Recent developments in the molecular genetics of the erythromycin-producing organism Saccharopolyspora erythraea

Recent Developments in the Molecular Genetics of the Erythromycin-Producing Organism

Saccharopolyspora erythraea THOMAS

J.

VANDEN BOOM

Abbott Laboratories Fermentation Microbiology Research and Development North Chicago, Illinois 60064

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Introduction Background Experimental Properties of S. erythraea Strains Characterization of the S. erythraea Genome A. Physical-Genetic Mapping of the Chromosome B. Genomic Polymorphisms in Industrially Improved S. erythraea Strains Introduction of DNA into S. erythraea A. Sonication-Dependent Electroporation B. Electroporation of Germinating Spores Transcriptional Organization and Regulation of the Erythromycin Biosynthetic Gene Cluster A. Previous Transcriptional Studies of the eryCI, ermE, and eryG Genes B. Construction and Analysis of Transcriptional Mutants in S. erythraea C. Erythromycin Biosynthetic Gene Cluster Promoters D. Transcriptional Overview of the ery Gene Cluster New Molecular Genetic Tools for Studying Gene Expression in S. erythraea Genetic-EngineeringApproaches to Industrial Strain Improvement A. Construction of High-Productivity Source Strains for Naturally Occurring Erythromycin Intermediates B. Two-Step Genetic-EngineeringApproaches for Optimization of Novel Macrolide Production in S. erythraea C. Introduction of the Vitreoscilla hemoglobin gene into S. erythraea Combinatorial Biosynthesis A. Manipulation of ery Biosynthetic Genes in Heterologous Streptomyces Hosts B. Manipulation of ery Biosynthetic Genes in S. erythraea Future Prospects References

I. Introduction N e a r l y 50 y e a r s s i n c e t h e m a c r o l i d e a n t i b i o t i c e r y t h r o m y c i n w a s first d e s c r i b e d ( M c G u i r e et al., 1952), t h e p r o d u c i n g m i c r o o r g a n i s m Saccharopolyspora erythraea r e m a i n s t h e s u b j e c t of k e e n i n d u s t r i a l i n t e r est. A n u m b e r of factors h a v e c o n t r i b u t e d to t h e o n g o i n g i n d u s t r i a l 79 ADVANCESINAPPLIEDMICROBIOLOGY.VOLUME47 Copyright©2000byAcademicPress Allrightsofreproductionin anyformreserved. 0065-2164/00$25.00

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THOMAS J. VANDENBOOM

research interest in both erythromycin and S. erythraea. Prominent among these is the fact that various dosage forms of erythromycin continue to enjoy widespread use globally for a variety of indications due to the excellent record of therapeutic efficacy and safety achieved by erythromycin-derived products. Moreover, the introduction of second-generation semisynthetic erythromycin derivatives in the early 1990s created additional demands for bulk erythromycin A as the starting raw material for these products. The two major commercial second-generation erythromycin species, clarithromycin and azithromycin, are shown in Figure 1. The emergence of clinical isolates resistant to the second-generation macrolide antibiotics (Weisblum, 1998) has fueled continuing research in a number of industrial laboratories to develop third-generation erythromycin derivatives (Ma et al., 1999; Agouridas et al., 1998; Phan et al., 1997). Perhaps the most promising class of third-generation candidates currently in clinical development is the 3-oxo-erythromycin derivatives, or "ketolides." Two leading clinical candidates in this class, ABT-773 and HMR-3647, are also shown in Figure 1. Finally, there appears to be growing interest in the genes involved in erythromycin biosynthesis in the emerging field of combinatorial biosynthesis. Both the type I polyketide synthase (PKS) from S. erythraea (for reviews, see Hutchinson, 1998, 1999; Cane et al., 1998) and the related desosamine deoxysugar biosynthesis genes from Streptomyces venezuelae (Zhao et aL, 1998) have been successfully manipulated to produce hybrid microbial metabolites. In this review, I discuss recent advances in the molecular genetics of S. erythraea, with particular emphasis on current topics of industrial interest. Our present knowledge of the S. erythraea genome, as well as recent advances in molecular genetic methods applicable to wild-type and industrially improved strains of this organism, are considered here. In addition, this review summarizes recent studies on the transcriptional organization and regulation of the erythromycin biosynthetic gene cluster. These studies have improved our understanding of erythromycin gene expression in this organism and provide a foundation for future genetic manipulations of this industrially significant metabolic pathway. Finally, I briefly consider genetic-engineering approaches to erythromycin strain improvement and the role of S. erythraea and erythromycin biosynthetic genes in the emerging field of combinatorial biosynthesis. S. erythraea has received considerable attention as a model system for the study of polyketide biosynthesis. This topic is beyond the scope of this review. A brief overview of the biosynthesis of the erythromycin polyketide backbone is included herein simply as background for this review. The interested reader is referred to several

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II. Background The Gram-positive actinomycete Saccharopolyspora erythraea produces the clinically significant macrolide antibiotic erythromycin A. The erythromycin biosynthetic gene cluster has been localized near one

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THOMAS J. VANDENBOOM

end of the linear S. erythraea chromosome (Reeves eta]., 1998). The gene cluster has been cloned and sequenced and contains at least 20 genes involved in the formation and modification of the 14-membered macrolide 6-deoxyerythronolide B (6-DEB) and in the synthesis, attachment, and modification of the two deoxysugars desosamine and mycarose (Salahbey et al., 1998; for reviews, see Staunton and Wilkinson, 1997; Katz, 1997). Functions for the majority of genes located in this cluster have been proposed based on an analysis of blocked mutants constructed through targeted gene inactivation. A schematic view of the erythromycin biosynthetic pathway through the first bioactive erythromycin intermediate erythromycin D is shown in Figure 2. During erythromycin biosynthesis, the aglycone backbone 6-DEB is produced by a type I modular PKS from one propionyl-CoA and six (2S)-methylmalonyl-CoA molecules in a process closely resembling fatty acid biosynthesis (Staunton and Wilkinson, 1997). The 6DEB synthase (DEBS) is encoded by three large genes, designated eryAI, eryAII, and eryAIII, located roughly in the center of the biosynthetic gene cluster. The three multifunctional enzymes encoded by these genes each contains two modules, or sets of enzymatic activities, responsible for a single round of polyketide chain extension. The catalytic activities present in these modules dictate the stereochemistry and extent of reduction during each round of chain extension. In addition, the specificity of the initial loading module dictates the preferred starter units used by the DEBS enzyme (Weissman et al., 1998b). Following synthesis of the 6-DEB polyketide backbone, a specific hydroxylation occurs at the C-6 position to produce erythronolide B (EB). The C-6 hydroxylase responsible for this reaction is encoded by the eryF gene (Weber et al., 1991). EB is then modified by sequential attachment of mycarose and desosamine at the C-3 and C-5 hydroxyl groups, respectively, to produce the first bioactive intermediate, erythromycin D. Mutations affecting the synthesis and attachment of mycarose define eryB genes and result in phenotypic accumulation of the aglycone EB, whereas mutations affecting the synthesis and attachment of desosamine define e ~ C genes and result in phenotypic accumulation of 3-a-mycarosyl erythronolide B. The terminal steps of the erythromycin biosynthetic pathway form a metabolic grid in which erythromycin D is converted to erythromycin A by two alternative pathways (Fig. 3). Two modification enzymes, a specific mycarosyl O-methyltransferase encoded by the eryG gene (Paulus et al., 1990; Haydock et al., 1991) and a C-12 hydroxylase encoded by the eryK gene (Stassi et al., 1993), compete for the erythromycin D

