Rational design for enzyme engineering of CYP153 family and its application to production of ω-hydroxy palmitic acid

Abstract

In this study, the ω–specific hydroxylation of fatty acids using cytochrome P450 monooxygenase (CYPs) was investigated. Among bacterial CYPs in CYP153 family which reported as fatty acid ω-hydroxylase, CYP153As from Marinobacter aquaeolei VT8 (CYP153A33), Alcanivorax borkumensis SK2 (CYP153A13) and Gordonia alkanivorans (CYP153A35) were selected, and compared their specific activities and product yields of ω-hydroxy palmitic acid based on whole-cell reactions toward palmitic acid. Using CamAB as redox partner, CYP153A35 and CYP153A13 showed the highest product yields of ω-hydroxy palmitic acid by whole-cell and in vitro reactions, respectively. To investigate electron transfer system for CYP153A35, artificial self-sufficient CYP153A35-BMR was constructed by fusing it to the reductase domain of CYP102A1 (i.e. BM3) from Bacillus megaterium, and its catalytic activity was compared with CYP153A35 and CamAB system. Unlike the expectations, the system with CamAB resulted 1.5 fold higher yield of ω-hydroxy palmitic acid than that using A35-BMR in whole-cell reaction, whereas the electron coupling efficiency of CYP153A35-BM3 reductase was 4 times higher than that of CYP153A35 and CamAB system. Furthermore, various CamAB expression systems according to gene arrangements of the three proteins and promoter strength in their gene expression were compared in terms of product yields and productivities. Tricistronic expression of the three proteins in the order of camB, cyp153A35 and camA, i.e. A35-AB2 construct, showed the highest product yield from 5 mM of palmitic acid within 9 h in batch reaction system owing to the concentration of CamB, which is the rate limiting factor for the activity of CYP153A35. However, in fed-batch reaction system, A35-AB1 construct, which expressed the three proteins individually using three T7 promoters, resulted the highest product yield of 17.0 mM (4.6 g/L) of ω-hydroxy palmitic acid from 20 mM (5.1 g/L) of palmitic acid in 30 h. For the improvement of hydroxylation activity of CYP153A35, the structures of CYP153A35 were predicted by homology modelling, and the major cavities and the amino acid interacting with the fatty acid were revealed by CAVER 3.0. In order to screen mutants, a powerful high-throughput screening assay was developed, which used Purpald to sense formaldehyde produced as a by-product during O-dealkylation reaction. Saturation mutagenesis on 19 amino acids was performed and D131S mutant showing 281.4 min-1mM-1 of catalytic constant which was more than 17 times higher value than that of wild-type (16.5 min-1mM-1). To optimize the linker sequence between fatty acid ω-hydroxylase (CYP153A33) and reductase domain of CYP102A1, repeated flexible or rigid sequence are designed randomly and screened. The best mutant, EAAAK-(GGGGS)3-EAAAK, showed the 50% higher specific activity than native BM3 linker, although poor expression level in E.coli.