Precursor-directed biosynthesis of 6-deoxyerythronolide B analogues is improved by removal of the initial catalytic sites of the polyketide synthase

Precursor-directed biosynthesis has been shown to be a powerful tool for the production of polyketide analogues that would be difficult or cost prohibitive to produce from medicinal chemistry efforts alone. It has been most extensively demonstrated using a KS1 null mutation (KS1⁰) to block the first round of condensation in the biosynthesis of the erythromycin polyketide synthase (DEBS) for the production of analogues of its aglycone, 6-deoxyerythronolide B (6-dEB). Here we show that removing the DEBS loading domain and first module (mod1Δ), rather than using the KS1⁰ system, can lead to an increase in the utilization of some chemical precursors and production of 6-dEB analogues (R-6dEB) in both Streptomyces coelicolor and Saccharopolyspora erythraea. While the difference in utilization of the precursor was diketide specific, in strains fed (2R*, 3S*)-5-fluoro-3-hydroxy-2-methylpentanoate N-propionylcysteamine thioester, twofold increases in both utilization of the diketide and 15-fluoro-6dEB (15F-6dEB) production were observed in S. coelicolor, and S. erythraea exhibited a tenfold increase in production of 15-fluoro-erythromycin when utilizing the mod1Δ rather than the KS1⁰ system.     

Erythromycin biosynthesis. The 4-pro-S hydride of NADPH is utilized for ketoreduction by both module 5 and module 6 of the 6-deoxyerythronolide B synthase.

Incubation of chirally deuterated NADPH with 6-deoxyerythronolide B synthase (DEBS) modules 5 and module 6 and analysis of the derived triketide lactones established that the two ketoreductase domains, KR5 and KR6, are both specific for the 4-pro-S hydride of the nicotinamide cofactor.     

Mechanism based protein crosslinking of domains from the 6-deoxyerythronolide B synthase

The critical role of protein-protein interactions in the chemistry of polyketide synthases is well established. However, the transient and weak nature of these interactions, in particular those involving the acyl carrier protein (ACP), has hindered efforts to structurally characterize these interactions. We describe a chemo-enzymatic approach that crosslinks the active sites of ACP and their cognate ketosynthase (KS) domains, resulting in the formation of a stable covalent adduct. This process is driven by specific protein-protein interactions between KS and ACP domains. Suitable manipulation of the reaction conditions enabled complete crosslinking of a representative KS and ACP, allowing isolation of a stable, conformationally constrained adduct suitable for high-resolution structural analysis.     

Reconstituting modular activity from separated domains of 6-deoxyerythronolide B synthase

The hallmark of a type I polyketide synthase (PKS), such as the 6-deoxyerythronolide B synthase (DEBS), is the presence of catalytic modules comprised of covalently fused domains acting together to catalyze one round of chain elongation. In addition to an obligate ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP), a module may also include a ketoreductase (KR), dehydratase (DH), and/or enoyl reductase (ER) domain. The size, flexibility, and fixed domain-domain stoichiometry of these PKS modules present challenges for structural, mechanistic, and protein-engineering studies. Here, we have harnessed the power of limited proteolysis and heterologous protein expression to isolate and characterize individual domains of module 3 of DEBS, a 150-kD protein consisting of a KS, an AT, an ACP, and an inactive KR domain. Two interdomain boundaries were identified via limited proteolysis, which led to the production of a 90-kD KS-AT, a 142-kD KS-AT-KR(0), and a 10-kD ACP as structurally stable stand-alone proteins. Each protein was shown to possess the requisite catalytic properties. In the presence of the ACP, both the KS-AT and the KS-AT-KR(0) proteins were able to catalyze chain elongation as well as the intact parent module. Separation of the KS from the ACP enabled direct interrogation of the KS specificity for both the nucleophilic substrate and the partner ACP. Malonyl and methylmalonyl extender units were found to be equivalent substrates for chain elongation. Whereas ACP2 and ACP4 of DEBS could be exchanged for ACP3, ACP6 was a substantially poorer partner for the KS. Remarkably, the newly identified proteolytic sites were conserved in many PKS modules, raising the prospect of developing improved methods for the construction of hybrid PKS modules by engineering domain fusions at these interdomain junctions.     

