Discovery,Engineering And Application Of Multi-halogenated Ketone Reductases

Multihalogenated ketones can be reduced to corresponding chiral alcohols which are being vital building blocks of drug synthesis,such as a series of important intermediates for antifungi,antidepressant and antiemetics.Reductases probably encounter various obstacles to catalyzing non-natural multihalogenated ketones,such as low activity,substrate inhibition,poor stability and so on,which greatly limit their industrial application.Herein,the aim of this research is to obtain novel multihalogenated ketone reductases with high performance and excellent robustness for industrial applications.Through genomic database mining and enzyme library screening,a ketoreductase was cloned from Scheffersomyces stipitis CBS 6045,named as SsCR,which could catalyze the asymmetric hydrogenation of a variety of aromatic ketones.SsCR exhibited a specific activity of 65 U mg-1 protein and excellent enantioselectivity(99.9%ee)towards the hydrophobic substrate 2-chloro-1-(2',4'-dichlorophenyl)ethanone(TCAP),which is a key intermediate in the synthesis of common antifungal agents such as miconazole,econazole and sertaconazole.The kinetic parameter kcat/Km for 2-chloro-l-(2',4'-dichlorophenyl)ethanone and 2-chloro-l-(2',4'-difluorophenyl)ethanone were 4.51×103 s-1 mM-1 and 2.65×104 s-1 mM-1 respectively,indicating that SsCR shows very high catalytic efficiency towards this kind of multihalogenated ketone substrates.Molecular dynamic simulation results demonstrate a higher substrate binding free energy change for this substrate as compared with other substrates.Based on the good catalytic properties of SsCR,(R)-2-chloro-1-(2',4'-dichlorophenyl)ethanol could be obtained with a space-time yield(STY)of up to 268 g L-1 d-1 without any addition of exogenous cofactor in the reductive reaction process.On scaling up the bioreaction,the(R)-alcohol was isolated with 88.2%yield and 99.9%ee,and the environmental factor(E factor)was 7.25.Meanwhile,the activity of reductase SsCR was severely inhibited by substrate with an inhibition constant(Ki)of 1.0 mM,which is not compatible with a practical reaction system containing more than 2.0 mM of substrate dissolved with over 10%DMSO(v/v).To cope with this problem,we resolved the crystal structures of native SsCR at 2.3 A resolution(PDB ID:5GMO)and SsCR-NADP+complex at 2.1 A resolution(PDB ID:5YW4).Alignment of the structures of SsCR and SsCR-NADP+suggests that the conformational changes of loop-80 and loop-200 are important for the cofactor binding,and contribute to the subsequent substrate binding and catalysis.Three key residues were selected from the tips of these two loops and substituted with amino acid residues having lower hydrophobicity,which could weaken the hydrophobic interactions that bridge the two loops,resulting in a remarkable reduction of substrate inhibition.Among these variants,L211H showed a significant attenuation of substrate inhibition,with a Ki of 16 mM,which was 16 times of the native enzyme.At the substrate loading of 100 mM,the space time yield of haloketone's asymmetric reduction using variant L211H was 3-fold higher than that using the wild-type enzyme.Subsequently,the crystal structures of mutant L211H and its complex with cofactor were solved,followed by the MD simulation of system L211H-NADP+-TCAP.The computational results showed the flexible loop-80 in mutant L211H occupied the same inhibitive site in the WT,presumably impeding the binding of the inhibitive substrate and cofactor.Thus,compared with the WT,the alleviation of substrate inhibition in L211H mutant could be attributed to the reduced formation of the quaternary complexes(E-NADPH/NADP+-S-S1).This work provides an instructive example that substrate inhibition is alleviated with a loop engineering approach.Besides substrate inhibition,the poor thermostability(5 min of t1/2 at 40?)and product tolerance(44 min of Pt1/2 at 5 mM product)of SsCR impeded its application.Therefore,we then engineered the thermostability and product tolerance of SsCR via directed evolution simultaneously.After four rounds of evolution,the mutant SsCRM4 was obtained with a thermostability parameter T5015 of 41.5?,which was 5.4? higher than that of the WT,and the half-life of SsCRM4 at 40? was enhanced 170-fold compared with the WT.Meanwhile,the product tolerance of SsCRM4 was also improved 52-fold compared with the WT.The robust and efficient mutant SsCRM4 showed good activity towards kinds of multihalogenated aromatic ketones,and can be applied to prepare chiral alchohol at 600 mM substrate loading with 98%ee.The STY reached up to 664 g L-1 d-1 with Substrate/Catalyst ratio of 107(g/g).In conclusion,we exploited a reductase SsCR that could efficiently catalyze the reduction of multi-halogenated aryl ketones.The substrate inhibition of SsCR was also alleviated with the guidance of crystal structure and computational simulation.Then the engineered robust reductase SsCRM4 with good thermostability and product tolerance was applied to the reduction of TCAP at higher substrate loading.By improving the catalytic performance of reductase SsCR for this non-natural substrate,a highly efficient green system was then established for biocatalytic reduction of multi-halogenated aryl ketones.