Engineering of Saccharomyces cerevisiae for the biosynthesis of fungal polyketides.

摘要

The overall goal of the research was to engineer Saccharomyces cerevisiae as a platform for the synthesis of polyketides. Polyketides are important pharmaceutical compounds naturally made in certain fungi and bacteria. The production in the native hosts is generally minute and these microorganisms may not be amenable to genetic manipulation. Therefore, it is advantageous to engineer yeast to synthesize polyketide products. We investigated engineering issues in this yeast, introduced new enzyme activities, and studied the synthesis of two fungal polyketides. We evaluated the S. cerevisiae ADH2 promoter, an inducer-free promoter, for regulation of gene expression in the yeast host. The promoter strength of the ADH2 promoter was also compared to the inducible CUP1 and GAL1 promoters using a single integrated copy of the lacZ gene. The ADH2 promoter gave 3 to 9 times higher beta-galactosidase specific activity at 48 hours than the two other promoters. We introduced the E. coli atoA and atoD genes into yeast for the synthesis of butyryl-CoA, an alternate starter unit for the creation of novel polyketide variants. Four copies of the dual-gene cassette were sequentially integrated into the yeast chromosomes and compared with plasmid-based systems. Active gene product (the tetrameric acetyl-CoA:acetoacetyl-CoA transferase) was synthesized and butyryl-CoA was detected using an in vivo cell assay. We also demonstrated that Aspergillus nidulans NpgA is an effective 4'-phosphopantetheinyl transferase (P-pant transferase) for post-translational activation of two polyketide synthases, 6-methylsalicylic acid synthase (6-MSAS) and lovastatin nonaketide synthase (LNKS), in yeast. Activation levels and polyketide synthesis were comparable to that observed with the Bacillus subtilis Sfp P-pant transferase. The polyketide 6-methylsalicylic acid (6-MSA) was synthesized and detected in the supernatant when 6-MSAS and NpgA (or Sfp) were co-expressed in yeast. Integration strains carrying one or two copies of npgA gave superior results relative to a 2mu-based plasmid system. As a first step in the synthesis of the polyketide lovastatin in S. cerevisiae, the LovB (LNKS), LovC (enoyl reductase) and NpgA (or Sfp) were coexpressed in the "protease deficient" strain BJ 5464. However, the expected lovastatin precursor, dihydromonacolin L could not be identified in cell extract or supernatant.