Eduardo Rodriguez

Combinatorial biosynthesis of antimicrobials and other natural products.

Combinatorial biosynthesis utilizes the enzymes from antibiotic (and other natural product) biosynthetic pathways to create novel chemical structures. The manipulation of modular polyketide synthases (PKSs) has been the major focus of this effort and has led to the production of, for example, several erythromycin analogs. Many new tools for manipulating and studying these multifunctional enzymes have been developed. These include multiple hosts and expression systems, enzymology tools for in vitro study, and ways to engineer pre-PKS and post-PKS pathways. The result is more rational and faster methods of engineering new compounds for the development of chemotherapeutic agents from natural products. The most significant recent advances in combinatorial biosynthesis are outlined.

Rapid engineering of polyketide overproduction by gene transfer to industrially optimized strains

Development of natural products for therapeutic use is often hindered by limited availability of material from producing organisms. The speed at which current technologies enable the cloning, sequencing, and manipulation of secondary metabolite genes for production of novel compounds has made it impractical to optimize each new organism by conventional strain improvement procedures. We have exploited the overproduction properties of two industrial organisms—Saccharopolyspora erythraea and Streptomyces fradiae, previously improved for erythromycin and tylosin production, respectively—to enhance titers of polyketides produced by genetically modified polyketide synthases (PKSs). An efficient method for delivering large PKS expression vectors into S. erythraea was achieved by insertion of a chromosomal attachment site (attB) for φC31-based integrating vectors. For both strains, it was discovered that only the native PKS-associated promoter was capable of sustaining high polyketide titers in that strain. Expression of PKS genes cloned from wild-type organisms in the overproduction strains resulted in high polyketide titers whereas expression of the PKS gene from the S. erythraea overproducer in heterologous hosts resulted in only normal titers. This demonstrated that the overproduction characteristics are primarily due to mutations in non-PKS genes and should therefore operate on other PKSs. Expression of genetically engineered erythromycin PKS genes resulted in production of erythromycin analogs in greatly superior quantity than obtained from previously used hosts. Further development of these hosts could bypass tedious mutagenesis and screening approaches to strain improvement and expedite development of compounds from this valuable class of natural products.

Engineered biosynthesis of 16-membered macrolides that require methoxymalonyl-ACP precursors in Streptomyces fradiae.

Development of host microorganisms for heterologous expression of polyketide synthases (PKS) that possess the intrinsic capacity to overproduce polyketides with a broad spectrum of precursors supports the current demand for new tools to create novel chemical structures by combinatorial engineering of modular and other classes of PKS. Streptomyces fradiae is an ideal host for development of generic polyketide-overproducing strains because it contains three of the most common precursors—malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA—used by modular PKS, and is a host that is amenable to genetic manipulation. We have expanded the utility of an overproducing S. fradiae strain for engineered biosynthesis of polyketides by engineering a biosynthetic pathway for methoxymalonyl-ACP, a fourth precursor used by many 16-membered macrolide PKS. This was achieved by introducing a set of five genes, fkbG–K from Streptomyces hygroscopicus, putatively encoding the methoxymalonyl-ACP biosynthetic pathway, into the S. fradiae chromosome. Heterologous expression of the midecamycin PKS genes in this strain resulted in 1xa0g/l production of a midecamycin analog. These results confirm the ability to engineer unusual precursor pathways to support high levels of polyketide production, and validate the use of S. fradiae for overproduction of 16-membered macrolides derived from heterologous PKS that require a broad range of precursors.

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.