title: Improving simvastatin bioconversion in Escherichia coli by deletion of bioH.
abstract: Simvastatin is an important cholesterol lowering compound and is currently synthesized from the natural product lovastatin via multistep chemical synthesis. We have previously reported the use of an Escherichia coli strain BL21(DE3)/pAW31 as the host for whole-cell biocatalytic conversion of monacolin J acid to simvastatin acid. During fermentation and bioconversion, unknown E. coli enzyme(s) hydrolyzed the membrane permeable thioester substrate dimethylbutyryl-S-methyl mercaptopropionate (DMB-S-MMP) to the free acid, significantly decreased the efficiencies of the whole-cell bioconversion and the downstream purification steps. Using the Keio K-12 Singe-Gene Knockout collection, we identified BioH as the sole enzyme responsible for the observed substrate hydrolysis. Purification and reconstitution of E. coli BioH activity in vitro confirmed its function. BioH catalyzed the rapid hydrolysis of DMB-S-MMP with kcat and Km values of 260+/-45 s(-1) and 229+/-26 microM, respectively. This is in agreement with previous reports that BioH can function as a carboxylesterase towards fatty acid esters. YT2, which is a delta bioH mutant of BL21(DE3), did not hydrolyze DMB-S-MMP during prolonged fermentation and was used as an alternative host for whole-cell biocatalysis. The rate of simvastatin acid synthesis in YT2 was significantly faster than in BL21(DE3) and 99% conversion of 15 mM simvastatin acid in less than 12 h was achieved. Furthermore, the engineered host required significantly less DMB-S-MMP to be added to accomplish complete conversion. Finally, simvastatin acid synthesized using YT2 can be readily purified from fermentation broth and no additional steps to remove the hydrolyzed dimethylbutyryl-S-mercaptopropionic acid is required. Together, the proteomic and metabolic engineering approaches render the whole-cell biocatalytic process more robust and economically attractive.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095, USA.
authors.forename: Xinkai
authors.initials: X
authors.lastname: Xie
authors.forename: Wilson W
authors.initials: WW
authors.lastname: Wong
authors.forename: Yi
authors.initials: Y
authors.lastname: Tang
mesh_terms: D015097:3-Mercaptopropionic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000378:metabolism*; D019821:Simvastatin / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; C523994:dimethylbutyryl-S-methyl mercaptopropionate; C061417:bioH protein, E coli; D019821:Simvastatin; D015097:3-Mercaptopropionic Acid