Engineering Saccharomyces cerevisiae for production of simvastatin.

Simvastatin is a semisynthetic cholesterol-lowering medication and one of the top-selling statins in the world. Currently, industrial production of simvastatin acid (SVA) is a multistep process starting from the natural product lovastatin. For this reason, there is significant interest in direct production of simvastatin from a microbial host. In this study, six heterologous biosynthetic genes were introduced into Saccharomyces cerevisiae and the acyl-donor dimethylbutyryl-S-methyl mercaptopropionate (DMB-SMMP) was added, resulting in initial production of 0.5 mg/L SVA. Switching the yeast strain from JHY686 to BJ5464-NpgA increased total polyketide production to over 60 mg/L and conversion from dihydromonacolin L acid to monacolin J acid (MJA) was increased from 60% to 90% by tuning the copy number of the P450 lovA. Increasing the media pH to 8.7 led to a further 10-fold increase in SVA production. Optimized chemical lysis of the cell walls in situ after maximum MJA production led to 55 mg/L SVA titer, representing nearly complete conversion from MJA and a 110-fold increase in titer from the initial SVA production strain. The yeast strains developed in this work can be used as an alternative production method for SVA, and the strategies employed can be broadly applied for heterologous production of other fungal polyketides and semisynthetic compounds in yeast.

Origins of stereoselectivity in evolved ketoreductases.

Mutants of Lactobacillus kefir short-chain alcohol dehydrogenase, used here as ketoreductases (KREDs), enantioselectively reduce the pharmaceutically relevant substrates 3-thiacyclopentanone and 3-oxacyclopentanone. These substrates differ by only the heteroatom (S or O) in the ring, but the KRED mutants reduce them with different enantioselectivities. Kinetic studies show that these enzymes are more efficient with 3-thiacyclopentanone than with 3-oxacyclopentanone. X-ray crystal structures of apo- and NADP(+)-bound selected mutants show that the substrate-binding loop conformational preferences are modified by these mutations. Quantum mechanical calculations and molecular dynamics (MD) simulations are used to investigate the mechanism of reduction by the enzyme. We have developed an MD-based method for studying the diastereomeric transition state complexes and rationalize different enantiomeric ratios. This method, which probes the stability of the catalytic arrangement within the theozyme, shows a correlation between the relative fractions of catalytically competent poses for the enantiomeric reductions and the experimental enantiomeric ratio. Some mutations, such as A94F and Y190F, induce conformational changes in the active site that enlarge the small binding pocket, facilitating accommodation of the larger S atom in this region and enhancing S-selectivity with 3-thiacyclopentanone. In contrast, in the E145S mutant and the final variant evolved for large-scale production of the intermediate for the antibiotic sulopenem, R-selectivity is promoted by shrinking the small binding pocket, thereby destabilizing the pro-S orientation.