Analysis of covalently bound polyketide intermediates on 6-deoxyerythronolide B synthase by tandem proteolysis-mass spectrometry

Polyketide natural products are biosynthesized via successive chain-elongation events mediated by elaborate protein assemblies. Facile detection of protein-bound intermediates in these systems will increase our understanding of enzyme reactivity and selectivity. We have developed a tandem proteolysis/mass spectrometric method for monitoring substrate loading and elongation in 6-deoxyerythronolide B synthase (DEBS), responsible for production of the macrolide precursor to erythromycin. Information regarding ketosynthase loading and polyketide unit elongation is readily acquired without need for complex protein or small molecule labels. A panel of structurally related substrates is evaluated through competition experiments and kinetic assays using LC-MS to resolve closely related species. Strong stereochemical effects are observed for ketosynthase substrate specificity. Semiquantitative kinetic analyses allow the resolution of the effects of structural and stereochemical changes on the individual ketosynthase-catalyzed steps of acyl-enzyme formation and polyketide chain extension.     

Chemobiosynthesis of Novel 6-Deoxyerythronolide B Analogues by Mutation of the Loading Module of 6-Deoxyerythronolide B Synthase 1

Chemobiosynthesis (J. R. Jacobsen, C. R. Hutchinson, D. E. Cane, and C. Khosla, Science 277:367-369, 1997) is an important route for the production of polyketide analogues and has been used extensively for the production of analogues of 6-deoxyerythronolide B (6-dEB). Here we describe a new route for chemobiosynthesis using a version of 6-deoxyerythronolide B synthase (DEBS) that lacks the loading module. When the engineered DEBS was expressed in both Escherichia coli and Streptomyces coelicolor and fed a variety of acyl-thioesters, several novel 15-R-6-dEB analogues were produced. The simpler “monoketide” acyl-thioester substrates required for this route of 15-R-6-dEB chemobiosynthesis allow greater flexibility and provide a cost-effective alternative to diketide-thioester feeding to DEBS KS1^o for the production of 15-R-6-dEB analogues. Moreover, the facile synthesis of the monoketide acyl-thioesters allowed investigation of alternative thioester carriers. Several alternatives to N-acetyl cysteamine were found to work efficiently, and one of these, methyl thioglycolate, was verified as a productive thioester carrier for mono- and diketide feeding in both E. coli and S. coelicolor.     

Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase

The assembly‐line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6‐deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein–protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF₃‐S‐ACP, was developed as a ¹⁹F NMR spectroscopic probe of protein–protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.     

6-deoxyerythronolide B production through chromosomal localization of the deoxyerythronolide B synthase genes in E. coli

Chromosomal engineering was used to localize the deoxyerythronolide B synthase (DEBS) genes and propionyl-CoA carboxylase (PCC) genes to the BAP1 Escherichia coli chromosome creating the new strain YW9. YW9 then featured a plasmid-free heterologous pathway for the production of the polyketide product 6-deoxyerythronolide B (6dEB, a precursor to the antibiotic erythromycin) highlighted by the successful chromosomal integration of five genes total and three DEBS genes each approximately 10 kb in length. The new strain was tested for small-scale 6dEB biosynthesis and compared to 6dEB production from plasmid-derived gene expression at 22, 30, and 37 degrees C. YW9 produced 6dEB at each temperature tested; whereas, the current plasmid-based system could only produce 6dEB at 22 and 30 degrees C. As determined by MS analysis, average production levels for YW9 were 0.47 (22 degrees C), 0.52 (30 degrees C), and 0.11 (37 degrees C)mg/L.     

Redesign, synthesis and functional expression of the 6-deoxyerythronolide B polyketide synthase gene cluster

A generic design of Type I polyketide synthase genes has been reported in which modules, and domains within modules, are flanked by sets of unique restriction sites that are repeated in every module [1]. Using the universal design, we synthesized the six-module DEBS gene cluster optimized for codon usage in E. coli, and cloned the three open reading frames into three compatible expression vectors. With one correctable exception, the amino acid substitutions required for restriction site placements were compatible with polyketide production. When expressed in E. coli the codon-optimized synthetic gene cluster produced significantly more protein than did the wild-type sequence. Indeed, for optimal polyketide production, PKS expression had to be down-regulated by promoter attenuation to achieve balance with expression of the accessory proteins needed to support polyketide biosynthesis.     

