Directed Evolution Of Enzymes For The Regulation Of Selectivity And Catalytic Promiscuity

Over the past 20 years,directed evolution of enzymes has gradually become one of the hottest research fields in biocatalysis.In contrast with natural enzymes.engineered enzymes achieved by directed evolution display varieties of advantages.such as higher activity,higher thermal stability and even better stereoselectivity.Recently,new catalytic activity of enzymes which cannot be accepted by natural enzymes and is so-called "catalytic promiscuity",have also been designed or discovered by directed evolution.These process offers a more attractive alternatives due to their low toxicity and mild reaction conditions.Thus,further research to obtain new engineered enzymes with better performance is still highly sought after.Candida antarctic lipase B(CAL-B)is one of the most studied lipases.The active site of CAL-B is composed of an acid pocket and an alcohol pocket.The hot spots lining the active site,which have important influence on the stereoselectivity of chiral acids and alcohols was found by screening a focused library.Through iterative mutation in the focused library,mutants with high activity and stereoselectivity were successfully evolved for the hydrolysis kinetic resolution of MBH ester and the esterification of 2-phenylpropionate.Using CAL-B as a model.we report the protein engineering of this enzyme into four highly stereocomplementary variants needed for obtaining all four stereoisomers in transesterification reactions between racemic acids and racemic alcohols in organic solvents.By generating and screening less than 100 variants.we achieved>90%selectivity for each of the four possible stereoisomers in the model reaction.MD simulation was implemented to gain the insight of the origin of selectivity.Moreover,we designed an enantiocomplementary decarboxylative hydroxylation that combines photocatalysts with(R)-or(S)-selective ketoreductases for the decarboxylative carbonylation of carboxylic acids to generate valuable chiral alcohols.Then,we described an engineered fatty acid photodecarboxylase(CvFAP)-catalyzed kinetic resolution of a-amino acids and a-hydroxy acids providing the unreacted(R)-configured substrates with high yields and excellent stereoselectivity(ee up to 99%).This efficient light-driven process requires neither NADPH recycling nor prerequisite preparation of esters,which are needed in previous biocatalytic approaches.The structure-guided engineering strategy(K,Q,F,Y)was based on large-sized amino acid scanning at hotspots to narrow the substrate binding tunnel.To the best of our knowledge,this is the first example of asymmetric catalysis by an engineered CvFAP.Cyclohexanone monooxygenases(CHMOs)show very high catalytic specificity for natural Baeyer-Villiger reaction.Mechanistically,flavin adenine dinucleotide(FAD),as the cofactor of CHMOs,is a versatile molecular that easy to switch between oxidation state and reduction state.Herein,we demonstrated a novel catalytically promiscuous activity converting the CHMO into a ketoreductase by employing the reduction state of FAD.Structure-guided engineering of CHMO by directed evolution was implemented to improve the catalytic activity(yield up to 95%)and stereoselectivity(ee up to 99%).This new transformation offers a new route for design and development of novel biocatalysts.