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pathway intermediate. Initial O-methylation of the mycarose moiety on erythromycin D leads to the bioactive intermediate erythromycin B. Subsequent C-12 hydroxylation of erythromycin B leads to erythromycin A. Alternatively, these reactions can be reversed. Initial C-12 hydroxylation of erythromycin D leads to the bioactive intermediate erythromycin C, which in turn is converted to erythromycin A by the specific mycarosyl O-methyltransferase. The latter route has been suggested as the preferred pathway based on kinetic studies of the C-12 hydroxylase enzyme (Lambalot eta]., 1995). II1. Experimental Properties of S.

erythraea Strains

Wild-type strains of S. erythraea (e.g., NRRL2338) are readily manipulated using minor variations of the molecular genetic methods devel-

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oped for the better-characterized Streptomyces species S. coelicolor and S. lividans (Hopwood et al., 1985). Strain NRRL2338, and closely related S. erythraea strains (e.g., ER720), have been widely used in molecular genetic studies to elucidate gene-function relationships in the erythromycin biosynthetic pathway. In addition, molecular genetic manipulations of the erythromycin polyketide synthase in these genetic backgrounds has led to isolation of a number of novel macrolide compounds (see below) and have provided some insights into the function of individual enzymatic domains in this complex enzyme system (Katz, 1997). Several variants of wild-type strain NRRL2338 are in use within the S. erythraea research community, including the NRRL2338 "red variant strain" used by Leadlay and coworkers (Hessler et al., 1997). These variants may differ slightly in their experimental handling properties (e.g., growth rate) and erythromycin productivities. In contrast to the wild-type variants noted above, a wide range of mutant strains of S. erythraea developed for the large-scale fermentative production of erythromycin have been less amenable to established streptomycete and wild-type S. erythraea molecular genetic methods (Fitzgerald eta]., 1998; Katz, 1997; Brunker et a]., 1998). Significant differences in protoplast formation and regeneration, recombination, genome structure, plasmid maintenance, drug resistance, and genetic stability have been observed between wild-type and certain industrially improved strains of this organism (unpublished observations). Industrially improved strains (e.g., CA340) represent the product of numerous cycles of mutagenesis and screening for improved erythromycin titers (or other desirable fermentation properties). Practical experimental differences between wild-type and industrially improved strains likely result from secondary mutations present in heavily mutagenized improved genetic backgrounds or reflect the pleiotropic nature of certain titer-enhancing mutations. These strain differences, although poorly understood, have provided an incentive for further development of molecular genetic tools applicable to the full range of wild-type and improved S. erythraea strains encountered in industrial applications. IV. Characterization of the S. erythraea Genome

A.

PHYSICAL-GENETIC MAPPING OF THE CHROMOSOME

A physical map of AseI and DraI restriction sites in the chromosome of S. er~hraea strain NRRL2338 was completed using high-resolution PFGE (Reeves et al., 1998). Summation of individual AseI, DraI, and AseI-DraI fragments revealed a chromosome size of roughly 8 Mb. This

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THOMAS J. VANDEN BOOM

genome size is comparable to several previously characterized Streptomyces chromosomes (Kieser et al., 1992; LeBlond et al., 1993; Lezhava eta]., 1995; Pandza et al., 1997). The S. erythraea chromosome also shares several other features in common with previously characterized Streptomyces chromosomes. These features include a linear topology, the localization of genes involved in secondary metabolism near one end of the chromosome, and evidence for large genetically unstable regions of DNA (LeBlond et al., 1996; Aigle et al., 1996). In contrast to previously described Streptomyces chromosomes, no readily detectable terminal-inverted-repeat (TIR) sequences were observed in the S. erythraea chromosome when chromosomal end restriction fragments were hybridized to total AseI- and DraI-digested genomic DNA. It remains to be determined if short TIR sequences are present in this organism. Such TIR sequences would likely be undetectable using the large end restriction fragment probes employed in these experiments (Reeves et al., 1998). A total of 15 genetic loci have been mapped to specific AseI and DraI restriction fragments. The erythromycin biosynthetic gene cluster has been localized to an approximately 700-kb AseI-DraI restriction fragment located within 1.25 Mb from one end of the linear S. erythraea chromosome. Interestingly, several additional unlinked genes possibly involved in erythromycin biosynthesis or resistance, including ertX, a putative ABC-type transporter (O'Neill et al., 1995), gdh, a thymidine diphosphoglucose-4,6-dehydratase, and kde, a putative thymidine diphospho-4-keto-6-deoxyglucose 3,5-epimerase (Linton et al., 1995), have been localized to the same 7O0-kb region of the chromosome. In addition, this region also contains the attB site for the integrative S. erythraea plasmid pSE101 (Brown et al., 1988). B. GENOMIC POLYMORPHISMS IN INDUSTRIALLY IMPROVED S. ERYTHRAEA STRAINS

The AseI, DraI, and AseI-DraI chromosomal restriction digest profiles appear similar between wild-type and industrially improved strains of S. erythraea, except for two notable genomic polymorphisms. AseI chromosomal digests of strain CA340 reveal the loss of the 48-kb AseIN fragment and the appearance of an approximately 75-kb novel restriction fragment (Reeves et al., 1998). Strain CA340 produces roughly 10-fold more erythromycin than wild-type strain NRRL2338. It is at present not known if this AseI polymorphism is related to this productivity increase. In addition, more recent industrially improved erythro-

