Gene Mining,molecular Engineering And Catalytic Application Of Camphor Reductase

Borneol is a widely used penetration enhancer in clinical practice and has the ability to pass through various physiological barriers,such as gastrointestinal mucosa and the blood-brain barrier（BBB）.Antibacterial,anti-inflammatory,and central nervous system protection are also among the properties of borneol that have received much attention.However,the current commercial borneol cannot meet the demand for high quality and low price,and the processing method does not conform to the sustainable environmental protection concept.The borneol market is in short supply,and how to efficiently obtain natural borneol with high purity and reasonable price in a green and environmentally friendly way is an urgent problem to be solved.This study proposed a method for the synthesis of（+）-borneol catalyzed by camphor reductase using low-cost scrambled camphor,and conducts research on gene mining,enzymatic property characterization,molecular engineering and catalytic application for screening camphor reductase.The main research results obtained are as follows:（1）A method for the synthesis of（+）-borneol from scrambled camphor catalyzed by camphor reductase was proposed.Using the amino acid sequence of Co TR with camphor reducing activity as a probe,38 candidate genes were screened by BLAST based on the NCBI database,and were successfully introduced into Escherichia coli for heterologous expression.A total of 3 camphor reductases were screened.After the enzyme activity test,we selected the camphor reductase with the highest activity（from T.hassleriana,named as ThCR）as the next research object.ThCR encodes 269 amino acids,and has the NCBI sequence number XP＿010521893.1.（2）Sequence analysis and enzymatic properties characterization of ThCR were carried out.Sequence analysis revealed that ThCR was a member of the Short-chain dehydrogenases/reductases（SDRs）family,with a conserved coenzyme-binding motif（TGXXXGXG）and a catalytic tetrad（N-S-Y-K）.ThCR was approximately 30 k Da,and a NADPH-dependent reductase.The optimum temperature and p H for the reduction of ThCR was 50°C and 5.0.ThCR was stable within 40°C,but the residual enzyme activity dropped at50°C.ThCR was stable when p H was between 6.0-8.5.The K_m and the k_cat/K_mof ThCR-catalyzed（+）-camphor were 992.38μmol·L^-1 and 0.71 s^-1·mmol^-1·L^-1.ThCR had the biocatalytic potential to synthesize（+）-borneol that could catalyze the formation of（+）-borneol with an e.e.value of 50.70%from scrambled camphor.（3）Molecularly engineering was carried out to increase stereoselectivity of ThCR.Based on homology modeling and molecular docking analysis of the substrate-binding pocket of ThCR,the catalytic key amino acids Ser155,Tyr168 and Lys172 were identified by site-directed mutagenesis.Phenylalanine and alanine scanning of amino acid residues within 5?of（+）-camphor（N107,I162,W199,F200,A206,Y209,K216）identified that Y209 and I162 was the core sites of ThCR for synthesis（+）-borneol.The optimal variant ThCR_Y209W was obtained by saturation mutation,and the substrate conversion rate,the e.e.and proportion of（+）-borneol were increased by 1.55,6.3 and 1.32-fold.ThCR_Y209W had improved affinity and catalytic efficiency for（+）-camphor(K_m was 826.71μmol·L^-1,and k_cat/K_mwas 2.40 s^-1·mmol^-1·L^-1).（4）In order to regenerate coenzyme NADPH,ThCR_Y209W was co-expressed with glucose dehydrogenase.After 10 h of whole-cell biotransformation,the conversion of scrambled camphor was 87.0%,and the production of（+）-borneol was 0.34 g·L^-1.This yield is superior to that reported so far for the biosynthesis of（+）-borneol.