Cloning, Expression, Characterization And Its Application Of A Novel P450 Sulfide Monooxygenase From Rhodococcus Sp. ECU0066

Chiral sulfoxides play very important roles in many areas. They were usually used as chiral auxiliaries or useful building blocks in the synthetic chemistry, and also as pharmaceuticals or natural products in biological chemistry. The asymmetric oxidation of a prochiral sulfide is undoubtedly the most direct and economical method for the synthesis of enantiomerically pure sulfoxides, as compared to the kinetic resolution of racemic sulfoxides. However, the asymmetric oxidation of prochiral sulfides with chemical methods is usually expensive and environmentally unfriendly. On the other hand, the biocatalytic methods that make use of whole cells or isolated enzymes are becoming advantageous alternatives for this purpose.In our previous work, a new strain ECU0066 was successfully isolated from soil samples, which was proven to be useful in enantioselectively oxidation of prochiral sulfides to produce optically pure sulfoxides. In this study, the strain was successfully identified by 16SrDNA sequence online BLAST. The enzymes which were active toward sulfides in the cells of strain ECU0066 were investigated, and a new cytochrome P450 monooxygenase was detected might involve in transformation of sulfides. Based on the sequence alignment, two degenerated primers were designed, and fragments of a new P450 monooxygenase were cloned from the genomic DNA of ECU0066. The flanking genomic sequences of the amplified fragment were obtained by genome walking. Then, the P450 monooxygenase was successfully expressed in E. coli BL21 (DE3), and the monooxygenase was purified by a nickel sepharose column, and characterized. The expression conditions of P450 monooxygenase in E. coli were further optimized in flasks and fermenter, then sufficient biocatalyst was obtained. To regenerate the NADPH in P450 monooxygenase reaction, a glucose dehydrogenase (GDH) gene was cloned from Bacillus subtilis genome DNA and expressed in E. coli BL21 (DE3). The highly active GDH was obtained and successfully used to regenerate the NADPH in P450 monooxygenase in vitro reaction system. At the same time, three systems for coexpression of the P450SMO and GDH gene (gdh) were constructed, which were used in whole-cell P450 reaction system for in vivo NADPH regeneration.First, a new and efficient sulfide oxidation strain ECU0066 was isolated and identified as Rhodococcus sp., which could transform phenyl methyl sulfide (PMS) to (S)-sulfoxide with 99% ee (enantiomeric excess) via two steps of enantioselective oxidations. With several organic compounds as potential inducer to improve the activity of Rhodococcus sp., phenyl methyl sulfide and naphthalene were found to be efficient inducer for sulfide oxidation. The imidazole was found to inhibit the activity of Rhodococcus sp., so we speculate that there may be a P450 monooxygenase responsible for sulfide oxidation in Rhodococcus sp.Second, according to the results of the P450 sequence alignment, two degenerated primers were designed and a new P450 monooxygenase fragments were cloned from Rhodococcus sp. genomic DNA. A 3.7 kb fragment containing the complete cytochrome P450 gene was obtained by genome walking method. The cytochrome P450 gene fragment (2361bp) encodes a protein of 786 amino acids. Theoretical pI and MW were 5.17 and 87.19 kD. A BLAST search revealed that this protein is a natural fusion protein consisting of a heme domain, a flavin-reductase domain, and a ferredoxin domain. A high level of homology to the thiocarbamate-inducible cytochrome P450 (CYP116) from Rhodococcus erythropolis NI86/21 was found (56% identity). The overall sequence identity of nucleotides and amino acids between the new P450 enzyme and P450RhF are 76% and 73%, respectively. No doubt, the newly identified protein is also a self-sufficient natural fusion protein similar to P450RhF, named as P450SMO. The enzyme was successfully expressed in E. coli BL21 (DE3), purified by a nickel sepharose column, and characterized. The purified enzyme can catalyze a number of sulfide substrates, yielding the (S)-sulfoxides with high enantiomeric excesses, and no sulfone formed. The absorption spectrum of the purified enzyme showed the heme Soret absorption maxima at 420 nm, which is typical for cytochrome P450 enzymes. The enzyme showed the maximum activity at 30℃. The optimum pH for activity of the purified enzyme was 7.0. P450SMO enzyme activity was obviously inhibited by imidazole.Third, expression conditions of P450 monooxygenase in E. coli were further optimized in shake flasks and fermenter, the optimized conditions in shake flasks were determined as followes:induction temperature 25℃, IPTG 0.5 mM, OD6000.7, induction time 18 h. Under these optimized conditions, the recombinant strain was cultured in a 5 L fermenter.400 nmol/L P450SMO was obtained when glycerol was used as carbon source, the DO was below 10% and the pH was maintained at 7.0. The specific activity of recombinant strain whole cells was enhanced to 1.5 U/g DCW. Intact cells were used to establish an efficient bioconversion system for the generation of sulfoxidation product. With p-chlorothioanisole as a representative substrate, the best productivity of 130 mg/L of desired product was achieved within 12 h with 99% ee.One limitation to the practical application of this P450 enzyme is the requirement for expensive cofactors such as NAD(P)H. Here, we cloned a glucose dehydrogenase (GDH) gene (786 bp) from Bacillus subtilis, which encodes a protein of 261 amino acids. The GDH was successfully expressed in E. coli BL21 (DE3), and the enzyme activity reached to 5 U/ml. With the GDH as NADPH regenerator, a catalytic reaction system of coupled P450SMO and GDH was constructed in in vitro, resulting in effective regeneration of the NADPH. The system was also used to transform sulfide to sulfoxide, and in the case of 1 mM p-chlorothioanisole,90% conversion was achieved after 12 h reaction, and the ee of product exceeded 98%.In terms of co-factors regeneration, the whole cells are often favored because the construction of a cofactor regeneration system is generally easier and less expensive in cells than in vitro. In this study, glucose dehydrogenase (GDH) gene was coexpressed with P450SMO gene by three recombinant E. coli systems, resulting in significant improvement in the substrate conversion. In the presence of glucose and 2 mM initial concentration of p-chlorothioanisole, the higher substrate conversion was obtained (nearly 10-folds) by using the recombinant E. coli BL21 (pET28a-P450-GDH) as biocatalyst. The expression conditions of P450SMO and GDH in E. coli BL21 (pET28a-P450-GDH) were further optimized in shake flasks, and the optimal conditions were determined as followes:induction temperature 25℃, IPTG 0.5 mM, OD6000.6, induction time 16 h. The specifc activity of recombinant strain whole cells was enhanced to 2.2 U/g DCW. In the case of 2 mM p-chlorothioanisole, the substrate conversion reached to 100% after 12 hour reaction, and the ee was above 98%. These results clearly showed that the addition of glucose to the reaction media further promoted sulfide sulfoxidation, suggesting that exogenous glucose is available for GDH in the host cell and actively participates in the catalytic cycle.