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mycin production strains, derived from strain CA340, harbor an additional roughly 150-kb chromosomal deletion (unpublished observation). Again, it is not known if this chromosomal polymorphism is directly related to the concomitant increase in erythromycin productivity observed in this genetic lineage. However, several genetic explanations seem plausible to explain the correlation between these genomic rearrangements and the phenotypic improvement in erythromycin biosynthesis: (1) competing secondary metabolic pathways might be deleted, (2) a negative trans-acting regulator might be deleted, or (3) a positive trans-acting regulator might be activated (or duplicated) as a result of the chromosomal rearrangement. Additional studies of these S. erythraea chromosomal polymorphisms should clarify the competing hypotheses outlined above, improve our understanding of the fluidity of the S. erythraea genome, and potentially facilitate future rational strain development efforts with this organism. The identification of the dispensable gene set present in the large 150-kb deleted region of the S. erythraea chromosome might also contribute to molecular genetic strain development approaches in other polyketide producing actinomycetes. V. Introduction of DNA into S. erythraea

Protoplast transformation techniques have proven effective for introduction of plasmid DNA into wild-type strains of S. erythraea at efficiencies of 105 to 106 transformants per ~g of replicating plasmid DNA (Yamamoto et al., 1986). Difficulties in extending this technique to industrially improved fermentation strains of S. erythraea led to the development of two complementary electroporation methods for introduction of DNA into this organism (Fitzgerald et al., 1998; English et aL, 1998). Although electroporation has found widespread application for introduction of DNA (and other macromolecules) into a broad range of cell types, there remain few reports describing application of this technology to industrially important filamentous organisms (Pigac and Schrempf, 1995; Tyurin and Livshits, 1996). A. SONICATION-DEPENDENTELECTROPORATION The development of electroporation techniques for S. erythraea was facilitated by the availability of a virulent bacteriophage for this host, designated CABT1, from the Abbott Laboratories Culture Collection (Fitzgerald et al., 1998). Concentrated preparations of the dsDNA

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THOMAS J. VANDEN BOOM

genome of this bacteriophage are readily obtained using standard phage propagation and purification techniques (Sambrook et al., 1989). This reagent permitted development of a sensitive electrotransfection assay and avoided potential problems with S. erythraea host restriction of shuttle vectors propagated in heterologous genetic backgrounds. During attempts to prepare well-dispersed homogeneous suspensions of vegetative S. erythraea cultures in our laboratory, we made the fortuitous observation that sonication treatment rendered this organism electrocompetent. Subsequent experimentation demonstrated that the observed electrocompetence was strictly sonication dependent for the vegetative cultures being examined. Culture preparation, sonication, and electroporation conditions were optimized using the electrotransfection assay to achieve electroporation efficiencies of 1.2 x 103 plaque forming units per microgram of CABT1 DNA. A plasmid-based electrotransformation assay was also developed to optimize sonicationdependent electroporation conditions for plasmid DNA uptake. This system utilized an Escherichia coli-Streptomyces shuttle vector, designated pCD1, derived from the pJV1 replicon of Streptomyces phaeochromogenes (Bailey et al., 1986; Fitzgerald et al., 1998). This vector is poorly maintained by S. erythraea but permits recovery and scoring of primary thiostrepton-resistant transformants prior to eventual loss of the plasmid. The electrotransformation efficiency obtained with this plasmid was 1.0 × 104 thiostrepton-resistant transformants per microgram of pCD1 DNA. Interestingly, the positive effect of sonication on electrocompetence was eliminated when the sonicated hyphal fragments were returned to culture tubes and incubated with shaking for 60 min prior to electroporation, suggesting that the physical alteration responsible for electrocompetence was eliminated or repaired during this period. It is tempting to speculate that the mechanical disruption of long vegetative hyphal fragments by ultrasound treatment in this procedure perhaps exposes interior hyphal membranes more susceptible to electroporation mediated DNA transfer. Alternatively, the sonication treatment might alter the normal outer cell wall to facilitate DNA entry into the cell. B. ELECTROPORATION OF GERMINATING SPORES

The amount of nonviable cellular material in the sonicated S. erythraea culture preparations prompted us to look for alternative (sonication-independent) conditions to achieve electrocompetence. Toward this end,

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cellular preparations from different stages of the developmental life cycle of S. erythraea were examined (English et al., 1998). The life cycle of S. erythraea is typical of other filamentous actinomycetes and has been reviewed elsewhere (Chater, 1998). This approach led to the discovery that germinating spores (germlings) of this organism move through a remarkably brief window of electrocompetence during outgrowth to vegetative hyphae. Under the culture conditions reported, the optimum outgrowth period for the harvest of electrocompetent germlings was between roughly 16 and 18 hours. The cellular physiological and/or morphological changes occurring during this period that result in electrocompetence are at present not understood. The utility of this method was demonstrated by constructing a targeted gene disruption of the pccA locus in the industrially improved S. erythraea strain CA340 (English et a]., 1998). The pccA gene encodes the biotinylated c~-subunit of the propionyl-CoA carboxylase from this organism. Electroporation efficiencies of 4-8 x 103 transformants per microgram of DNA were obtained with the suicide vector pJAY4 used in these experiments. Taken together with the protoplast transformation technique of Yamamoto et al. (1986), the two electroporation procedures reviewed here permit introduction of DNA into the full range of S. erj/thraea strains we have examined.

Vl. Transcriptional Organization and Regulation of the Erythromycin Biosynthetic Gene Cluster A. PREVIOUS TRANSCRIPTIONAL STUDIES OF THE ERYCI, ERME, AND ERYG GENES

Although DNA sequence analysis of the erythromycin biosynthetic gene cluster has provided some insights into the potential transcriptional organization of this gene cluster (Dhillon et al., 1989; Cortes et al., 1990; Donadio et al., 1991; Haydock et al., 1991; Donadio and Katz, 1992; Stassi et a]., 1993; Gaisser et al., 1997, 1998; Summers et al., 1997), detailed transcriptional studies have been lacking. Previous detailed transcriptional studies of ery genes have been limited to analysis of the eryCI-ermE region of the ery gene cluster reported by Bibb et al. (1994). A preliminary transcriptional study of the eryG gene has also been reported (Weber eta]., 1989). The regulatory region involved in divergent transcription of the eryCI and ermE genes contains two ermE and two eryCI promoters (Bibb et al., 1994). The ermE gene encodes an N-methyltransferase, which confers resistance to erythromycin in the producing host through methyla-

