Screening And Engineering Of Carbonyl Reductases For The Synthesis Of Important Pharmaceutical Intermediates

Carbonyl reductases catalyze the reduction reaction of numerous ketones to their corresponding chiral alcohols,making considerable contributions to industrial manufacturing of valuable intermediates for the pharmaceutical and chemical industry.Although the industrial production of some chemicals and pharmaceuticals at high yields and low costs has been made possible by carbonyl reductases,there are many more waiting for a better biocatalyst.In the scope of this thesis,three studies were performed to achieve carbonyl reductases with the desired property by engineering or screening method.Enzyme engineering is an emerging method to overcome almost all challenges in biocatalysis and make the biocatalyst applicable for industrial applications.To improve the catalytic performance in synthesizing pharmaceutically important compounds,a carbonyl reductase from Bacillus subtilis WB600 was engineered by semi-rational design based on in silico docking and alanine screening.Optically active 1-phenylethanol and its derivatives are versatile chiral precursor for many pharmaceuticals.Although several biosynthesis strategies have been conducted to convert halogenated acetophenones to corresponding(R)and(S)alcohols,the catalytic efficiency and stereoselectivity are still far below the requirement of industrial applications.Due to the increasing demand of enantio pure alcohols,it is essential to explore more robust biocatalyst capable of preparing enatiomerically active alcohols efficiently The carbonyl reductase YueD was engineered successfully by site-directed mutagenesis,generating a mutant Val181Ala,which reduced acetophenone and its derivatives remarkably.An enzyme coupled cofactor regeneration system was constructed for proficient and economical synthesis of alcohol products,using xylose as the cosubstrate to regenerate NADPH in situ,and employed in the asymmetric reduction of halogenated acetophenones.In the presence of 0.18 g/mL of Xyleose,500 mM of halogenated acetophenones were reduced to(S)and(R)-alcohols with>99%ee and a high conversion rate of 85%to 99%by mutant VAL181Ala.The enzyme substrate docking analysis provided some insights into the structural basis for the high substrate load transformation by mutating Val181.These data demonstrate the promising prospect of the mutant Val181Ala in practical synthesis of valuable chiral alcohols.Keeping in mind the important role of cofactor in the catalytic performance of dehydrogenases,another effort was made to engineer a dehydrogenase from Pseudomonas putida for improved catalytic activity by introducing an additional hydrogen bond between the enzyme and its cofactor.Short-chain Alcohol Dehydrogenases(SDRs)are NADPH-dependent enzymes,which catalyze the redox reaction in the presence of the corresponding cofactor.To explore how the binding with cofactor affects the activity of short-chain alcohol dehydrogenases,SDH_P.p-4654 was engineered in this study.Computational analysis suggested that Aln89,Vln140 and Aln141 locate in the proximity to the cofactor,so they were replaced by Trp with long side chain to increase the hydrogen interactions.Further activity analysis revealed that the Aln89Trp mutation enhanced the kcat/Km up to 3.4 folds and the maximum conversion of isobutyraldehyde achieved by the Aln89Trp mutant was 67%.The molecular dynamics simulation also showed that the hydrogen interactions between the enzyme and NADPH were stronger for the Aln89Trp mutant and the binding energy increased.Moreover,the RMSD and RMSF results indicated that the Ala89Trp mutant was more stable than the wild-type.The results demonstrate mutant Aln89Trp as a good candidate for the production of biofuels such as isobutanol and may provide hints for engineering of other NAD(P)H-dependent enzymes for higher catalytic performance.Similarly,protein engineering strategy was applied to redesign the PpySDR from Pseudomonas putida ATCC 12633 for improved catalytic activity toward substrate 4-hydroxy-2-butanone for the efficient biosynthesis of chiral 1,3-butandiol,a very important intermediate for the synthesis of pharmaceutically valuable compounds,but the results were not satisfactory.Screening is an alternative powerful approach to new robust biocatalysts.Screening of a library of 20 carbonyl reductases identified two carbonyl reductases,PFODH from Pichia finlandica and CpSADH from Candida parapsilosis with satisfactory catalytic activity and enantioselectivity toward 4-hydroxy-2-butanone.PFODH and CpSADH respectively delivered optically pure R-and S-1,3-butanediol,and both showed high substrate/product tolerance and good catalytic activity(with conversions of 81-90%)without requirement of external cofactors Regeneration of the cofactor NADH was facilitated by a substrate-coupled system using isopropyl alcohol as the co-substrate.The substrate spectra of PFODH and CpSADH were further investigated by expanding to various substituted aryl ketones.Almost all of the analyzed ketones were reduced asymmetrically into their corresponding chiral alcohols with excellent ee values of 97-99%.PFODH easily reduced the substrates which are substituted adjacent to the carbonyl group or those substituted on the meta-position of the phenyl ring,while CpSADH hardly reduced those with substituents adjacent to the carbonyl group.These results demonstrate the industrial potential of PFODH and CpSADH in biosynthesis of optically pure 1,3-butanediol and other valuable chiral alcohols.The above results demonstrate the power of protein engineering and screening method in delivering efficient biocatalysts.Protein engineering could in many cases reshape the enzyme for high catalytic efficiency and tolerance toward non-natural substrates,whereas enzyme screening acts as a useful backup in case that protein engineering fails to achieve satisfactory results,which together inaugurate a preparative scale up process for producing chiral alcohols with excellent enantiopurity.