Studies On Biocatalytic Reduction Of Keto Esters And Carboxylic Acids

Chiral secondary alcohols are frequently required as important intermediates in the synthesis of numerous pharmaceuticals, fine and agricultural chemicals, and specialty materials. As an efficient and straightforward access to chiral alcohols, biocatalytic asymmetric reduction of prochiral ketones offers some advantages in comparison with traditional chemical methods, such as mild and environmentally benign reaction conditions and remarkable selectivity, and is increasingly applied for the industrial production of some valuable chiral alcohols. Reductions of carboxylic acids are interesting reactions, since the generated products, aldehydes and alcohols are potentially applicable in the flavor and aroma industry. It is therefore of great interest and importance to develop biocatalytic reduction of carboxylic acids which constitutes an area underrepresented in current research efforts.This dissertation, on one hand, describes the genome hunting, purification and biocatalytic properties of carbonyl reductases. Construction of coexpressing systems with high NADPH-regeneration capacities and their application for the highly efficient and enantioselective synthesis of several optically active hydroxyl esters were studied in detail. On the other hand, a new and simple biocatalytic reduction procedure for carboxylic acids by using H2as reductant is presented.Firstly, three carbonyl reductases from Bacillus sp. ECU0013were identified and their biocatalytic properties were characterized. In the screening of11E. coli strains overexpressing recombinant reductases from Bacillus sp. ECU0013, three NADPH-dependent carbonyl reductases (YtbE, FabG and YtbE) were identified with capability of producing chiral alcohols and purified to homogeneity. YueD showed an optimal pH of7.5-8.0, FabG of7.0and YtbE of6.5, while all of them exhibited the highest activity at45-50℃. The recombinant carbonyl reductases could reduce a broad spectrum of prochiral ketones including aromatic ketones, a-and β-keto esters. YueD showed the greatest preference for ethyl pyruvate and a relatively superior preference for ethyl4-chloro-3-oxobutanoate (COBE). All the tested aromatic ketones and β-keto esters were reduced to the corresponding chiral alcohols in enantiomerically pure forms catalyzed by YueD. FabG was dramatically active in the conversion of ethyl2-oxo-4-phenylbutyrate (OPBE) to the corresponding alcohols with>99%ee. YtbE showed high activity and splendid enantioselectivity in the reduction of methyl o-chloro-benzoylformate (CBFM), giving enantiopure methyl (R)-o-chloromandelate [(R)-CMM]. Secondly, coexpression systems were constructed and applied for the efficient reduction of keto esters. A glucose dehydrogenase (GDH) from Bacillus subtilis was coexpressed with YueD, FabG and YtbE respectively on two different styles. Higher substrate conversion was observed by using E. coli harboring a coexpression plasmid in the presense of glucose and NADP+. After optimizing the expression condition of carbonyl reductases and GDH and the reaction condition, highly efficient and enantioselective reduction of prochiral keto esters were achieved by using recombinant E. coli. Ethyl (S)-2-hydroxy-4-phenylbutyrate [(S)-HPBE] with>99%ee was produced via reduction of OPBE at3.0M (620g L-1) by using E. coli BL21(pET28a-FabG-GDH) without addition of external cofactor, giving space-time yield (STY) of615g L-1d-1. In an aqueous/toluene biphasic system, E. coli BL21(pET28a-YueD-GDH) gave a substrate conversion efficiency of100%and an enantiomeric purity of ethyl (R)-4-chloro-3-hydroxybutanoate [(R)-CHBE] of>99%ee when subjected to the reduction of COBE at a total concentration of1.3M (215g L-1) by using substrate fed-batch strategy, resulting in STY of956g L-1d-1. Using E. coli BL21(pET28a-YtbE-GDH), as much as2.5M (500g L-1) of CBFM was stoichiometrically converted into enantiopure (R)-CMM at20℃in the absence of external cofactor, giving STY of815g L-1d-1.Thirdly, a new method for the biocatalytic reduction of carboxylic acids was developed. Rest cells of Pyrococcus furiosus as biocatalysts enabled us to utilize hydrogen (H2) as a clean reductant to promote a challenging reduction of carboxylic acids. A borad range of aliphatic and aromatic carboxylic acids turned out to be suitable substrates, among which3-phenylpropionic acid and medium-chain aliphatic acids were converted most smoothly. A rough estimation of the catalytic performance of aldehyde oxidoreductase (AOR) for the reduction of3-phenlpropionic acid yielded a TOF of389h-1. The reaction proceeded highly chemoselectively to the corresponding alcohols under low hydrogen pressure and temperature compared with previously reported catalytic hydrogenation processes. More importantly, other functional groups such as keto groups and isolated C=C double bonds remained untouched, representing another clear advantage over most chemocatalytic methods. P. furiosus exhibits high tolerance to organic solvents and the chemoselectivity of the carboxylate reduction could be directed towards aldehydes by simple medium engineering. The aldehyde selectivity seemed to correlate with the solvent hydrophobicity and increased from0%in aqueous reaction media to more than70%in hexadecane. Furthermore, instead of H2, CO can also serve as stoichiometric reductant for the bioreduction of carboxylic acids.