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THOMAS J. VANDENBOOM

tion of the S. erythraea 23S rRNA (Thompson et al., 1982). $1 nuclease and exonuclease VII transcript mapping experiments identified two transcriptional start sites for ermE, designated ermEpl and ermEp2. These experiments were performed using RNA isolated from Streptomyces lividans strain TK24 containing the eryCI-ermE promoter region cloned on high-copy-number plasmids. The divergently transcribed erythromycin biosynthetic gene eryCI is also transcribed from two promoters, designated eryCIpl and eryCIp2. The eryCI gene is thought to encode an aminotransferase involved in the synthesis of desosamine. Interestingly, transcription from ermEpl is initiated at the same position as eryCIp2, but on the opposing DNA strand. The existence of tandemly arranged, divergently transcribed overlapping promoters in this region led to the suggestion by these workers that a high degree of coordinate regulation occurs with these genes. The transcriptional start site for ermEpl is located immediately adjacent to the ermE translational start codon. This results in transcription of a leaderless message for the ermE gene. The ermEpl, ermEp2, and eryCIpl promoters contain recognizable -10 and -35 regions. In contrast, no recognizable -10 or -35 sequences were evident in the eryCIp2 promoter region, suggesting the involvement of alternative transcription factors in initiation of transcription from this promoter. Transcription of the eryG gene, which encodes the mycarosyl Omethyltransferase enzyme, was previously examined by Weber et al. (1989). Using Northern hybridization experiments, these workers identified an approximately 1.3-kb eryG transcript. A possible promoter region immediately upstream was reported based on an analysis of a cloned S. erythraea DNA fragment in a luxAB reporter system in S. lividans. More recent evidence from our laboratory has identified, in addition to the approximately 1.3-kb transcript described earlier, an additional 2.6-kb eryG transcript identifiable in Northern hybridization experiments (Reeves et al., 1999). This larger transcript contains the eryBII gene located immediately upstream of eryG. From our analysis of transcriptional terminator mutations in this region (see below), the stable eryG and eryBII-eryG messages detected in Northern hybridization experiments appear to be derived primarily from one of two very large overlapping polycistronic transcripts in this region. There is no evidence for a functional promoter in S. erythraea immediately upstream of either the erzG or eryBII gene. The previous results from Weber and coworkers (1989) might be attributable to the use of the high-copy-number luxAB reporter group plasmid and heterologous genetic background used in the earlier experiments.

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B. CONSTRUCTIONAND ANALYSISOF TRANSCRIPTIONAL MUTANTSIN S. ERYTHRAEA The development of a novel transcriptional terminator cartridge for the targeted construction of polar transcriptional mutants in S. erythraea has facilitated transcriptional studies of the erythromycin gene cluster (Reeves et al., 1999). These studies have improved our understanding of the transcriptional organization of this industrially significant biosynthetic gene locus. The transcriptional terminator cartridge, designated trm, contains a 227-bp transcriptional terminator sequence obtained from the cloned S. erythraea ribosomal RNA rrnD operon. This terminator sequence is flanked by convenient multiple cloning site sequences to permit in-vitro insertion of the cartridge into targeted genes of interest. A native S. erythraea terminator sequence was selected to ensure functionality in this host. Two regions of predicted secondary structure are present in the trm sequence. The first is a stem-loop structure consisting of a 17-bp stem with a short 4-base loop. This stem-loop is immediately followed by a thymidine-rich region, characteristic of rho-independent terminators (Deng et al., 1987). The calculated AG of the stem-loop is -30 kcal/mol. The second predicted region of secondary structure in this sequence is a stem-loop consisting of an 18-bp stem with a 4-base loop, located 30 bp downstream from the first stem-loop. This structure has a predicted AG o f - 2 4 kcal/mol. The t~m cartridge was cloned into targeted ery genes and introduced into the corresponding S. erythraea chromosomal loci via homologous recombination using either a pWHM3-derived (Vara et al., 1989) or pJVI-derived (Bailey et al., 1986) integration vector. Several additional features make S. erythraea an attractive experimental system for molecular genetic studies of the erythromycin biosynthetic pathway. These include: (1) the availability of cloned ery genes and DNA sequences, (2) the availability of DNA transformation and gene replacement methods for this organism, (3) the availability of purified erythromycin pathway intermediates, and (4) the ability of this organism to utilize exogenously supplied pathway intermediates in erythromycin biosynthesis. In our genetic studies of the ery gene cluster, biotransformation experiments with erythromycin pathway intermediates provided a simple, but powerful method to analyze the effects of specific trn~ transcriptional terminator insertion mutations (designated ery::t~n~) on expression of downstream ery genes. A variety of molecular genetic and biochemical methods, including Northern hybridizations, Western blotting, and $1 nuclease protection assays, were

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also employed in the characterization of the ery::trnn mutants. Two examples of the recently described S. erythraea tm~ insertion mutants (Reeves et a]., 1999) are reviewed here to illustrate the use of this methodology in the study of ery gene expression in wild-type and industrially improved genetic backgrounds. Based on these studies, the tm~ genetic element should also be of general use for the study of other genetic loci in this host. The tmn transcriptional terminator element was introduced in vitro into a cloned segment of the eryAI gene. This disrupted gene sequence was then introduced into the chromosome of wild-type strain NRRL2338 via homologous recombination to generate mutant NRRL2338 eryAI::tm~. The eryAI::tmn insertion was also introduced into the industrially improved genetic background of strain CA340 to generate mutant CA340 eryAI::tm~. As predicted, these eryA blocked mutants do not produce erythromycin A or any erythromycin pathway intermediates, consistent with previously characterized blocked mutants in the polyketide synthase genes. The central region of the erythromycin biosynthetic gene cluster contains, in addition to the three large eryA genes, four additional downstream genes. These seven genes, which include (in order) eryAL eryAII, eryAIII, eryCII, eryCIII, eryBII, and eryG, span approximately 35 kb of the erythromycin gene cluster. Several lines of evidence obtained from our analyses of the eryAI::tm~ mutants suggest that this set of genes is transcribed as a very large 35-kb polycistronic message. In biotransformation experiments, the eryAI::tmn mutation in both NRRL2338 and CA340 shows a polar effect on downstream ery genes, including the eryG Oomethyltransferase gene. Cultures of the eryAI::t~ mutant, when supplemented with erythromycin C, show greatly reduced levels of bioconversion of the erythromycin C to erythromycin A. Similar results were obtained in feeding experiments with other pathway intermediates, indicating an additional polar effect on both downstream eryB and eryC genes, as predicted. $1 nuclease protection assays using an eryG probe were also performed using total RNA extracted from the NRRL2338 eryAI::trnn and CA340 eryAI::i~nn mutant strains. A significant reduction of the eryG signal was observed in the insertion mutant strains relative to the parental controls, hterestingly, the polar effect of eryAI::tr~ insertion on eryG transcript levels appeared to be more pronounced in the CA340 industrially improved genetic background. This suggests that the promoter region immediately upstream of eryAI plays a more significant role in expression of the downstream eryG gene in the industrially improved strain CA340 than in the wild-type strain NRRL2338. Western blot experiments were also performed on wild-type strain NRRL2338 and the

