Molecular Engineering And Application Of Candida Glabrata Keto Ester Reductase For Bioreduction Of Aromatic α-keto Esters

Enantiometrically pure secondary alcohols are an important class of chiral building blocks in the synthesis of numerous pharmaceuticals, fine and agricultural chemicals and specialty materials. As an important part of optically pure secondary alcohols, hydroxyl esters are frequently required in organic synthesis. Asymmetric reduction of prochiral ketones with carbonyl reductases is an effective approach to producing chiral secondary alcohols since it can offer advantages of mild and environmentally reaction conditions and remarkable selectivity. However, enzymes from nature rarely have the combined properties necessary for industrial chemical production such as high substrate concentration, excellent selectivity, time saving, low biocatalyst loading, highly efficient cofactor regeneration system etc.This dissertation describes the progress of engineering a keto ester reductase from Candida glabrata (CgKRl) by protein engineering, resulting in a variant with increased thermostability and activity toward aromatic a-keto esters.Firstly, a semi-rational design strategy combining homology modeling and docking analysis was employed to alter the substrate specificity of CgKRl by engineering its active pocket. The substrate specificity of CgKRl was significantly altered and this tailor-made variant M3(F92L/F94V) showed much higher activity toward aromatic a-keto esters. CgKR1-M3showed the highest activities toward methyl ortho-chlorobenzoylformate (CBFM,109U/mg protein) and ethyl2-oxo-4-phenylbutyrate (OPBE,110U/mg protein), in contrast to CgKRl-WT, which showed the highest activity toward ethyl4-chloro-3-oxobutanoate (COBE,122U/mg protein). Also, CgKRl-M3showed the same trend for other substrates in the substrate spectrum.Secondly, the thermostability of CgKRl was improved by several protein engineering strategies. Several thermostability-related sites were identified using amino acid consensus approach based on sequence alignment. These sites were investigated by site directed mutagenesis and combinatorial mutagenesis, and a double variant M4(I99Y/G174A) that enhanced the T1550by2.6℃over wild type was obtained. M3was then chosen as the new parent of next evolution. After one round of random mutagenesis, followed by site-saturation mutagenesis of residue138, M5(I99Y/D138Y/G174A) and M6(I99Y/D138N/G174A) were identified with7.0℃and9.0℃increase in T1550as compared with M4respectively. At last, combination of M6and M3yielded a last variant M8harboring five mutations (F92L/F94V/I99Y/D138N/G174A), showing not only higher catalytic efficiency toward aromatic a-keto esters, such as CBFM and OPBE, but also the highest value in T1550Thirdly, the asymmetric reduction of CBFM by CgKRl-WT and M8was investigated. The keto esters reductases were coexpressed with BmGDH, which resulted in about80%lose of their expression quantity. So the coexpression strategy didn’t fit CgKR1. Under the conditions of1.0g/L lyophilized cells of CgKR1and4.0g/L lyophilized BmGDH powders, CBFM at0.5M (100g/L) could be nearly completely converted to product by M8with addition of0.2mM external NADP+within10h while WT resulted in84%conversation because of its rapid inactivation.