title: 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 approximately 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 h, 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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, 5531 Boelter Hall, 420 Westwood Plaza, UCLA, Los Angeles, California 90095, USA.
authors.forename: Xinkai
authors.initials: X
authors.lastname: Xie
authors.forename: Inna
authors.initials: I
authors.lastname: Pashkov
authors.forename: Xue
authors.initials: X
authors.lastname: Gao
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Guerrero
authors.forename: Todd O
authors.initials: TO
authors.lastname: Yeates
authors.forename: Yi
authors.initials: Y
authors.lastname: Tang
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D055162:Biocatalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009281:Naphthalenes / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019821:Simvastatin / Q000378:metabolism*; D012995:Solubility; D013997:Time Factors
chemical_list: D009281:Naphthalenes; D011994:Recombinant Proteins; D019821:Simvastatin; C045376:monacolin J