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NRRL2338 eryAI::tm~ mutant to determine if the eryG O-methyltransferase protein was present in cell free extracts of these strains. A crossreacting band was readily detected in extracts of the parental control strain, but not in similarly prepared extracts of the eryAI::trnn mutant. In order to examine whether the eryAI::tm, insertion was having an effect other than on termination of transcription, an independent mutant strain was constructed using oligonucleotide-directed mutagenesis. In this mutant, designated ARR50, the predicted eryAI promoter ATrich -10 hexamer sequence (TATTGT) was replaced with the Sinai restriction enzyme recognition sequence CCCGGG. The polar phenotype of this strain was identical to that of the eryAI::trnn insertion mutant. Taken together, these experiments provide strong evidence that the eryAI, eryAII, eryAIII, eryBII, eryCII, eryCIII, and eryG genes are primarily cotranscribed from a promoter upstream of eryAL In addition, these experiments demonstrate the general utility of this new genetic element in the study of complex transcriptional units in this organism. C. ERYTtIROMYCINBIOSYNTHETICGENECLUSTERPROMOTERS The transcription start sites for seven additional ery gene cluster promoters, located in four ery gene cluster regulatory regions, have been reported (Reeves et al., 1999). These promoter regions include the 224-bp eryAI-eryBIV intergenic region, the 188-bp eryBI-eryBIII intergenic region, the 83-bp eryCVI-eryBVIintergenic region, and the region immediately upstream of the eryK gene. The 224-bp eryAI-eryBIV intergenic region contains an eryAI promoter divergently transcribed from two eryBIV promoters, designated eryBIVP1 and eryBIVP2. The eryAI transcription start site was located 27 bp upstream of the translation start codon for this gene. Based on an analysis of the eryAI::trnnmutant described above, the eryAI-containing transcript extends from eryAI to eryG. The transcription start sites for the eryBIVP1 and eryBIVP2 promoters are located 84-88 and 132 bp upstream of the predicted translational start codon for eryBIV, respectively. The eryBIVP1 promoter appears to be the major rightward promoter (as shown in Fig. 4) for this region based on the relative abundance of the respective Sl-protected fragments. The eryBIV-containing transcript is thought to extend from eryBIVto eryBVII, based on an analysis of ery::tmn insertion mutations in this region. This segment of the ery gene cluster also contains a smaller overlapping polycistronic transcript that extends from eryBVI through eryCVI. The promoter for this transcript is located in the 83-bp eryBVI-eryCVIintergenic region. The -35 region of the minor eryBIV promoter, eryBIVP2, is predicted

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to overlap with the -35 region of the divergent eryAI promoter. On the basis of our transcriptional analysis of the cry gene cluster, the three promoters identified in this 224-bp regulatory region account for the transcription of roughly 80% of the gene cluster, or 14 of the 20 identified cry genes. The sequence of this regulatory region was determined for an industrially improved strain of S. erythraea and compared to the wild-type strain NRRL2338 sequence. Surprisingly, no mutational changes were identified in this regulatory region in the industrially improved genetic background (unpublished observation) despite the significant increase in cry transcript levels and erythromycin titers observed in this genetic strain lineage (Reeves eta]., 1999). The transcriptional activity of DNA fragments containing the 224-bp eryAI-eryBIV regulatory region was examined using a kanamycin/neomycin phosphotransferase (APH) reporter group in both S. erythraea strain NRRL2338 and S. lividans strain TK24 (Atkins and Baumberg, 1998). In S. lividans, appreciable APH activity was detected in APH fusion strains regardless of the orientation of the S. erythraea eryAI-eryBIV DNA fragment. This finding is consistent with the presence of divergently transcribed promoters in this region. The function of these promoters in S. lividans further supports the notion that a pathway-specific activator is not involved in regulation of the cry gene cluster and is consistent with the previous observation that the ermE and eryCI promoters also function in this heterologous host (Bibb et al., 1994). The 188-bp eryBI-eryBIII intergenic region contains two divergently transcribed promoters. Two potential transcriptional start sites, located 1-2 bp upstream of the predicted start codon, were identified upstream of the eryBIII gene. An eryBIII::tr,,n insertion mutant displayed a polar effect on the downstream eryF gene, indicating that the eryF gene is cotranscribed with eryBIII. The start site for the divergently transcribed monocistronic eryBI message is located 17-18 bp upstream of the predicted translational start for the eryBI gene. The transcription start site for the eryK gene, which encodes the P450-dependent C-12 hydroxylase, was localized to a region 45-50 bp upstream of the predicted TTG start codon for this gene and 9-14 bp from the predicted termination codon of the adjacent orf21 coding region (Pereda et al., 1997). This gene, which is located on one end of the cry gene cluster, is transcribed as a monocistronic message. D. TRANSCRIPTIONAL OVERVIEW OF THE ERY GENE CLUSTER

A current transcriptional map of the erythromycin biosynthetic gene cluster is shown in Figure 4. The monocistronic messages for eryCI and

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ermE, described earlier, along with the more recently characterized eryBI and eryK transcripts (Reeves et al., 1999), represent the exception in this gene cluster, as the majority of ery genes appear to be transcribed as large polycistronic messages. This includes the previously reported eryG gene described above. Four polycistronic messages, including a large transcript of approximately 35 kb, account for the expression of roughly 85% of the gene cluster (Reeves et al., 1999). The two largest transcripts in the gene cluster extend divergently from the eryA-eryBIV intergenic region. The large 35-kb transcript includes the eryAL eryAII, eryAIII, eryCII, eryCIII, eryBII, and eryG genes. The divergently expressed transcript includes the eryBIV, eryBV, eryCVI, eryBVI, eryCIV, eryCV, and eryBVII genes. A second promoter upstream of the eryBVI gene produces an overlapping transcript that includes the eryBVI, eryCIV, eryCV, and eryBVII genes. Finally, a bicistronic message appears to be involved in expression of the eryBIII and eryF genes, and is divergently expressed from the monocistronic eryBI message.

