxidations and three O-methylations)

involved in converting pre-rapamycin into rapamycin.

This allows for 24 different theoretical combinations of

the tailoring modifications, and although a specific route

from pre-rapamycin is preferred during native rapamycin

biosynthesis, the deletion of all genes responsible pro-

vides an opportunity to add back all possible combina-

tions, providing specific access to structures not usually

obtainable [3�,4�]. In addition, the deletion strain also

lacks a pair of genes (rapK and rapL) that are responsible

for the production of two key precursors of pre-rapamy-

cin. The rapK gene is essential for biosynthesis of the

trans-3,4-dihydroxycyclohex-1-ene carboxylic acid ‘star-

ter unit’ required to initiate polyketide chain synthesis [2]

and the rapL gene is involved in providing elevated levels

of the unnatural amino acid L-pipecolic acid [5]; this is

required as the final component of the macrocyclic ring

and is involved in forming the lactone and lactam linkages

of pre-rapamycin.

As the deletion strain lacks these two essential precursor

supply genes it provided an opportunity for incorporating

structural diversity through mutasynthesis [3�,4�]. When

synthetically generated analogues of these two precursor

units were supplied exogenously, biosynthesis of rapa-

mycin analogues was established. These rapamycin ana-

logues contained structural features determined by the

exogenously fed compounds and by the combination of


170 Biocatalysis and biotransformation

Figure 1

Biosynthetic engineering of rapamycin. (a) Rapamycin is biosynthesised from pre-rapamycin, the first enzyme-free intermediate of the pathway,

through regiospecific oxidations and methylations. (b) Representative rapalogue libraries generated by combinatorial biosynthesis and derived

from modified starter units (cycloheptane- and 3-fluoro-4-hydroxycyclohexane carboxylic acids) with optional combinations of modification

(R1 = H,H or keto; R2 = H or CH3; R3 = H, OH or OCH3). S. hyg WT = Streptomyces hygroscopicus wild type.

macrocycle-processing genes encoded within the particu-

lar mutant strain utilized. This method is truly combina-

torial for focused library production, as the 24 mutant

strains can be fed any combination of synthetic carboxylic

acids and amino acids accepted by the biosynthetic

machinery. Examples of ‘rapalogues’ successfully pro-

duced by the method are shown in Figure 1b and cover

broad chemical space, addressing issues of potency and

physiochemical properties that remedy limitations of solu-

bility and metabolism inherent to the parent compound

and its simple semi-synthetic analogues. The approach has

led to the production of promising drug candidates, with

properties distinct from other rapamycin analogues.

Several other instructive examples of focused library

generation through these methods have been reported


for antibiotics such as the aminocoumarins [6] and cyclic

lipopeptides [7], and for anti-cancer agents such as the

indolocarbazoles [8] and epothilones [9].

In vivo biosynthetic engineering of naturalproduct glycosylation patternsGlycosylation of natural product templates is often essen-

tial for their biological activity. This had led to tremen-

dous interest in developing biocatalytic methods for

modifying both natural and engineered aglycone sub-

strates.

The exploitation of glycosyltransferases (GTs) for in vitroelaboration of core templates has at times been hampered

because of problems isolating functional enzymes and the

availability of the activated sugar substrates, although the


Biocatalysis in pharmaceutical preparation and alteration Wilkinson and Bachmann 171

approach has been powerfully exemplified for the glyco-

peptide antibiotics [10,11]. This ‘chemoenzymatic gly-

corandomization’ approach for elaborating core templates

has also been reviewed extensively elsewhere [12,13].

We focus here on in vivo methods that utilize a ‘cell

factory’ approach for producing both the activated sugar

and the GT of choice in a host organism of choice [14].

Such cell factories are capable of accumulating an acti-

vated sugar and expressing any required GT. The agly-

cone component of the reaction can then be generated invivo, by genetic introduction of appropriate biosynthetic

machinery, or through exogenous addition. Both of these

approaches have their merits, the latter allowing particu-

lar flexibility as multiple aglycone analogues may be fed

to any particular strain.

A significant advance in this area came with an approach

for generating biosynthetic gene cassettes [15]. These

plasmid cassettes carry all of the genetic information

required for the biosynthesis of a specific activated sugar

and are modular in nature, allowing biosynthetic genes to

be readily removed or added. Such an approach relies on a

flexible substrate tolerance for the various enzymes

encoded by the genes that are to be ‘mixed and matched’

from different sugar pathways. The resulting biosynthetic

cassettes are transformed into host cells that already

contain a heterologous plasmid containing any specific

GT of choice. For demonstration purposes ElmGT of the

elloromycin biosynthetic pathway was selected. This GT

possesses broad substrate tolerance towards the activated

sugar component of the reaction. When the resulting

strains were grown in the presence of 8-demethyl-tetra-

cenomycin C, the cassettes produced the expected acti-

vated sugars as demonstrated by the production of novel

compounds containing the expected sugar moieties

attached to 8-demethyl-tetracenomycin through the

expected glycosidic linkage.

