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Biocatalysis in pharmaceutical preparation and alteration
Biocatalysis in pharmaceutical preparation and alteration Barrie 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. Addresses 1 Biotica technology Ltd, Chesterford Research Park, Little Chesterford, Essex CB10 1XL, UK 2 College of Arts and Science, Vanderbilt University, 7921 Stevenson Center, Nashville, TN 37235-1822, USA
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 disciplines, underlining significant overlapping opportunities for both drug discovery and production.
In vivo biosynthetic engineering of natural product core structures
Engineering-based methods for ‘improving’ natural product structures have succeeded in generating focused libraries of structurally related analogues anticipated to have improved pharmaceutical properties. These alterations 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 structures, such as by oxidation, methylation and glycosylation. We report on recent ambitious efforts toward the expression and engineering of natural and non-natural biosynthetic pathways.
Biosynthetic engineering has been utilized to produce analogues of rapamycin. This is the only validated inhibitor of mTOR (mammalian target of rapamycin), a serine/threonine kinase and central controller of eukaryotic cellular processes related to growth and proliferation. The analogues were created by deleting multiple genes responsible for the tailoring of the pre-rapamycin macrolactone from the biosynthetic gene cluster of Streptomyces hygroscopicus; pre-rapamycin is the first enzymefree 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 provides an opportunity to add back all possible combinations, 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-rapamycin. The rapK gene is essential for biosynthesis of the trans-3,4-dihydroxycyclohex-1-ene carboxylic acid ‘starter 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.
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 pathways, 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
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 rapamycin analogues was established. These rapamycin analogues contained structural features determined by the exogenously fed compounds and by the combination of
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
Introduction
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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 particular mutant strain utilized. This method is truly combinatorial 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 produced by the method are shown in Figure 1b and cover broad chemical space, addressing issues of potency and physiochemical properties that remedy limitations of solubility 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 Current Opinion in Chemical Biology 2006, 10:169–176
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 natural product glycosylation patterns Glycosylation of natural product templates is often essential for their biological activity. This had led to tremendous interest in developing biocatalytic methods for modifying both natural and engineered aglycone substrates. The exploitation of glycosyltransferases (GTs) for in vitro elaboration of core templates has at times been hampered because of problems isolating functional enzymes and the availability of the activated sugar substrates, although the www.sciencedirect.com
Biocatalysis in pharmaceutical preparation and alteration Wilkinson and Bachmann 171
approach has been powerfully exemplified for the glycopeptide antibiotics [10,11]. This ‘chemoenzymatic glycorandomization’ 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 activated sugar and expressing any required GT. The aglycone component of the reaction can then be generated in vivo, by genetic introduction of appropriate biosynthetic machinery, or through exogenous addition. Both of these approaches have their merits, the latter allowing particular 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-tetracenomycin C, the cassettes produced the expected activated sugars as demonstrated by the production of novel compounds containing the expected sugar moieties attached to 8-demethyl-tetracenomycin through the expected glycosidic linkage.
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-mycaminose and D-angolosamine in place of D-desosamine [20].
Pathway heterologous expression: a platform for drug modification and production Heterologous 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 nonribosomally encoded peptides, isoprenoids and alkaloids (Table 1) [21]. Gene clusters of up to 128 kilobases have been successfully reconstituted in a variety of heterologous hosts [22]. We highlight two applications of heterologous expression that illustrate the broad utility of this technology.
This approach was subsequently extended with new cassettes able to direct the biosynthesis of further sugars including L-digitoxose [16], L-mycarose, 4-deacetyl-Lchromose 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.
Heide and co-workers have developed a system to introduce cosmids containing biosynthetic gene clusters directly into the genome of a heterologous host (Figure 3). This technique was demonstrated for novobiocin 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. Subsequently, 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 chromosome. 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 deletion or substitution. An impressive reduction to practice of this strategy has been demonstrated by Wenzel and coworkers [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.
This modular approach has been recently applied to the modification of other chemical series, as demonstrated through their combination with GTs and aglycone substrates 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
The utility of heterologous expression extends beyond functional analysis of biosynthetic genes and clusters. Heterologous expression can also provide access to compounds from organisms that are difficult to culture or endangered, and offers a starting point for improving the production of natural products produced at low abundance. For example, the patellamides (Figure 4) are cytotoxic compounds originally isolated from a marine
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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 Prochloron 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 marine ecosystem from which they were isolated.
Biosynthetic pathways for biopharmaceutical precursors and intermediates In addition to modifying existing natural products by core and tailoring enzyme alterations, new efforts in biocatalysis focus on the concatenation of separate heterologous biocatalytic enzymes into new biosynthetic pathways for the production of valuable small molecules. These pathways 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
Table 1 Selected heterologous expression from recent literature. Compound
Ref.