Saccharopolyspora erythraea-catalyzed bioconversion of 6-deoxyerythronolide B analogs for production of novel erythromycins.

A method was developed for the large-scale bioconversion of novel 6-deoxyerythronolide B (6-dEB) analogs into erythromycin analogs. Erythromycin biosynthesis in Saccharopolyspora erythraea proceeds via the formation of a polyketide aglycone, 6-dEB, which is subsequently glycosylated, hydroxylated and methylated to yield the antibiotic erythromycin A. A modular polyketide synthase (PKS) directs 6-dEB synthesis using a dedicated set of active sites for the condensation of each of seven propionate units. Strategies based on genetic manipulation and precursor feeding are available for the efficient generation of novel 6-dEB analogs using a plasmid-based system in Streptomyces coelicolor. 6-dEB and 13-substituted 6-dEB analogs produced in this manner were fed to S. erythraea mutants which could not produce 6-dEB, yet retained their 6-dEB modification systems, and resulted in the generation of erythromycin A and 13-substituted erythromycin A analogs. Erythromycin B, C and D analogs were observed as intermediates of the process. Dissolved oxygen, temperature, the specific aglycone feed concentration, and pH were found to be important for obtaining a high yield of erythromycin A analogs. Cultivation conditions were identified which resulted in the efficient bioconversion of 6-dEB analogs into erythromycin A analogs, which this process demonstrated at the 100 l scale.     

Combining classical, genetic, and process strategies for improved precursor-directed production of 6-deoxyerythronolide B analogues

A process for the production of erythromycin aglycone analogues has been developed by combining classical strain mutagenesis techniques with modern recombinant DNA methods and traditional process improvement strategies. A Streptomyces coelicolor strain expressing the heterologous 6-deoxyerythronolide B (6-dEB) synthase (DEBS) for the production of erythromycin aglycones was subjected to random mutagenesis and selection. Several strains exhibiting 2-fold higher productivities and reaching >3 g/L total macrolide aglycones were developed. These mutagenized strains were cured of the plasmid carrying the DEBS genes and a KS1 degrees mutant DEBS operon was introduced for the production of novel analogues when supplemented with a synthetic diketide precursor. The strains expressing the mutant DEBS were screened for improved 15-methyl-6-dEB production, and the best clone, strain B9, was found to be 50% more productive as compared to the parent host strain used for 15-methyl-6-dEB production. Strain B9 was evaluated in 5-L fermenters to confirm productivity in a scalable process. Although peak titers of 0.85 g/L 15-methyl-6-dEB by strain B9 confirmed improved productivity, it was hypothesized that the low solubility of 15-methyl-6-dEB limited productivity. The solubility of 15-methyl-6-dEB in water was determined to be 0.25-0.40 g/L, although higher titers are possible in fermentation medium. The incorporation of the hydrophobic resin XAD-16HP resulted in both the in situ adsorption of the product and the slow release of the diketide precursor. The resin-containing fermentation achieved 1.3 g/L 15-methyl-6-dEB, 50% higher than the resin-free process. By combining classical mutagenesis, recombinant DNA techniques, and process development, 15-methyl-6-dEB productivity was increased by over 100% in a scalable fermentation process.     

An acyl-carrier-protein-thioesterase domain from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea. High-level production, purification and characterisation in Escherichia coli

The C-terminal region of a multifunctional polypeptide from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea is predicted to contain an acyl carrier protein and a thioesterase or acyltransferase activity [Cortes, J., Haydock, S. F., Roberts, G. A., Bevitt, D. J. & Leadlay, P. F. (1990) Nature 348, 176-178]. Site-directed mutagenesis by means of the polymerase chain reaction was used to construct an efficient pT7-based expression plasmid for this domain. The recently developed technique of electrospray mass spectrometry was used to demonstrate that the purified protein had not been post-translationally modified by attachment of a 4'-phosphopantetheine group. However, treatment with the serine proteinase inhibitor phenylmethylsulphonyl fluoride led to highly selective labelling of the predicted active site of the thioesterase or acyltransferase.     