This approach was subsequently extended with new

cassettes able to direct the biosynthesis of further sugars

including L-digitoxose [16], L-mycarose, 4-deacetyl-L-

chromose B and 2,3,4-tridemethyl-L-nogalose [17], and

L- and D-amicetose [18]. It is noteworthy that these latter

reports identified certain substrate tolerance limitations

within the range of sugar biosynthetic enzymes utilized.

By harnessing numerous pathways, however, enzymes

with the appropriate activities were identified.

This modular approach has been recently applied to the

modification of other chemical series, as demonstrated

through their combination with GTs and aglycone sub-

strates from the indolocarbazole biosynthetic pathway

[19��]. Derivatives of the protein kinase C inhibitor

staurosporine have been generated in which the native

sugar moiety was replaced with L-rhamnose, L-olivose or

L-digitoxose, attached through either one or two N–C


linkages, and with D-olivose attached through a single

linkage (Figure 2). A similar approach has been reported

in the patent literature for producing erythromycin and

tylosin analogues carrying the aminosugars D-mycami-

nose and D-angolosamine in place of D-desosamine [20].

Pathway heterologous expression: a platformfor drug modification and productionHeterologous expression of whole metabolic pathways

has become an increasingly common technique in natural

product biosynthetic studies, and the method of choice

for providing experimental evidence for the complete

cloning of a new biosynthetic cluster. Representative

examples can be found in nearly all classes of natural

products including polyketides, ribosomally and non-

ribosomally encoded peptides, isoprenoids and alkaloids

(Table 1) [21]. Gene clusters of up to 128 kilobases have

been successfully reconstituted in a variety of heterolo-

gous hosts [22]. We highlight two applications of hetero-

logous expression that illustrate the broad utility of this

technology.

Heide and co-workers have developed a system to intro-

duce cosmids containing biosynthetic gene clusters

directly into the genome of a heterologous host

(Figure 3). This technique was demonstrated for novo-

biocin and chlorobiocin from Streptomyces spheroids. l-Red

mediated homologous recombination was used to

exchange the b-lactam resistance gene in a cosmid with

the attachment site and integrase of phage fC31. Subse-

quently, this cosmid is competent to integrate into the

genome of the desired recipient. The advantage of this

method is that potentially any cosmid, fosmid or Bac clone

containing a gene cluster, or a portion of a gene cluster, can

be similarly modified and integrated into a bacterial chro-

mosome. Once in hand, heterologous constructs can often

be more easily manipulated in a well-defined heterologous

host than in the original organism, providing the ability to

rapidly and functionally analyze individual genes by dele-

tion or substitution. An impressive reduction to practice of

this strategy has been demonstrated by Wenzel and co-

workers [23]. In their approach, a similar recombination

principle (RedET) was employed to ‘stitch’ together

multiple cosmids containing myxobacterial genomic

DNA and integrate them into Pseudomonas putida. A

1000-fold increase in myxochromide S production was

observed, providing a platform for subsequent pathway

investigation and modification.

The utility of heterologous expression extends beyond

functional analysis of biosynthetic genes and clusters.

Heterologous expression can also provide access to com-

pounds from organisms that are difficult to culture or

endangered, and offers a starting point for improving the

production of natural products produced at low abun-

dance. For example, the patellamides (Figure 4) are

cytotoxic compounds originally isolated from a marine



Figure 2

Staurosporine and biosynthetically engineered analogues. (a) Structure of staurosporine. (b) Staurosporine analogues carrying modified

deoxysugars produced by engineered biosynthesis.

ascidian from the Republic of Palau [24]. Interestingly,

these compounds are not produced by the ascidian but by

an obligate symbiont (unculturable) cyanobacterial Pro-chloron species. Schmidt and co-workers have recently

identified the patellamide biosynthetic gene cluster by

metagenomic analysis of the symbiont [25]. Surprisingly,

these highly modified cyclic peptides were found to be

ribosomally encoded. The gene cluster was subsequently

detected in a fosmid clone by a PCR-based method,

which was found to be competent in the production of

patellamides in an E. coli host. Successful expression in a

heterologous E. coli host was also independently reported

by Long et al. [26]. These results form the basis of both a

new patellamide engineering platform and a means of

producing patellamides that no longer relies on the mar-

ine ecosystem from which they were isolated.

Table 1

Selected heterologous expression from recent literature.

