A carbonyl reductase from Gluconobacter oxydans ( Go CR) exhibited good activity and strict enantioselectivity for the asymmetric reduction of a series of halogenated acetophenones, resulting in the corresponding S -alcohols. For efficient synthesis of alcohol products, an economical and satisfactory substrate-coupled cofactor regeneration system was constructed employing isopropanol as the co-substrate to regenerate NADH in situ . In the presence of 2.0 molar eq. of isopropanol, 500 mM o -chloroacetophenone was reduced to ( S )-1-(2-chlorophenyl)-ethanol with >99% ee and a high conversion rate of 96% by recombinant Escherichia coli BL21 cells overexpressing Go CR. In this reaction system, a slight excess of isopropanol as hydride source could drive the reduction reaction to near completion due to the speculated forming of an intramolecular hydrogen bond between the Cl group and the new obtained alcohol moiety of ( S )-1-(2-chlorophenyl)-ethanol. Furthermore, other tested halogenated acetophenones at 100 or 200 mM substrate load were also reduced at 71–96% conversion, affording S -configuration alcohols with >99% ee . Homology modeling and molecular dynamics simulation were performed to uncover the structural basis for the excellent enantioselectivity of Go CR toward halogenated acetophenones. These results imply the high potential of Go CR in the production of halogenated 1-phenylethanols, such as ( S )-1-(2-chlorophenyl)-ethanol, an important chiral building block with wide application in pharmaceutical chemistry and fine chemicals.
A basic scheme of using 2,4-dinitrophenol (2,4-DNP) as an uncoupler between electron transport chain and oxidative phosphorylation for the biochemical regulation of Gluconobacter oxydans (G. oxydans) is raised. Under the weakly acidic environment, the lipophilic 2,4-DNP as an uncoupling agent destroys the electrochemical proton (H+) gradient in mitochondria, which leads to a loss of the driving force for adenosine triphosphate (ATP) synthesis. Based on the main enzyme system of G. oxydans, four typical substrates were studied for exploring the biochemical effects of uncoupling on the cells. As a result, it was found that under uncoupling conditions, cells receive a feedback signal of ATP deficiency, which promotes the utilization of catabolized substrates such as sorbitol and glucose through the substrate level phosphorylation pathway in order to make up for the deficiency of ATP. The proposal of strategy has a further understanding of the basic biochemical knowledge of G. oxydans and lays a theoretical foundation for the directional regulation of G. oxydans.
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