VII. New Molecular Genetic Tools for Studying Gene Expression in S. erythraea

The gene encoding the native S. erythraea c¢-galactosidase enzyme, designated melA, has been cloned and sequenced (manuscript in preparation). The MelA enzyme was purified to near homogeneity and the N-terminal amino-acid sequence determined. Oligonucleotide primers based on the N-terminal amino-acid sequence and a conserved downstream region within the a-galactosidase gene were used to generate a 640-bp PCR product probe. Using this probe, the complete ct-galactosidase gene was identified in a lambda phage library of S. erythraea chromosomal DNA. The S. erythraea melA gene has been used to construct a reporter system for the study of gene expression in this organism. This system includes two components: (1) a parental S. erythraea strain containing a chromosomal deletion of the melA gene, and (2) a promoterless melA gene engineered into a pJVl-based integration vector. The melA integration plasmid is designated pDPE185. The promoterless melA gene cartridge present in pDPE185 is preceded by stop codons engineered in all three frames and includes a convenient upstream multiple cloning site. A ribosomal RNA terminator is present both upstream and downstream of the melA gene. Expression of the melA gene is conveniently monitored in liquid and solid agar cultures using either p-nitrophenol-

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0~-D-galactopyranoside (pNP{xG) or 5-bromo-4-chloro-3-indoyl-~-D-galactoside (X-~-gal), respectively. The function of this reporter group system was examined using the ermE* promoter region. The ermE* promoter fragment was inserted into the multiple cloning site of pDPE185. The resulting plasmid was introduced into the S. erythraea melA deletion host strain. Strains containing the ermE* promoter upstream of the melA gene had 10- to 15-fold more o~-galactosidase activity, as measured using the pNPRGbased assay, than the parental control strain. The melA reporter group system should be useful for constructing new S. erythraea vectors (e.g., insertional inactivation applications), for identifying new promoters, and in the study of gene expression in this organism (Satter, 1998).

VIII. Genetic-Engineering Approaches to Industrial Strain Improvement A. CONSTRUCTION OF HIGH-PRODUCTIVITY SOURCE STRAINS FOR NATURALLY OCCURRING ERYTHROMYCIN INTERMEDIATES

Genetic-engineering approaches have been used to introduce null mutations in the eryG and eryK genes, both individually and in combination, into various wild-type and industrially improved strains of S. erythraea. The analysis of mutants harboring disruptions of these genetic loci have permitted assignment of biochemical functions to these gene products (Paulus et al., 1990; Stassi eta]., 1993). In addition, genetically engineered eryG, eryK, and eryG/eryK null mutants represent important fermentation source strains for erythromycins C, B, and D, respectively (English et al., 1998). These erythromycin intermediates represent potential starting materials for development of new semisynthetic fermentation-based products (Faghih eta]., 1998). Naturally occurring erythromycin intermediates also find routine use among erythromycin manufacturers as analytical reference standards. Introduction of these mutant alleles into industrially improved erythromycin A-producing strains had little or no impact on overall macrolide titers {unpublished observation). This approach illustrates the utility of exploiting macrolide productivity levels of existing erythromycin A-producing industrial strains for production of other naturally occurring erythromycin intermediates. Moreover, this approach should also be applicable to the wide range of novel polyketide derivatives being generated through combinatorial biosynthesis methods with this organism (see below).

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B. TWO-STEP GENETIC ENGINEERING APPROACHES FOR OPTIMIZATION OF NOVEL MACROLIDE PRODUCTION IN S. ERYTHRAEA

Two reports by Stassi and coworkers (1998a, 1998b) illustrate the potential of rational strain development approaches for optimization of novel polyketide yields in genetically engineered strains of S. erythraea. The first report involves construction of a double mutant with disruptions in both the eryF gene, encoding the erythromycin C-6 hydroxylase, and the eryK gene, encoding the erythromycin C-12 hydroxylase. The predicted product of this double mutant is 6,12-dideoxyerythromycin A. Fermentations of the double eryF/eryK S. erythraea mutant produced a mixture of the desired product--6,12-dideoxyerythromycin A--and the immediate biosynthetic precursor--6,12-dideoxyerythromycin D--with the precursor representing the dominant product in the fermentation. In order to facilitate conversion of the 6,12-dideoxyerythromycin D precursor to the desired end-product, an additional copy of the eryG gene, encoding the 3"-O-methyltransferase enzyme responsible for this final biosynthetic step, was engineered into the eryF/eryK double mutant. The additional copy of the eryG gene under the control of the ermE* promoter was integrated into a chromosomal locus unlinked to the erythromycin gene cluster. The eryG diploid strain had significantly higher specific activities of EryG throughout the course of a 5-day fermentation. This increase in EryG activity resulted in quantitative conversion of the 6,12-dideoxyerythromycin D precursor to the desired 6,12-dideoxyerythromycin A product in fermentations of the eryF/eryK eryC--diploid strain. The second report involves addition of a heterologous primary metabolic enzyme to S. erythraea to improve precursor availability for a genetically engineered hybrid PKS. The ethylmalonate-specific acyltransferase domain from module five of the niddamycin PKS of Streptomyces caelestis (Kakavas et al., 1997) was substituted for the methylmalonate-specific acyltransferase domain present in module four of the erythromycin PKS (Stassi et al., 1998b). The predicted product of this mutant is 6-desmethyl-6-ethylerythromycin A. Interestingly, S. erythraea strains harboring this hybrid PKS still produced erythromycin A instead of the predicted hybrid polyketide product. The authors attributed this surprising result to the relaxed substrate specificity of the niddamycin acyltransferase domain used in this mutant construction and the limited intracellular availability of the ethylmalonyl-CoA precursor (relative to methylmalonyl-CoA) in this organism. In order to increase the available ethylmalonyl-CoA pools in this hybrid PKS mutant, the ccr gene from Streptomyces collinus expressed from the ermE*

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promoter was integrated at the same unlinked genetic locus used in the eryF/eryK mutant described above (Stassi et al., 1998a). The ccr gene encodes the crotonyl-CoA reductase enzyme responsible for the last step in the reductive biosynthesis of butyryl-CoA from two molecules of acetyl-CoA (Wallace et al., 1996) in S. collinus. Butyryl-CoA can then be carboxylated to form the desired ethyl malonyl-CoA precursor (Wallace et al., 1997). S. erythraea strains containing the S. collinus ccr gene had roughly 20-fold higher Ccr activity than the parental control strain. The addition of this activity to the PKS mutant led to production of the desired 6-desmethyl-6-ethylerythromycin A compound as the predominant product in fermentations of this strain. Taken together, these examples clearly demonstrate the utility of two-step genetic-engineering approaches to optimize both primary and secondary metabolic activities required for production of novel polyketides in S. erythraea. C. INTRODUCTION OF THE VITREOSCILLA HEMOGLOBIN GENE INTO S. ERYTHRAEA