Compound Ref.

Novobiocin [22]

Daptomycin [39]

Chartreusin [40]

Phosphinothricin [41]

Patellamide [25]

Palmitoyl- putrescine [42]

Safracin [43]

Isocyanide antibiotic [44]

Myxochromide [23]


Biosynthetic pathways for biopharmaceuticalprecursors and intermediatesIn addition to modifying existing natural products by core

and tailoring enzyme alterations, new efforts in biocata-

lysis focus on the concatenation of separate heterologous

biocatalytic enzymes into new biosynthetic pathways for

the production of valuable small molecules. These path-

ways have the potential to provide routes to complex

compounds that are difficult to obtain by synthetic means

or existing biosynthetic pathways. In addition to the

discovery of new antibiotics by combinatorial application

of heterologous GTs, the development of biocatalytic

means of production of the substrates of GTs enables

the aforementioned discovery efforts and the subsequent

production/supply demands of a recombinant pathway.

Further, the synthesis of glycosides and glycoconjugates

Original producer Heterologous host

S. spheroides S. coelicolor/lividans

S. roseosporus S. lividans

S. Chartreusis S. albus

S. viridochromogenes S. lividans

Prochloron didemni E. coli

Metagenomic DNA E. coli

P. fluorescens A2-2 Pseudomonas

Metagenomic DNA E. coli

Stigmatella aurentica Pseudomonas



Figure 3

l-Red mediated strategy for introducing a gene cluster of interest

(shown in blue) into a heterologous chromosome. The b-lactamase (bla)

gene in SuperCos1 is retrofitted with an integrase (int) and attachment

site (attP) of phage fC31, permitting facile integration of retrofitted

cosmid in attB site of the genomes of Streptomyces coelicolor and

S. lividans.

Figure 4

Patellamides.

is in great demand because of their importance as anti-

biotics, in addition to the increasing awareness of the role

of glycobiology (glycomics) in human development and

pathology. Glycosides and glycoconjugates are excellent

targets for synthetic biology as sugars and polysaccharides

are challenging synthetic targets. Controlling the multi-


ple stereocenters in individual sugars and directing the

linkage between them represents a unique challenge to

synthetic chemistry.

An increasingly viable alternative to chemical synthesis is

a biosynthetic approach, which utilizes cloned and

expressed enzymes of existing pathways. The biosyn-

thetic pathway for the conversion of TDP-4-keto-6-

deoxy-d-glucose to TDP-L-epivancosamine has been

reconstructed in vitro by cloning and overexpressing five

separate genes from Amycolatopsis orientalis in E. coli [27]

(Figure 5a). Sequential application of the His-tagged

purified enzyme resulted in the biosynthesis of this highly

modified deoxysugar. This pathway provides an alterna-

tive to the synthetic route, which is an eight-step synth-

esis from a starting methyl glycoside, itself derived

semisynthetically via degradation of Di-N-CBz vancomy-

cin. Although cloning genes and obtaining the soluble

expressed enzymes for a given pathway represents an

initial investment, once in hand, His-tag purified

enzymes can be obtained with relative ease and

employed for the synthesis of many interesting glyco-

sides.

One practical drawback of the biocatalytic approach to

sugar synthesis is that it requires chemical synthesis of

precursor sugar nucleotide diphosphates [28]. Wang et al.have demonstrated a way to overcome some of these

difficulties with an in situ recycling system. Two artificial

gene clusters were constructed comprising three genes

each for the production of globotriose and gala1,3Lac,

two immunologicaly important oligosaccharides

(Figure 5b). Isolated yields were 22% and 18% respec-

tively. A distinct multi-gene approach by Elling et al. uses

gene cassettes, in analogy to the examples above, to



Figure 5

Biosynthetic pathways for biopharmaceutical precursors and

intermediates. (a) Sequential in vitro biotransformation for the production

of dTDP-L-epi-vancosamine. (b) Batch-scale glycoconjugate synthesis

by a multi-enzyme pathway. (c) Two pathways for microbial

overproduction of shikimic acid by recombinant Escherichia coli or a

two-step two-bacteria process.

synthesize dTDP-activated deoxysugars and precursors

[29]. Another strategy, pioneered by Thorson and co-

workers, is to engineer promiscuous kinase and nucleo-

tidyltransferase activities to activate a broad range of

sugar precursors for subsequent glycorandomization

experiments [30].

Small-molecule pathways from primary metabolism can

also be harnessed for the synthesis of drug precursors.

Tamiflu is a neuramidase inhibitor of growing importance

because of its potent antiviral activity against prominent

emergent viral infections such as avian influenza and

SARS. The starting point for Tamiflu synthesis is shiki-

mate (Figure 5c), an intermediate in primary metabolism.

