Rational improvement of simvastatin synthase solubility in Escherichia coli leads to higher whole‐cell biocatalytic activity

Abstract

Simvastatin is the active pharmaceutical ingredient of the blockbuster cholesterol lowering drug Zocor. We have previously developed an Escherichia coli based whole-cell biocatalytic platform towards the synthesis of simvastatin sodium salt (SS) starting from the precursor monacolin J sodium salt (MJSS). The centerpiece of the biocatalytic approach is the simvastatin synthase LovD, which is highly prone to misfolding and aggregation when overexpressed from E. coli. Increasing the solubility of LovD without decreasing its catalytic activity can therefore elevate the performance of the whole-cell biocatalyst. Using a combination of homology structural prediction and site-directed mutagenesis, we identified two cysteine residues in LovD that are responsible for nonspecific intermolecular crosslinking, which leads to oligomer formation and protein aggregation. Replacement of Cys40 and Cys60 with alanine residues resulted in marked gain in both protein solubility and whole-cell biocatalytic activities. Further mutagenesis experiments converting these two residues to small or polar natural amino acids showed that C40A and C60N are the most beneficial, affording 27% and 26% increase in whole cell activities, respectively. The double mutant C40A/C60N combines the individual improvements and displayed ~50% increase in protein solubility and whole-cell activity. Optimized fed-batch high-cell-density fermentation of the double mutant in an E. coli strain engineered for simvastatin production quantitatively (>99%) converted 45 mM MJSS to SS within 18 hours, which represents a significant improvement over the performance of wild type LovD under identical conditions. The high efficiency of the improved whole-cell platform renders the biocatalytic synthesis of SS an attractive substitute over the existing semisynthetic routes.

Cysteine mutagenesis improves the production without abrogating antigenicity of a recombinant protein vaccine candidate for human chagas disease

“…The approach selected to minimize or eliminate the aggregation observed was to modify the cysteine residues present in the molecule to serine residues which would eliminate aberrant disulfide bond formation without introducing local structural changes within the molecule. Mutagenesis of cysteine residues has been shown to reduce aggregation of Cu, Zn-superoxide dismutase, 31 as well as improve the solubility of human renal dipeptidase, 32 human galectin-2, 34 and simvastatin synthase LovD 35 expressed in E. coli. Expression of the gingipain adhesion/hemagglutinin domain (Hgp44) on the surface of Lactococcus lactis, as a vaccine candidate for periodontitis, was improved without eliminating antigenic properties.…”

“…Just as important, we wanted to verify that the structure of the mutated protein was similar to the wild type protein in order to maintain equivalent antigenicity. Many other proteins have likewise been modified by cysteine mutagenesis in order to reduce aggregation and improve solubility while maintaining conformation and/or biological activity or antigenicity including human renal dipeptidase, 32 simvastatin synthase, 35 and a 44 kDa gingipain adhesion/hemagglutinin domain (Hgp44) which shows promise as a candidate antigen for Porphyromonas gingivalis induced periodontitis. 36 Therefore we hypothesized that the cysteine mutations would not significantly impact protein structure.…”

“…Cysteine mutagenesis has been performed to increase expression levels and solubility, reduce mis-folding and aggregation, enhance stability, and improve the bioactivity of the target protein. [31][32][33][34][35][36] Two modified expression cassettes were designed as follows: the first construct, Tc24-C2, had mutations at residues C4S and C66S, and the second, Tc24-C4, at C4S, C66S, C74S, and C124S. The C4S and C66S mutations were selected for the Tc24-C2 construct over cysteine residues C74 and C124 due to their location on the surface of the molecule as determined by X-ray crystallography.…”

Asymmetric Synthesis with Recombinant Whole‐Cell Catalysts

“…It is not clear which single chaperone or which combination of chaper ones is the most suitable to improve the protein folding process optimally, thus a set of genes coding for different chaperones needs to be tested. A more targeted strategy was described by Tang et al [9]. Applying a combination of homology structural pre diction and site-directed mutagenesis, they identified two cysteine residues in the simvastatin synthase LovD that are responsible for nonspecific intermolecular crosslinking, leading to the formation of oligomers and aggregation.…”

Lovastatin, Compactin, and Related Anticholesterolemic Agents

“…Deletion of the gene responsible for the production of BioH in E. coli and over-expression of LovD in the same strain signifi cantly improved the stability of the DMB-S-MMP donor in cell cultures [ 47 ]. The whole-cell bioconversion of monacolin J to simvastatin was even further improved through both a rational design [ 48 ] and directed evolution approach [ 49 ]. In these experiments, the activity of LovD was improved such that LovD mutants increased the overall rate of transesterifi cation of DMB-S-MMP onto monacolin J.…”