Enantioselective reduction of carbonyl compounds by whole-cell biotransformation, combining a formate dehydrogenase and a (R)-specific alcohol dehydrogenase

• Published in: Applied microbiology and biotechnology Vol. 66; no. 6; pp. 629 - 634
• Main Authors: Ernst, Marianne, Kaup, Björn, Müller, Michael, Bringer-Meyer, Stephanie, Sahm, Hermann
• Format: Journal Article
• Language: English
• Published: Berlin Springer-Verlag 01.03.2005; Springer; Springer Nature B.V
• Subjects: acetoacetates; Acetoacetates - metabolism; ADH gene; Alcohol; Alcohol dehydrogenase; Alcohol Dehydrogenase - genetics; Alcohol Dehydrogenase - metabolism; Alcohol Oxidoreductases - metabolism; alcohols; Bioconversions. Hemisynthesis; Biological and medical sciences; Biotechnology; Biotransformation; carbonyl compounds; Coenzymes - pharmacology; Dehydrogenases; E coli; enantiomers; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - metabolism; Esters; FDH gene; Formate dehydrogenase; Formate Dehydrogenases - genetics; Formate Dehydrogenases - metabolism; Fundamental and applied biological sciences. Psychology; genes; ketones; Kinases; Lactobacillus - enzymology; Lactobacillus - genetics; Lactobacillus brevis; Methods. Procedures. Technologies; Mycobacterium - enzymology; Mycobacterium - genetics; Mycobacterium vaccae; NAD (coenzyme); NADH; NADP; NADP (coenzyme); Organic Chemicals - metabolism; Oxidation; Oxidation-Reduction; Proteins; Q1; Q2; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Stereoisomerism; Substrate Specificity; Chemical reduction; Carbonyl compounds; Enantioselectivity; Enzyme; Biotransformation; Escherichia coli; Bacteria; Formate dehydrogenase; Oxidoreductases; Alcohol dehydrogenase; Genetically modified microorganism; Enterobacteriaceae
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-004-1765-5

A whole-cell biotransformation system for the reduction of prochiral carbonyl compounds, such as methyl acetoacetate, to chiral hydroxy acid derivatives [methyl (R)-3-hydroxy butanoate] was developed in Escherichia coli by construction of a recombinant oxidation/reduction cycle. Alcohol dehydrogenase from Lactobacillus brevis catalyzes a highly regioselective and enantioselective reduction of several ketones or keto acid derivatives to chiral alcohols or hydroxy acid esters. The adh gene encoding for the alcohol dehydrogenase of L. brevis was expressed in E. coli. As expected, whole cells of the recombinant strain produced only low quantities of methyl (R)-3-hydroxy butanoate from the substrate methyl acetoacetate. Therefore, the fdh gene from Mycobacterium vaccae N10, encoding NAD⁺-dependent formate dehydrogenase, was functionally coexpressed. The resulting two-fold recombinant strain exhibited an in vitro catalytic alcohol dehydrogenase activity of 6.5 units mg⁻¹ protein in reducing methyl acetoacetate to methyl (R)-3-hydroxy butanoate with NADPH as the cofactor and 0.7 units mg⁻¹ protein with NADH. The in vitro formate dehydrogenase activity was 1.3 units mg⁻¹ protein. Whole resting cells of this strain catalyzed the formation of 40 mM methyl (R)-3-hydroxy butanoate from methyl acetoacetate. The product yield was 100 mol% at a productivity of 200 μmol g⁻¹ (cell dry weight) min⁻¹. In the presence of formate, the intracellular [NADH]/[NAD⁺] ratio of the cells increased seven-fold. Thus, the functional overexpression of alcohol dehydrogenase in the presence of formate dehydrogenase was sufficient to enable and sustain the desired reduction reaction via the relatively low specific activity of alcohol dehydrogenase with NADH, instead of NADPH, as a cofactor.