Modular polyketide synthases: Investigating intermodular communication using 6 deoxyerythronolide B synthase module 2

A novel variant of 6-deoxyerythronolide B synthase (DEBS) module 2 was constructed to explore the balance between protein-protein-mediated intermodular channeling and intrinsic substrate specificity within DEBS. This construct, termed (N3)Mod2+TE, was co-incubated with a complementary, donor form of the same module, (N5)Mod2(C2), as well as with a mutant of (N5)Mod2(C2) with an inactive ketosynthase domain, in order to determine the extent of intermediate channeling versus substrate diffusion into the downstream module.     

Process and metabolic strategies for improved production of Escherichia coli-derived 6-deoxyerythronolide B

Recently, the feasibility of using Escherichia coli for the heterologous biosynthesis of complex polyketides has been demonstrated. In this report, the development of a robust high-cell-density fed-batch procedure for the efficient production of complex polyketides is described. The effects of various physiological conditions on the productivity and titers of 6-deoxyerythronolide B (6dEB; the macrocyclic core of the antibiotic erythromycin) in recombinant cultures of E. coli were studied in shake flask cultures. The resulting data were used as a foundation to develop a high-cell-density fermentation procedure by building upon procedures reported earlier for recombinant protein production in E. coli. The fermentation strategy employed consistently produced approximately 100 mg of 6dEB per liter, whereas shake flask conditions generated between 1 and 10 mg per liter. The utility of an accessory thioesterase (TEII from Saccharopolyspora erythraea) for enhancing the productivity of 6dEB in E. coli was also demonstrated (increasing the final titer of 6dEB to 180 mg per liter). In addition to reinforcing the potential for using E. coli as a heterologous host for wild-type- and engineered-polyketide biosynthesis, the procedures described in this study may be useful for the production of secondary metabolites that are difficult to access by other routes.     

The role of serine-246 in cytochrome P450eryF-catalyzed hydroxylation of 6-deoxyerythronolide B

A strongly conserved threonine residue in the I-helix of cytochrome P450 enzymes participates in a proton delivery system for binding and cleavage of dioxygen molecules. 6-Deoxyerythronolide B hydroxylase (P450eryF) is unusual in that the conserved threonine residue is replaced by alanine in this enzyme. On the basis of the crystal structures of substrate-bound P450eryF, it has been proposed that the C-5 hydroxyl group of the substrate and serine-246 of the enzyme form hydrogen bonds with water molecules 519 and 564, respectively. This hydrogen bonding network constitutes the proton delivery system whereby P450eryF maintains its catalytic activity in the absence of a threonine hydroxyl group in the conserved position. To further assess the role in the proton delivery system of hydroxyl groups around the active site, three mutant forms of P450eryF (A245S, S246A, and A245S/S246A) were constructed and characterized. In each case, decreased catalytic activity and increased uncoupling could be correlated with changes in the hydrogen bonding environment. These results suggest that Ser-246 does indeed indirectly participate in the proton shuttling pathway, and also strongly support our previous hypothesis that the C-5 hydroxyl group of the substrate participates in the acid-catalyzed dioxygen bond cleavage reaction.     

Precursor-directed biosynthesis of 6-deoxyerythronolide B analogs in Streptomyces coelicolor: understanding precursor effects

A fermentation process employing precursor-directed biosynthesis is being developed for the manufacture of 6-deoxyerythronolide B (6-dEB) analogues. Through a plasmid-based system in Streptomyces coelicolor, 6-dEB synthesis is catalyzed by 6-dEB synthase (DEBS). 6-dEB synthesis is abolished by inactivation of the ketosynthase (KS) 1 domain of DEBS but can be restored by providing synthetic activated diketides. Because of its inherent catalytic flexibility, the KS1-deficient DEBS is capable of utilizing unnatural diketides to form various 13-substituted 6-dEBs. Here we characterize process variables associated with diketide feeding in shake-flask experiments. 13-R-6-dEB production was found to depend strongly on diketide feed concentrations, on the growth phase of cultures at feeding time, and on the R-group present in the diketide moiety. In all cases a major portion of the fed diketides was degraded by the cells.     