Original producer
Heterologous host
Novobiocin Daptomycin Chartreusin Phosphinothricin Patellamide Palmitoyl- putrescine Safracin Isocyanide antibiotic Myxochromide
[22] [39] [40] [41] [25] [42] [43] [44] [23]
S. spheroides S. roseosporus S. Chartreusis S. viridochromogenes Prochloron didemni Metagenomic DNA P. fluorescens A2-2 Metagenomic DNA Stigmatella aurentica
S. coelicolor/lividans S. lividans S. albus S. lividans E. coli E. coli Pseudomonas E. coli Pseudomonas
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Biocatalysis in pharmaceutical preparation and alteration Wilkinson and Bachmann 173
Figure 3
Figure 4
Patellamides.
ple stereocenters in individual sugars and directing the linkage between them represents a unique challenge to synthetic chemistry.
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.
is in great demand because of their importance as antibiotics, 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 multiwww.sciencedirect.com
An increasingly viable alternative to chemical synthesis is a biosynthetic approach, which utilizes cloned and expressed enzymes of existing pathways. The biosynthetic pathway for the conversion of TDP-4-keto-6deoxy-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 alternative to the synthetic route, which is an eight-step synthesis from a starting methyl glycoside, itself derived semisynthetically via degradation of Di-N-CBz vancomycin. 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 glycosides. 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% respectively. A distinct multi-gene approach by Elling et al. uses gene cassettes, in analogy to the examples above, to Current Opinion in Chemical Biology 2006, 10:169–176
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Figure 5
way to shikimic acid by evolving the primary metabolic enzyme 2-keto-3-deoxy-6-phosphogalactonate aldolase to accept an alternative substrate, ethythrose-4-phosphate [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 biosynthetic 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 converted to aminoshikimate by recruiting the secondary metabolic enzyme aminoshikimate dehydrogenase [33].
Enabling technologies define the future of synthetic biology
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 coworkers, is to engineer promiscuous kinase and nucleotidyltransferase 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 shikimate (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 pathCurrent Opinion in Chemical Biology 2006, 10:169–176
Recent 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 production 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 sequencing 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 thousands 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 shotgun sequencing methods. Firstly, a disadvantage of pyrophosphate 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 problematic 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 www.sciencedirect.com
Biocatalysis in pharmaceutical preparation and alteration Wilkinson and Bachmann 175
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 biosynthetic 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 bacterial genes in recombinant hosts. This new bottleneck is being addressed by a shift in paradigms from restriction/ ligase-based cloning to recombination-based gene manipulation [38]. These advances, in combination with commercially 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 biosynthetic studies, and, furthermore, demonstrate the tremendous 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 construction using tools of both metabolic engineering and natural product biosynthetic studies. We can envision the production of unusual small peptides, polyketides, glycoconFigure 6
jugates containing highly modified sugars, and pathways for unnatural small molecules, among others.
Acknowledgement BOB acknowledges the Vanderbilt Institute of Chemical Biology for financial support.
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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 disciplines, underlining significant overlapping opportunities for both drug discovery and production.
In vivo biosynthetic engineering of natural product core structures
Engineering-based methods for ‘improving’ natural product structures have succeeded in generating focused libraries of structurally related analogues anticipated to have improved pharmaceutical properties. These alterations 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 structures, such as by oxidation, methylation and glycosylation. We report on recent ambitious efforts toward the expression and engineering of natural and non-natural biosynthetic pathways.
Biosynthetic engineering has been utilized to produce analogues of rapamycin. This is the only validated inhibitor of mTOR (mammalian target of rapamycin), a serine/threonine kinase and central controller of eukaryotic cellular processes related to growth and proliferation. The analogues were created by deleting multiple genes responsible for the tailoring of the pre-rapamycin macrolactone from the biosynthetic gene cluster of Streptomyces hygroscopicus; pre-rapamycin is the first enzymefree 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 provides an opportunity to add back all possible combinations, 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-rapamycin. The rapK gene is essential for biosynthesis of the trans-3,4-dihydroxycyclohex-1-ene carboxylic acid ‘starter 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.
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 pathways, 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
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 rapamycin analogues was established. These rapamycin analogues contained structural features determined by the exogenously fed compounds and by the combination of
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
Introduction
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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 particular mutant strain utilized. This method is truly combinatorial 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 produced by the method are shown in Figure 1b and cover broad chemical space, addressing issues of potency and physiochemical properties that remedy limitations of solubility 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 Current Opinion in Chemical Biology 2006, 10:169–176
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 natural product glycosylation patterns Glycosylation of natural product templates is often essential for their biological activity. This had led to tremendous interest in developing biocatalytic methods for modifying both natural and engineered aglycone substrates. The exploitation of glycosyltransferases (GTs) for in vitro elaboration of core templates has at times been hampered because of problems isolating functional enzymes and the availability of the activated sugar substrates, although the www.sciencedirect.com
Biocatalysis in pharmaceutical preparation and alteration Wilkinson and Bachmann 171
approach has been powerfully exemplified for the glycopeptide antibiotics [10,11]. This ‘chemoenzymatic glycorandomization’ 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 activated sugar and expressing any required GT. The aglycone component of the reaction can then be generated in vivo, by genetic introduction of appropriate biosynthetic machinery, or through exogenous addition. Both of these approaches have their merits, the latter allowing particular 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-tetracenomycin C, the cassettes produced the expected activated sugars as demonstrated by the production of novel compounds containing the expected sugar moieties attached to 8-demethyl-tetracenomycin through the expected glycosidic linkage.
