Expression Optimization Of Carbonyl Reductase Gene In Bacillus Subtilis

Methamphetamine(ephedrine) is a kind of alkaloids in ephedra, whose commonstereoisomers are l-ephedrine and d-pseudoephedrine. In clinic, ephedrine is widely usedbecause of its pharmacological effects on blood and nervous systems. Comparing with plantextraction and chemical synthesis, biotransformation has such characters as high reaction rateand mild conditions, which makes it have more space for potential development.Based on previous studies, in order to further improve the expression level of mldh inB.subtilis, the promoter was optimized firstly by constructing different expression vectors.Recombinant with the higherst-level of expression was chosen and studied further in codons.By site-directed mutation and gene synthesis, rare codons were substituted by preferencecodons for host in order to realize its heterologous expression. The main research work isdescribed as follows:⑴Using the genome DNA of Morganella morganii CMCC(B)49208as template, thegene mldh was cloned by PCR amplification, and then ligased with promoter PQ and p43toget different expression vectors PHY-p43-mldh、PHY-PQ-mldh、PHY-p43-p43-mldh andPHY-p43-PQ-mldh. Subsequently, these recombined vectors were transformed into B.subtilisWb600. SDS-PAGE from recombinant and whole cellular biotransformation showed thatthere was distinctive difference among the four recombinants, in which the B.subtilisWb600(PHY-p43-p43-mldh) got the highest concentration of d-pseudoephedrine inconversion solution and mole ratio of substrate was142.1mg/L and78.25%respectively.⑵Using frequency of mldh codons was analyzed by http://www.jcat.de/, and aminoacid codon with lower frequency and more concentrated was chosen. Primers T1-up andT1-down was designed and PCR was operated using PHY-p43-p43-mldh as template.Through digestion of DpnI、 transformation and screening, expression vectorPHY-p43-p43-mldh-mut was obtained by sequencing. It was shown by chemicaltransformation for B.subtilis Wb600and whole cellular biotransformation that theconcentration of d-pseudoephedrine in conversion solution reached139.2mg/L and the moleratio of MAK was76.6%. Compared with the situation before it was nearly unchanged, whichshowed that site-directed mutation had little effect on mldh expression.⑶In order to maximize the expression of carbonyl reduction, we substitute its rarecodons with preference ones by gene-synthesis. During this process,220bp was changed, theoriginal sequence remained identity of79.93%, and GC content was reduced from46.2%to39.41%. After chemical transformation and whole cellular biotransformation, theconcentration of d-pseudoephedrine in conversion solution reached174.2mg/L and the moleratio of MAK was94.9%. By comparing with B.subtilis Wb600（PHY-p43-p43-mldh），wefound that the yield of d-pseudoephedrine and the mole ratio of MAK was increased by21%and16.65%respectively.⑷The research of enzyme properties for the B.subtilis Wb600(PHY-p43-p43-mldh-syn)found that the optimal pH was7.0and temperature37℃. In buffer solution at ph6-8after12h, its activity could remain over70%, which could reach90%after10minutes of treatment under20-25℃. Most of mental ions had inhibitory effect on the enzyme, except Li+; Bycomparing enzyme characters of B.subtilis Wb600(PHY-p43-p43-mldh-syn) with M.morganiiJ-8, we found that their respective optimal pH and temperature were of no big difference. ThepH stable boundary of the recombinant was less than that of M.morganii, and both of the twostrains were thermostable under30℃。...