Cloning And Expression Of A Novel Hydroxylase Gene From Amycolatopsis Sp. CGMCC1149in The Hydroxylation Of Lovastatin

Wuxistatin, a novel inhibitor of3-hydroxy-3-methyl-glutaryl coenzyme A reductase anda potential anticholesterolemic drug, is converted from lovastatin by Amycolatopsis sp.CGMCC1149. In the bioconversion, lovastatin is firstly hydroxylated by a hydroxylase，which is a key rate-limiting enzyme. In this paper, a novel hydroxylase gene was isolatedfrom Amycolatopsis sp. CGMCC1149by Degenerate PCR and Self-Formed Adaptor PCR(SEFA PCR) and expressed in Escherichia coli. Furthermore, a lovastatin catalytic system invitro was constructed. These would be helpful for further studies in large-scale production ofwuxistatin.A hydroxylase gene was isolated by Degenerate PCR and SEFA PCR on the basis ofprevious studies. It contains1209bp, which can encode a403-amino-acid protein with amolecular weight of44.8kDa. Bioinformatics analysis showed that the gene was a novel geneand it belongs to cytochrome P450gene superfamily CYP105family and has a close ofphylogenetic relationship with P450Um-1from Amycolatopsis azurea. The secondarystructure showed that this protein comprises many typical functional regions of P450, such asoxygen binding site, ion-pair region and heme binding region. Meanwhile, a3D model wasconstructed based on the known crystallographic structure using protein homology modelingtechnology. The structure comprises12α-helices and4β-strands, and it is roughly dividedinto α-rich and β-rich regions. αFG and BC loop may be form a channel of substrates entranceto the active site. Moreover, the gene has a preference for codon usage, which trends to use Gor C in the third codon position.In order to establish a lovastatin catalytic system in vitro, a recombinant strain E. coliDH5α (pEtac-p450lov) was constructed. The results showed that it has well plasmid stabilityand the plasmid retention rate is92%after subculture for60generations. CO differencespectrum showed that there has an absorption peak at450nm, which indicated that thehydroxylase were correctly expressed in an active form. When the cell density reached0.6ofOD600,subsequenctly0.1mM IPTG was added for incubating another8h at20°C, thehydroxylase yield reached maximization. Besides, adding0.5mg/L FeCl2into the mediumwas in favor of hydroxylase expression.In order to achieve lovastatin catalyzing in vitro, three lovastatin catalytic systems in vitrowere established on the basis of different electron transport systems. Lovastatin can not becatalyzed when E. coli electron transport system was used. When using Amycolatopsis sp.CGMCC1149electron transport system, it could catalyse lovastatin hydroxylation, but thecatalytic efficiency was very low. The lovastatin catalytic system in vitro constructed byferredoxin and ferredoxin reductase could catalyse lovastatin hydroxylation and had thehighest catalytic efficiency. Therefore, it is choosed as the object of study and the optimumreaction conditions were as follows: temperature30°C, pH7.5, reaction time6h, NADH600μM, lovastatin30μM.