Asymetric Reduction Of COBE With Combinant E.coli Strains

Optically active (R)-4-chloro-3-hydroxybutanoate ethyl ester ((R)-CHBE) is a key chiral intermediate in the enantioselective synthesis of L-ptomaine, (R)-γ-amino-8-hydroxy acetic acid, Negamicyn, 2,5-hexofibrin and so on. The aim of this work was to produce (R)-CHBE from ethyl 4-chloro-3-oxobutanoate (COBE) by whole cell catalyzed reduction.In this work, an expression vector, pQE30-ALRgdh223, which harboring an NADPH-dependent aldehyde reductase gene from Sporobolomyces salmonicolor ZJU0307 and a glucose dehydrogenase gene from Bacillus megaterium ZJU0310, was constructed. The recombinant strain, E. coli M15 (pQE30-ALRgdh223), was cultivated and applied in the asymmetric reduction of ethyl 4-chloro-3- oxobutanoate to form (R)-4-chloro-3-hydroxylbutanoate. The results showed that 92.5% of 30mM COBE was reduced to (R)-CHBE with 99% e.e. value by 100g/L the recombinant cells at optimal conditions. When the concentration of COBE increased to 90mM, the yield reduced to 30%. It indicated that the substrate inhibition was serious in aqueous phase reaction. Dividing the addition of substrate to several fractions could decrease substrate inhibition in certain extent.To reduce the product inhibition, the asymmetric reduction of COBE was carried out in a water/n-butyl acetate two-phase system by using whole-cells of the recombinant cells- E. coli M15 (pQE30-ALRgdh223). At the optimal conditions, 90mM COBE was reduced to (R)-CHBE with an e.e. value of 99% and a yield of 74% by 100 g/L cells of E. coli M15 (pQE30-ALRgdh223). Comparing with the reaction in aqueous system, the yield of (R)-CHBE was increased from 74% to 30% at the same substrate concentration.In comparison, another reaction system with two different cells as catalysts was also investigated. E. coli M15 (pQE30-ALR) and E. coli M15 (pQE30-gdh223) were recombinant strains harboring the ALR gene from Sporobolomyces salmonicolor ZJU0307 and the gdh223 gene from Bacillus megaterium. The cells of these two strains were mixed to construct a reaction system with cofactor regeneration. To reduce the inhibition of the substrate and the product, the reaction was carried in a water/n-butyl acetate two-phase system. At the optimal conditions, 360mM COBE was reduced, with an e.e. value of 87% and a yield of 78.5% by using 80g/L cells of E.coli M15 (pQE30-ALR) and 20 g/L cells of E. coli M15 (pQE30-gdh223).In this two systems, the biotransformation reaction from COBE to (R)-CHBE was completed effectively without any addition of glucose dehydrogenase or NADPH/NADP*. This new strategy has the potential to be employed in other biotransformation systems requiring coenzyme regeneration.