Glycogen synthase (GYS1) mutation causes a novel skeletal muscle glycogenosis

Polysaccharide storage myopathy (PSSM) is a novel glycogenosis in horses characterized by abnormal glycogen accumulation in skeletal muscle and muscle damage with exertion. It is unlike glycogen storage diseases resulting from known defects in glycogenolysis, glycolysis, and glycogen synthesis that have been described in humans and domestic animals. A genome-wide association identified GYS1, encoding skeletal muscle glycogen synthase (GS), as a candidate gene for PSSM. DNA sequence analysis revealed a mutation resulting in an arginine-to-histidine substitution in a highly conserved region of GS. Functional analysis demonstrated an elevated GS activity in PSSM horses, and haplotype analysis and allele age estimation demonstrated that this mutation is identical by descent among horse breeds. This is the first report of a gain-of-function mutation in GYS1 resulting in a glycogenosis.     

Improved bioconversion of 15-fluoro-6-deoxyerythronolide B to 15-fluoro-erythromycin A by overexpression of the eryK Gene in Saccharopolyspora erythraea

The bioconversion of a 6-deoxyerythronolide B analogue to the corresponding erythromycin A analogue (R-EryA) by a Saccharopolyspora erythraea mutant lacking the ketosynthase in the first polyketide synthase module was significantly improved by changing fluxes at a key branch point affecting the erythromycin congener distribution. This was achieved by integrating an additional copy of the eryK gene into the chromosome under control of the eryAIp promoter. Real-time PCR analysis of RNA confirmed higher expression of eryK in the resulting strain, S. erythraea K301-105B, compared to its parent. In shake flasks, K301-105B produced less of the shunt product 15-fluoro-erythromycin B (15F-EryB), suggesting a shift in congener distribution toward the desired product, 15-fluoro-erythromycin A (15F-EryA). In bioreactor studies, K301-105B produced 1.3 g/L of 15F-EryA with 75-80% molar yield on fed precursor, compared with 0.9 g/L 15F-EryA with 50-55% molar yield on fed precursor by the parent strain. At higher precursor feed rates, K301-105B produced 3.5 g/L of 15F-EryA while maintaining 75-80% molar yield on fed precursor.     

Characterization of Saccharopolyspora erythraea cytochrome P-450 genes and enzymes, including 6-deoxyerythronolide B hydroxylase.

Previous studies of erythromycin biosynthesis have indicated that a cytochrome P-450 monooxygenase system is responsible for hydroxylation of 6-deoxyerythronolide B to erythronolide B as part of erythromycin biosynthesis in Saccharopolyspora erythraea (A. Shafiee and C. R. Hutchinson, Biochemistry 26:6204-6210 1987). The enzyme was previously purified to apparent homogeneity and found to have a catalytic turnover number of approximately 10(-3) min-1. More recently, disruption of a P-450-encoding sequence (eryF) in the region of ermE, the erythromycin resistance gene of S. erythraea, produced a 6-deoxyerythronolide B hydroxylation-deficient mutant (J. M. Weber, J. O. Leung, S. J. Swanson, K. B. Idler, and J. B. McAlpine, Science 252:114-116, 1991). In this study we purified the catalytically active cytochrome P-450 fraction from S. erythraea and found by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis that it consists of a major and a minor P-450 species. The gene encoding the major species (orf405) was cloned from genomic DNA and found to be distinct from eryF. Both the orf405 and eryF genes were expressed in Escherichia coli, and the properties of the proteins were compared. Heterologously expressed EryF and Orf405 both reacted with antisera prepared against the 6-deoxyerythronolide B hydroxylase described by Shafiee and Hutchinson (1987), and the EryF polypeptide comigrated with the minor P-450 species from S. erythraea on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. In comparisons of enzymatic activity, EryF hydroxylated a substrate with a turnover number of 53 min-1, whereas Orf405 showed no detectable activity with a 6-deoxyerythronolide B analog. Both enzymes showed weak activity in the O-dealkylation of 7-ethoxycoumarin. We conclude that the previously isolated 6-deoxyerythronolide B hydroxylase was a mixture of two P-450 enzymes and that only the minor form shows 6-deoxyerythronolide B hydroxylase activity.