Introduction of the Vitreoscilla hemoglobin gene (vhb) into an industrial strain of S. erythraea has been reported (Minas et al., 1998; Brunker et al., 1998). The vhb gene has been introduced into a variety of microorganisms, including Acremonium chrysogenum (DeModena et al., 1993), Escherichia coli (Khosla and Bailey, 1988), Corynebacterium glutamicum (Sander et al., 1994), Bacillus subtilis (Kallio and Bailey, 1996), and S. coelicolor (Magnolo et al., 1991) in attempts to improve oxygen metabolism and product yields in these organisms. However, the role of this gene in enhancing specific product yields in these systems remains speculative. The dramatic 70% improvement in erythromycin titers reported by these authors is difficult to assess due to the lack of information provided on the industrially improved S. erythraea strain used in these experiments. Nonetheless, this work represents the first reported attempt to improve erythromycin titers in S. erythraea through introduction of a heterologous gene into this organism. Functional expression of the vhb gene in the genetically engineered S. erythraea strain was confirmed by CO-difference spectrum assays. In the absence of selective pressure, the vhb integrant appeared to be genetically stable through at least a 9-day fermentation cycle. The molecular role of the Vhb protein in improving erythromycin titers in this strain is at present poorly understood. However, the improvement in erythromycin production observed in the genetically modified industrial strain did not appear to be due to simply an increase in biomass yield or to the pattern of mycelial fragmentation. The commercial

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utility of the S. erythraea::vhb strain described by these workers remains to be determined. Due to differences in industrial S. erythraea strain lineages, it will be of interest to determine whether the observed improvement in erythromycin yields is of general utility or limited to the model strain used by these workers.

IX. Combinatorial Biosynthesis In the past decade, intensive work in a number of laboratories (for reviews, see Staunton and Wilkinson, 1997; Katz, 1997) has led to significant advances in our understanding of the genetics and biochemistry of the erythromycin type I polyketide synthase in S. erythraea. Early molecular genetic studies of this system revealed the modular structure of this complex enzyme system and provided significant insights into the functional roles of individual enzymatic domains within the eryA-encoded PKS (Cortes eta]., 1990; Donadio et al., 1991). These studies also led to a series of successful genetic-engineering efforts involving targeted gene replacements of specific PKS modules to produce a number of novel erythromycin derivatives (reviewed by Katz, 1997). Although providing an important validation of this genetic-engineering approach to produce new molecular structures, this methodology suffered from several significant limitations. The construction of individual mutants was labor intensive and involved single mutational changes. In addition, mutants were produced in wild-type S. erythraea genetic backgrounds, leading to relatively low titers of the novel erythromycin derivatives produced in these strains. The development of combinatorial genetic-engineering approaches that permit introduction of multiple genetic alterations into the ery PKS represents a significant technical advance and illustrates the potential of this gene cluster for production of a wide range of polyketide compounds (McDaniel et al., 1999). Parallel advances in precursor-directed biosynthesis of novel polyketide derivatives using genetically engineered variants of erythromycin PKS further demonstrate the promise of S. erythraea and the role of erythromycin biosynthetic genes in the emerging field of combinatorial biosynthesis (Marsden et a]., 1998; Pacey et al., 1998; Weissman et al., 1998a). These technical developments have generated considerable interest of late, as reflected in the number of reviews that have appeared on this topic (Hutchinson, 1998, 1999; Cane et al., 1998; Hallis and Liu, 1999). In the context of this review, I consider briefly here the complementary combinatorial biosynthesis experimental platforms that have emerged for manipulation of erythromycin biosynthetic genes in both the native host S. erythraea

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and heterologous bacterial hosts. Continued development of appropriate experimental platforms to fully exploit the potential of S. erythraea and the ery gene cluster in combinatorial biosynthesis applications will play a significant role in successfully translating this exciting new technology into therapeutically useful and commercially viable products. Ultimately, the success of these approaches in drug discovery programs will be measured not by the number of new chemical entities produced in submilligram quantities, but rather by the number of viable therapeutic leads that move into preclinical and clinical testing. A. MANIPULATIONOF ERY BIOSYNTHETICGENESIN HETEROLOGOUSSTREPTOMYCESHOSTS The milestone report by McDaniel et al. (1999) illustrates the successful use of a heterologous system to manipulate the erythromycin PKS system. These authors report production of >100 6-DEB derivatives through construction of erythromycin PKS mutants harboring multiple combinations of individual eryA mutations in either S. coelicolor or S. lividans. The mutations include AT substitution, KR deletion, KR gainof-function, and KR stereochemical alterations. The use of S. coelicolor and S. lividans offers numerous experimental advantages because of the well-developed molecular genetic systems in these organisms (Hopwood et al., 1985; Gusek and Kinsela, 1992; Hopwood, 1997). This includes a wide range of plasmid vectors and gene expression tools, well-established protoplast transformation methods, and available genomic information (Kieser et al., 1992). These genetic tools facilitate construction of the numerous polyketide synthase mutants required to produce a library of 6-DEB polyketide derivatives. The host strains used by McDaniel and coworkers, S. coelicolor strain CH999 and S. lividans strain K4-114, contain a chromosomal deletion of the entire actinorhodin polyketide gene cluster (Ziermann and Betlach, 1999). Deletion of the actinorhodin biosynthetic genes in these hosts simplifies quantitative analysis of the novel polyketides produced and reduces competing metabolic demands for production of extrachromosomally encoded PKS proteins. The erythromycin PKS genes are expressed in these strains from the pCK7 expression plasmid, which has been reviewed elsewhere (Katz, 1997). Several limitations of this heterologous experimental platform should also be considered. First, the S. coelicolor system described by McDaniel et al. (1999) involved only the polyketide synthase genes from S. erythraea to produce 6-DEB derivatives. The erythromycin biosynthetic genes required for biosynthesis and attachment of the two

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deoxysugars mycarose and desosamine, and for the hydroxylation and O-methylation modification reactions were not provided in the heterologous host. This required separate introduction of the PKS mutations into S. erythraea to examine the impact of the polyketide modifications on the downstream biosynthetic reactions. The importance of the glycosylations and further macrolide ring and sugar modifications on the bioactivity of erythromycin derivatives underscores the importance of this step in evaluating this combinatorial library. Presumably, the genetic versatility of S. coelicolor and S. ]ividans would permit further genetic engineering of these hosts to include these ancillary reactions if desired. However, further development of the native S. erythraea host might represent a more attractive option since this organism already contains the required secondary metabolic pathways necessary to supply biosynthetic precursors for these pathways. Second, the yield of 6-DEB in the S. coelicolor control strain harboring the wild-type erythromycin PKS genes was approximately 20 mg/liter. All mutational changes in the PKS genes resulted in lower yields from this parental baseline, presumably reflecting differences in substrate specificity and processivity in the downstream polyketide biosynthetic reactions. The authors also noted that yield losses correlated with particular single mutations appeared to be additive when engineered in multiple combinations into the hybrid PKS. The resulting yields of the 6-DEB derivatives produced in the S. coelicolor library ranged from <0.5 to 70% of the baseline 20 mg/liter yield. Further productivity losses are also likely at the post-PKS glycosylation and modification steps. Clearly, improving the relatively low yields of the novel polyketide compounds reported in this system represents an important technical challenge if this technology is to become a viable drug discovery tool. B.

