Efficient synthesis of simvastatin by use of whole-cell biocatalysis

Abstract

Simvastatin is a semisynthetic derivative of the fungal polyketide lovastatin and is an important drug for lowering cholesterol levels in adults. We have developed a one-step, whole-cell biocatalytic process for the synthesis of simvastatin from monacolin J. By using an Escherichia coli strain overexpressing the previously discovered acyltransferase LovD (X. Xie, K. Watanabe, W. A. Wojcicki, C. C. Wang, and Y. Tang, Chem. Biol. 13:1161-1169, 2006), we were able to achieve >99% conversion of monacolin J to simvastatin without the use of any chemical protection steps. The key finding was a membrane-permeable substrate, alpha-dimethylbutyryl-S-methyl-mercaptopropionate, that was efficiently utilized by LovD as the acyl donor. The process was scaled up for gram-scale synthesis of simvastatin. We also demonstrated that simvastatin synthesized via this method can be readily purified from the fermentation broth with >90% recovery and >98% purity as determined by high-performance liquid chromatography. Bioconversion using high-cell-density, fed-batch fermentation was also examined. The whole-cell biocatalysis can therefore be an attractive alternative to currently used multistep semisynthetic transformations.

FIG. 1.

Chemical structures of lovastatin acid, monacolin J acid, and simvastatin acid. The biocatalytic reaction studied is the enzymatic conversion of monacolin J to simvastatin (thick arrow). LovD is able to regioselectively acylate the C₈ hydroxyl group. Two commonly used semisynthetic processes are shown with dashed arrows.

FIG. 2.

Kinetic analysis of LovD-catalyzed acylation of monacolin J to yield simvastatin using DMB-S-MMP as the acyl thioester. The y axis is expressed as the catalytic turnover (V/E_o). (A) Michaelis-Menten kinetics of LovD as a function of monacolin J concentration at a fixed DMB-SMMP concentration of 2 mM. No substrate inhibition was observed. The K_m (monacolin J) was 0.78 ± 0.12 mM. (B) Michaelis-Menten kinetics of LovD as a function of DMB-S-MMP concentration at a fixed monacolin J concentration of 2 mM. The K_m (DMB-S-MMP) was 0.67 ± 0.04 mM. In both assays, the k_cat was estimated to be 0.66 ± 0.03 min⁻¹.

FIG. 3.

Low-density fermentation and biocatalysis. E. coli strain BL21(DE3)/pAW31 expressing LovD overnight was concentrated 10 times to a final OD₆₀₀ of 22. The substrates monacolin J and DMB-S-MMP were added to final concentrations of 15 and 25 mM, respectively. The conversion was monitored as a function of time by HPLC analysis. (A) HPLC traces of the time course study. The labeled peaks are monacolin J (lactonized form) (1), DMB-S-MPA as a result of DMB-S-MMP hydrolysis (2), DMB-S-MMP (3), and simvastatin (lactonized form) (4). (B) Conversion of monacolin J to simvastatin as a function of time. The final conversion at 24 h was 99%. The data points are averaged values of two runs.

FIG. 4.

High-density fermentation and biocatalysis. (A) Fed-batch fermentation (500 ml) with F1 minimal medium. At an OD₆₀₀ (circles) of 5.93 (I), the temperature of the fermentation was decreased to and maintained at RT. IPTG was added to a final concentration of 200 μM, and the feeding was initiated and maintained at 0.08 ml/min. The effective concentrations of LovD (μM) at different points of the fermentation were measured (squares). (B) Rate of conversion of monacolin J to simvastatin by cells at four different points during the fermentation (indicated by 1, 2, 3, and 4) in A. The cells are either made “resting” by shifting to 50 mM HEPES (pH 7.9) or “nonresting” without medium change.