Broader Substrate Specificity Of Carbonyl Reductase And Co-expression Of Dual Enzymes

It is well recognized that asymmetric synthesis of chiral aromatic alcohols with carbonyl reductases has gained increasing popular, due to mild reaction condition, high yield, excellent enantioselectivity, and environmental compatibility. Herein, the function for catalyzing acetophenone derivatives of (S)-carbonyl reductase II (SCRII) was modified by site-directed mutagenesis, which overcome the wild enzyme deficiency and broadened the substrate specificity and catalytic function of SCRII. Meanwhile, in order to establish the efficient asymmetric bioreduction of acetophenone, a glucose dehydrogenase (GDH) from Bacillus sp. YX-1 was introducted to maintain abundant NADPH supplied. Highly efficient synthesis of chiral aromatic alcohols was achieved with recombinant E. coli co-expressing E228S and GDH. Furthermore, whole-cell system and cell-free extract system were investigated with acetophenone as substrate. The main results were shown as follows:(1) An important site (E228) was selected for mutagenesis through amino acid sequence and protein structure alignment, and the corresponding variant E228S was constructed in E. coli. Using the acetophenone derivatives as substrates, specific activities and biotransformation function of the variant were determined. The specific activity of the variant E228S increased to approximately 7-20 times for acetophenone, 4'-methylacetophenone, 4'-chloroacetophenone and 4'-bromoacetophenone. The biotransformation results showed that the substrate specificity for catalyzing acetophenone derivatives by the variant was broadened. The variant exhibited excellent performance to afford (R)-1-phenylethanol, (R)- 1-(4-methylpheyl) ethanol, (R)-1-(4-chlorophenyl) ethanol and (R)-1-(4-bromophenyl) ethanol from acetophenone, 4'-methylacetophenone, 4'-chloroacetophenone and 4'-bromoacetophenone with high optical purity of >99% in a yield of over 80%.(2) According to the conserved sequences of glucose dehydrogenase (GDH) derived from Bacillus sp, gdh was amplified from Bacillus sp. YX-1 genome and the corresponding inducible expression plasmid pET-gdh was constructed. Purified enzyme was obtained by HisTrap HP affinity column chromatography, subsequently. The characterization of purified enzyme indicated that GDH was highly resistant to organic solvents. It retained more than 90% activity when incubated in 50% cyclohexane, octane, decane at home temperature for one hour, respectively. Besides, it displayed broad substrate specificity and could catalyze a variety of sugars to afford reduced coenzymes. In addition, it exhibited similar capability to regenerate either NADH or NADPH with specific activity of 8.37 U/mg and 8.62 U/mg for NAD+ and NADP+, accordingly. The maximum specific activity of purified enzyme was achieved under the condition of 45℃and pH 8.0.(3) Co-expression plasmids harboring both E228S and GDH and fusion expression plasmids carrying both E228S and GDH were constructed with overlap extension PCR. When using acetophenone (10 g/L) as substrate, recombinant E. coli BL21/pET-S-SD-AS-G showed excellent performance to give (R)-1-phenylethanol with high optical purity of >99% in a yield of 92% in 12 h.(4) In order to further improve the substrate concentration and catalytic efficiency, cell-free system harboring in situ cofactor regeneration was constructed. This cell-free system containing 0.03 mmol/L NADP+ exhibited better performance on reduction acetophenone (10 g/L) to produce (R)-enantiomers with >99% e.e. and 89% yield in 1 h. Besides, a substrate fed-batch strategy was introduced into cell-free system for reduction acetophenone (20 g/L) in 4 h, and the optical purity and yield of (R)-enantiomers were kept at >99% and 86%, respectively.