| Cytochrome P450 monooxygenase(CYPs)is a superfamily of heme-dependent monooxygenases,the enzymes are commonly found in eukaryotes,archaea,bacteria and viruses,which can carry out chemically difficult biotransformations on a variety of substrates,CYPs catalyze a wide range of chemical reactions including hydroxylation,decarboxylation,epoxidation,reductive dehalogenation,dealkylation,sulfoxidation,and anti-Markovnikov oxidation.CYPs mediated enzymatic hydroxylation of fatty acids present a green alternative to chemical synthesis of hydroxy fatty acids(HFAs),which are high-value oleochemicals with various application in materials industry and medical field.Although many CYPs require the presence of additional reductase proteins for catalytic activity,self-sufficient CYPs have their reductase partner naturally fused into their catalytic domain,leading to a greatly simplified biotransformation process.However,the poor thermal stability and limited regioselectivity are main drawbacks in their application.Therefore,it is of great value to obtain CYPs with excellent performance and new regioselectivity.Two genes encoding for self-sufficient CYP102 family enzymes(bamf2522 and bamf0695)in Bacillus amyloliquefaciens DSM 7 have been cloned through genome mining technology,purified,and subjected to biochemical characterization.Activity of both enzymes with various saturated and unsaturated fatty acid substrates were assessed by using whole cells.In addition,through homology modeling,rational and semi-rational design of Bamf2522 and Bamf0695,a series of mutants with regioselective changes to fatty acids were obtained.The possible reasons for the regioselective changes in fatty acids catalyzed by some mutants were analyzed by molecular docking.Finally,this study initially explored the catalytic ability of Bamf2522 and Bamf0695 on fatty acid mixtures in renewable resources microalgae.The main content and results of this study are as follows:1.In this study,according to the existing bacterial resources in the laboratory,based on genome mining technology,amino acids sequence of P450 BM3(CYP102A1)from Bacillus megaterium was used as the template,two genes bamf2522 and bamf0695 encoding for self-sufficient enzymes in Bacillus amyloliquefaciens DSM 7 was cloned.Based on sequence alignment and phylogenetic tree analysis,it was found that they belong to the CYP102A subfamily.The study on the characterization of these two enzymes indicated that both enzymes could hydroxylate various saturated and unsaturated fatty acids with distinct regioselectivity.Bamf0695 mainly catalyzes hydroxylation at the ω-1,ω-2 and ω-3 positions of most saturated linear fatty acids.In contrast,Bamf2522 exhibits broader products diversity by carrying out hydroxylation at different fatty acids,especially for palmitic acid(C16:0),which exhibits novel regio selectivity by hydroxylating in-chain positions of palmitic acid generating ω-1 toω-7 HFAs,making it the only studied wild-type CYP102 enzyme that hydroxylates ω-6 and ω-7 positions for this substrate.2.Both enzymes were analyzed for their spectroscopy characterization.Upon addition of CO to the reduced enzymes.A soret band from 450 nm can be observed for both enzymes,as expected for heme-containing CYP proteins.This study further characterized these enzymes by determining optimum conditions(temperature and pH)and thermal stability.The results demonstrate that optimal temperature and pH conditions are 30℃ and pH 7.0 for Bamf2522,and 35℃ and pH 7.5 for Bamf0695.Bamf0695 has a high thermostability even at 50℃,which presents an ideal catalyst option for enzymatic bioproduction.3.Based on amino acid sequence alignment,homology modeling,rational and semi-rational design,site-directed was applied to the active site amino acids F89,1266,and A331 in the Bamf0695 substrate binding pocket.After screening by many fatty acids,the results showed that although single mutant F89I or A331Vof Bamf0695 had very little effect on the regioselectivity towards palmitic acid.The distribution of the products is almost same as the wild-type,however,F89I/A331 V mutant of Bamf0695 yields much higher ω-1 hydroxy product than the wild-type enzyme(61%vs.31%),indicating the interaction between two amino acids plays an important role in the regioselectivity of palmitic acid.The site-saturation mutagenesis at A331 was performed,it showed that the A331I mutant exhibited 81.9%of ω-1 product from C 12:0 and 89.6%of ω-1 product from C 16:0,respectively,it demonstrated that the regio selectivity shifts favoring the formation of a single HFA regioisomer were obtained.Besides,many other mutants were able to achieve over 50%regioselectivities with various chain length fatty acids(C 12～C18),making them good candidates for production of pure single HFA isomer.In order to find the possible reasons for the change in fatty acid regioselectivity,the palmitic acid was docked into the homology model of wild-type and mutant enzymes,it is clearly seen in the docking models thatω-1 carbon is much closer to the iron atom in the mutant F89I/A331 V compared to wild-type enzyme(4.1 A vs.6.1 A),suggesting a possible explanation for increased ω-1 hydroxylation.4.Based on the previous work on P450 BM3(CYP102A1)and on the structural analysis of Bamf2522 homology model,we determined hot-spot residues F89,1266 and A331 at the active site as well as at distant locations hot-spot residues S49,F53,N72,M187,V218 and M240 for site-directed mutagenesis,Exploring its influence on fatty acid regioselectivity by performing site-directed mutagenesis at a single site or at multiple sites(in various combinations of the selected residues),the results showed that a single mutant F89I of Bamf2522 extends the product scope of palmitic acid from(ω-1)～(ω-7)to(ω-1)～(ω-9),we obtained Bamf2522 variants that demonstrate greatly increased regioselectivity for in-chain positions(ω-4 to ω-9)of various medium to long chain fatty acids,Remarkably,when a six-residue mutant R41(A331V/F891/N72S/M187T/V218A/M240L)was reacted with palmitic acid,84%of total product content was the sum of ω-7,ω-8 and ω-9 HFA products,that can achieve product profiles highly enriched with in-chain HFAs,this is the highest in-chain selectivity observed to date with a self-sufficient CYP.Similarly,in order to explore the reasons for the change in fatty acid regioselectivity,the substrate palmitic acid was docked into the homology model of wild-type and mutant enzymes,it is clearly show that ω-8 and ω-9 carbons are in closer proximity to the iron atom of the heme for Bamf2522 F89I than for wild-type Bamf2522,suggesting a possible explanation for regioselectivity of palmitic acid.5.In order to explore the catalytic performance of CYPs on fatty acids in renewable resources,so as to expand the source of CYPs fatty acid substrates,this study tested Bamf2522 and Bamf0695 for their ability to convert microalgae fatty acids into HFAs.Enzymatic reactions show that Bamf0695 can convert microalgae fatty acids extracts into high-value HFAs,However,Bamf2522 did not show the enzyme activity towards microalgae fatty acids extracts.In short,this study demonstrates the potential of recently discovered CYPs Bamf2522,Bamf0695 and their variants for green and sustainable production of a variety of high-value HFAs. |
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