MANIPULATION OF ERY BIOSYNTHETIC GENES IN S. ERYTHRAEA

Several laboratories have successfully engineered wild-type strains of S. erythraea to produce novel erythromycin derivatives using gene replacement methods to introduce mutations in the PKS genes in this organism (for review, see Katz, 1997). Although providing an important conceptual validation of genetic engineering strategies to produce new polyketides using this organism, these studies proved to be labor intensive and produced a relatively small number of new compounds. Several PKS mutant constructions failed to produce detectable levels of the desired polyketide compounds, although the genetic information was confirmed present in the chromosomal gene locus (Ruan et al., 1997;

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Stassi et al., 1998b). In addition, the dependence on wild-type S. erythraea strains to perform molecular genetic manipulations in these experiments significantly limited the yields of novel erythromycin derivatives even in successful genetic constructions (Katz, 1997). Taken together, these factors, including the relatively low throughput of compounds, the poor predictive ability in regard to permissible genetic alterations in the erythromycin PKS, and the low product yields of new erythromycin derivatives, were inconsistent with the demands of modern drug discovery programs and led to a reduction or elimination of drug discovery efforts utilizing these directed genetic approaches at several pharmaceutical houses in the late 1990s. The development of improved S. erythraea host strains, DNA transformation methods, and expression plasmids should stimulate renewed interest in these genetic-engineering approaches. The S. erythraea strain described by Rowe et al. (1998) should prove useful in combinatorial biosynthesis applications in this organism. This strain, designated JC2, harbors a deletion of almost the entire eryA region. The region of eryAIII encoding the chain-terminating TE domain has been retained at the chromosomal ery gene locus. This deletion mutant permits expression of genetically engineered PKS derivatives in the absence of a wild-type PKS. A homologous deletion construct introduced into an industrially improved genetic background would represent a particularly useful improvement in this technology. These authors also describe the construction and use of an expression vector, designated pCJR24, that permits expression of genetically engineered PKS genes under the control of the actI promoter and cognate activator protein ActII-ORF4 in strain JC2. The extension of precursor-directed polyketide biosynthesis in S. erflhraea by Leadlay and coworkers (Marsden et al., 1998; Pacey et al., 1998; Parsons et al., 1999) offers a promising complement to other combinatorial biosynthesis strategies in this organism. These workers substituted the S. avermitilis PKS loading domain involved in avermectin biosynthesis for the eryAI-encoded loading domain involved in erythromycin biosynthesis. Previous studies (Dutton eta]., 1991) demonstrated the broad substrate specificity of the avermectin PKS loading domain. This broad substrate specificity was exploited to incorporate a variety of substrates into the avermectin polyketide backbone, resulting in production of a series of novel C-25 substituted avermectins. The genetically engineered strain of S. erythraea containing this modified PKS, when supplemented with various exogenous fatty acids, produced a variety of novel C-13 erythromycin species as predicted.

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X. Future Prospects

The continued interest in erythromycin and semisynthetic second- and third-generation erythromycin derivatives as antiinfectives will continue to justify ongoing research in this molecule and in the producing organism, S. erythraea. Since large-scale production of semisynthetic compounds necessarily involves losses of the parent natural product in the synthetic reaction steps, there will be continued demand for improved fermentation manufacturing technologies to economically produce high-quality erythromycin A or advanced intermediates for these products. This is particularly evident from the structural complexity (and related complexity of the synthetic chemistries involved) of the lead third-generation ketolide drug candidates in clinical development (Fig. 1). The recent advances in the development of molecular genetic tools for manipulation of S. erythraea offer significant promise for further metabolic engineering improvements in the full range of fermentation strains supporting the manufacture of these products. Transcriptional studies of erythromycin biosynthesis have provided new insights to define rational strain improvement strategies for this pathway. These efforts should continue to provide improvements in erythromycin production technology in the years ahead. The recent developments in combinatorial biosynthesis using S. erythraea and erythromycin biosynthetic genes have significantly improved the prospects for discovery of new therapeutic candidates derived from this system. Two key issues will need to be addressed if we are to realize the potential of this technology. In order to compete with combinatorial chemistry and other alternative drug discovery approaches, combinatorial biosynthesis systems capable of producing significantly larger compound libraries will be necessary The progress in this area, reviewed by Hutchinson (1999), is encouraging in this regard. In addition, the productivity of genetically engineered S. erythraea mutants, used in either genetic or precursor-directed combinatorial biosynthesis applications, must be improved. This is essential both in order to support the drug discovery process and to facilitate timely preclinical and clinical development of lead drug candidates. Although further development of heterologous hosts like S. coelicolor is likely to improve the current baseline productivity of these systems, this is unlikely to be competitive with approaches utilizing the native host S. erythraea. Existing high-productivity mutants have not been fully exploited in this regard, probably owing to the separation of process

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development and drug discovery groups within large pharmaceutical houses and the general unavailability of these proprietary strains to academic laboratories and small biotechnology companies engaged in the field. This would perhaps be most effectively addressed through exploration of potential licensing and collaborative opportunities in this area. Existing technologies developed for large-scale fermentation of S. erythraea and the experience with this organism in industry represents a significant commercial, manufacturing, and regulatory advantage for development of new drug candidates using this platform. It seems likely that S. erythraea mutants producing several orders of magnitude higher polyketide levels would open a larger window of observation for new compounds during the drug discovery process and facilitate the isolation of gram to kilogram quantities for further preclinical and clinical testing. As an alternative, the combinatorial drug discovery phase could perhaps be supported by moderately improved yields in the heterologous host systems noted above. Appropriate vectors for transfer of the genetically engineered biosynthetic genes into higher-productivity S. erythraea genetic backgrounds could then be developed to support the demands of the later stages of the drug development process. The continuing advances in our ability to genetically manipulate S. erythraea, along with the highly developed fermentation technologies available for this organism, will ensure a continuing role for S. erythraea in the development and production of new drug candidates in the years ahead.

Acknowledgments I am grateful to the many researchers who have contributed their time and talents to the study of erythromycin and Saccharopolyspora erythraea over the past four decades. I am especially indebted to my Abbott colleagues, past and present, who have enriched my working life through their insights into this organism. In particular, I would like to acknowledge David Post, Diane Stassi, Leonard Katz, Martin Babcock, Jay Lampel, Mark Satter, Andrew Reeves, Samuel English, James Petzel, Bill Ellefson, Nancy Fitzgerald, Sandra Splinter, Donna Santucci, Tom Paulus, Janet DeWitt, and Vicki Luebke for their contributions toward improving our understanding of the genetics and physiology of this industrially important microorganism.

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