As a result, shikimate metabolic engineering has become

the subject of extensive investigation [31]. Recently,

Frost and co-workers have developed an alternative path-


way to shikimic acid by evolving the primary metabolic

enzyme 2-keto-3-deoxy-6-phosphogalactonate aldolase

to accept an alternative substrate, ethythrose-4-phos-

phate [32��]. The result is an E. coli strain capable of

producing 8.3 g/l of shikimate from glucose (5% yield).

Subsequently, the Frost group has constructed a biosyn-

thetic system for the production of aminoshikimic acid in

Bacillus subtilis and recombinant E. coli. In this sequence,

3-amino-3-deoxy glucose from Bacillus subtilis is con-

verted to aminoshikimate by recruiting the secondary

metabolic enzyme aminoshikimate dehydrogenase [33].

Enabling technologies define the future ofsynthetic biologyRecent technological advances are having a profound

effect on the rate of discovery of antibiotic biosynthetic

pathways and modifying enzymes. The construction of

‘superhosts’, bacterial hosts that are optimized for pro-

duction of secondary metabolites and hosts that contain

cassettes of biosynthetic genes for producing discreet

intermediates of primary and secondary metabolism, will

provide the building blocks for combinatorial synthesis

and biosynthesis [34]. Recently, whole genome sequen-

cing of bacterial genomes has moved within reach of

many investigators because of the commercial availability

of high density picoliter reactors [35�]. This technology

utilizes sheered genomic DNA, which is bound to beads

under conditions favoring one DNA molecule per bead.

The beads are suspended in PCR reaction mixture in oil

emulsion, and amplified. The beads, containing thou-

sands of copies of the DNA, are then deposited on an

optical slide containing 1.6 million wells. Pyrophosphate

sequencing of the beads results in reads of an average

length of 100 bp. The advantage of this system is that it

can produce a draft bacterial genome in less than a week

(20 MBp/4.5 h), including sample preparation time.

There are two considerations when comparing this

method, in its current embodiment, to traditional shot-

gun sequencing methods. Firstly, a disadvantage of pyr-

ophosphate sequencing is that it is often unable to read

through homopolymer repeat regions (e.g. poly A). This is

a relatively minor disadvantage with regard to small

bacterial genome sequencing, but may be more proble-

matic in eukaryotes. Secondly, shot-gun library based

sequencing methods have a distinct practical advantage

of yielding a library of genomic DNA for subsequent

DNA manipulation. In some cases, the shot-gun library

has been constructed of fosmid DNA, resulting in an

immediate entry point for sub-cloning and heterologous

expression of a biosynthetic gene cluster [25].

In any event, the ability to rapidly obtain draft bacterial

genomes will accelerate natural product gene cluster

identification and biosynthetic gene discovery. It has been

demonstrated that genome mining of microorganisms

results in the rapid identification of gene clusters, natural

product pathways and, in some cases, the discovery of new



natural product structures [36,37]. This technology

changes the current paradigm of primary and secondary

biosynthetic gene discovery and investigation (Figure 6).

In the ‘pre-genomics’ era a typical flow chart of biosyn-

thetic gene discovery and investigation was limited by

both cloning and sequencing biosynthetic genes and the

production of gene knockouts for functional analysis. In

the ‘post-genomics’ era the relief of the labor-intensive

sequencing bottleneck directs new emphasis on tools for

the rapid manipulation and expression of multiple bacter-

ial genes in recombinant hosts. This new bottleneck is

being addressed by a shift in paradigms from restriction/

ligase-based cloning to recombination-based gene manip-

ulation [38]. These advances, in combination with com-

mercially available multi-gene expression systems, offer

significant opportunities in the synthetic biology of natural

and unnatural product biosynthetic pathway engineering.

Recent studies demonstrate a trend in the blurring of

metabolic engineering and secondary metabolism biosyn-

thetic studies, and, furthermore, demonstrate the tremen-

dous potential for the synergy between synthetic

chemistry and biocatalytic pathways for the production

of intermediates and products. These studies precede

what is sure to be an explosion of drug pathway construc-

tion using tools of both metabolic engineering and natural

product biosynthetic studies. We can envision the pro-

duction of unusual small peptides, polyketides, glycocon-

Figure 6

Genomic sequencing technology changes the paradigm for

biosynthetic gene discovery. Red shaded steps are often the rate

limiting steps.


jugates containing highly modified sugars, and pathways

for unnatural small molecules, among others.

AcknowledgementBOB acknowledges the Vanderbilt Institute of Chemical Biology forfinancial support.

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� of special interest

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Biocatalysis in pharmaceutical preparation and alteration