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-mycaminose and D-angolosamine in place of D-desosamine [20].
Pathway heterologous expression: a platform for drug modification and production Heterologous 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 nonribosomally encoded peptides, isoprenoids and alkaloids (Table 1) [21]. Gene clusters of up to 128 kilobases have been successfully reconstituted in a variety of heterologous hosts [22]. We highlight two applications of heterologous expression that illustrate the broad utility of this technology.
This approach was subsequently extended with new cassettes able to direct the biosynthesis of further sugars including L-digitoxose [16], L-mycarose, 4-deacetyl-Lchromose 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.
Heide and co-workers have developed a system to introduce cosmids containing biosynthetic gene clusters directly into the genome of a heterologous host (Figure 3). This technique was demonstrated for novobiocin 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. Subsequently, 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 chromosome. 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 deletion or substitution. An impressive reduction to practice of this strategy has been demonstrated by Wenzel and coworkers [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.
This modular approach has been recently applied to the modification of other chemical series, as demonstrated through their combination with GTs and aglycone substrates 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
The utility of heterologous expression extends beyond functional analysis of biosynthetic genes and clusters. Heterologous expression can also provide access to compounds from organisms that are difficult to culture or endangered, and offers a starting point for improving the production of natural products produced at low abundance. For example, the patellamides (Figure 4) are cytotoxic compounds originally isolated from a marine
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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 Prochloron 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 marine ecosystem from which they were isolated.
Biosynthetic pathways for biopharmaceutical precursors and intermediates In addition to modifying existing natural products by core and tailoring enzyme alterations, new efforts in biocatalysis focus on the concatenation of separate heterologous biocatalytic enzymes into new biosynthetic pathways for the production of valuable small molecules. These pathways 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
Table 1 Selected heterologous expression from recent literature. Compound
Ref.
Original producer
Heterologous host
Novobiocin Daptomycin Chartreusin Phosphinothricin Patellamide Palmitoyl- putrescine Safracin Isocyanide antibiotic Myxochromide
[22] [39] [40] [41] [25] [42] [43] [44] [23]
S. spheroides S. roseosporus S. Chartreusis S. viridochromogenes Prochloron didemni Metagenomic DNA P. fluorescens A2-2 Metagenomic DNA Stigmatella aurentica
S. coelicolor/lividans S. lividans S. albus S. lividans E. coli E. coli Pseudomonas E. coli Pseudomonas
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Figure 3
Figure 4
Patellamides.
ple stereocenters in individual sugars and directing the linkage between them represents a unique challenge to synthetic chemistry.
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.
is in great demand because of their importance as antibiotics, 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 multiwww.sciencedirect.com
An increasingly viable alternative to chemical synthesis is a biosynthetic approach, which utilizes cloned and expressed enzymes of existing pathways. The biosynthetic pathway for the conversion of TDP-4-keto-6deoxy-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 alternative to the synthetic route, which is an eight-step synthesis from a starting methyl glycoside, itself derived semisynthetically via degradation of Di-N-CBz vancomycin. 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 glycosides. 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% respectively. A distinct multi-gene approach by Elling et al. uses gene cassettes, in analogy to the examples above, to Current Opinion in Chemical Biology 2006, 10:169–176
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Figure 5
way to shikimic acid by evolving the primary metabolic enzyme 2-keto-3-deoxy-6-phosphogalactonate aldolase to accept an alternative substrate, ethythrose-4-phosphate [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 biosynthetic 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 converted to aminoshikimate by recruiting the secondary metabolic enzyme aminoshikimate dehydrogenase [33].
Enabling technologies define the future of synthetic biology
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 coworkers, is to engineer promiscuous kinase and nucleotidyltransferase 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 shikimate (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 pathCurrent Opinion in Chemical Biology 2006, 10:169–176
Recent 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 production 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 sequencing 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 thousands 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 shotgun sequencing methods. Firstly, a disadvantage of pyrophosphate 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 problematic 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 www.sciencedirect.com
Biocatalysis in pharmaceutical preparation and alteration Wilkinson and Bachmann 175
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 biosynthetic 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 bacterial genes in recombinant hosts. This new bottleneck is being addressed by a shift in paradigms from restriction/ ligase-based cloning to recombination-based gene manipulation [38]. These advances, in combination with commercially 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 biosynthetic studies, and, furthermore, demonstrate the tremendous 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 construction using tools of both metabolic engineering and natural product biosynthetic studies. We can envision the production of unusual small peptides, polyketides, glycoconFigure 6
jugates containing highly modified sugars, and pathways for unnatural small molecules, among others.
Acknowledgement BOB acknowledges the Vanderbilt Institute of Chemical Biology for financial support.
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