Screening Of Key Catalytic Enzymes For Biosynthesis Of 10-HDA And Rational Design Of P450 Enzyme

10-Hydroxy-2-Decenoic acid(10-HDA)is an important functional component of royal jelly,It can not only enhance the body's immunity,but also has significant effects in anti-tumor,anti radiation,antibacterial and other aspects.It has broad development potential and large market demand.At present,10-HDA is only found in royal jelly,the content is only 1.4-2.4 g/L,and the extraction cost is high,so the supply is difficult to meet the market demand.Although a variety of chemical synthesis methods of 10-HDA have been designed,these methods are difficult to achieve large-scale production due to their long process route and harsh synthesis conditions.With the continuous improvement of people's living standards,the demand for functional natural health care products is growing rapidly,Biosynthesis of 10-HDA will have a huge market prospect.In this study,we used cheap decanoic acid as the substrate,based on the transformation of E.coli?-oxidation pathway by our previous research group,that is,knockout the genes(fad B?fad J?fad R)that affect the synthesis of trans-2-decenoic acid,optimized the selection of trans-2-decenoic acid synthesis elements:acyl CoA synthase(FadD),acyl CoA dehydrogenase(fadE),acyl COA thioesterase(YdiI),and optimized the synthesis of P450 enzymes(CYP153A33-CPR_BM3)was designed rationally to improve the conversion rate of trans-2-decenoic acid terminal hydroxylation.For the first time,a two-step biotransformation pathway of trans-2-decenoic acid synthesis from decanoic acid,trans-2-decenoic acid terminal hydroxylation and finally10-HDA synthesis was constructed.The first step of this pathway is to synthesize trans-2-decenoic acid from decanoic acid.When(BL21(DE3)-?fad B-?fad J-?fad R/p ET28a-sumo-Ecydi I/p ETDuet-1-Ecfad D-EcfadE)was used as substrate,the yield of trans-2-decenoic acid was 37 mg/L.We used mamacs from Mycobacterium avium instead of Ecfad D and Pp FadE from Pseudomonas putida KT2440 instead of EcfadE,At the same time,six homologous proteins of acyl CoA thioesterase YdiI from different sources were screened out.Finally,Ct YdiI from Cronobacter turicensis and Se YdiI from Salmonella enterica were selected to replace acyl CoA thioesterase Ec YdiI from E.coli.Then,the optimized key components were combined to construct the engineering strain(BL21(DE3)-?fad B-?fad J-?fad R/p ET28a-sumo-Ctydi I/p CDFDuet-1-Ma MACS-PpfadE).Through the whole cell catalytic experiment,the engineering strain could catalyze 0.5 g/L decanoic acid to obtain 0.31 g/L trans-2-decenoic acid,and the conversion rate reached 62.3%,which was 8.67 times of the engineering strain before optimization.The second step of this synthetic pathway is to synthesize 10-Hydroxy-2-Decenoic acid(10-HDA)from trans-2-decenoic acid catalyzed by cytochrome P450 enzyme(CYP153A33-CPR_BM3).Due to the low trans-2-decenoic acid conversion rate of wild-type CYP153A33-CPR_BM3,which is only 23%,in this project,we used the method of rational protein design to study the key amino acids(Gln131?Ile132?Ile131?Val141?Glu142?Met143?Ile145?Ala146?Met228?Ala229?Leu303?Val306?Gly307?Thr311?Leu354?Met357?Cys418 and Phe455)interacting with the substrate by molecular docking simulation.The binding free energy of the key amino acids(Gln129?Ile131?Val141?Met143?Met228 and Ala229)with the substrate was calculated.Based on this,the site directed mutagenesis of the key amino acids(Iie131?Met228 and Ala229)was carried out.The mutant(BL21(DE3)-p ET21b-CYP153A33/M228L-CPR_BM3)constructed by CYP153A33/M228L gene catalyzed 500 mg/L trans-2-decenoic acid to439 mg/L 10-Hydroxy-2-Decenoic acid,with a conversion rate of 87%.Compared with wild-type(BL21(DE3)-p ET21b-CYP153A33-CPR_BM3),the catalytic efficiency was increased by 3.82 times.The K_D values of CYP153A33?CYP153A33/M228L and trans-2-decenoic acid were 3.583×10^-6 M and 3.754×10^-7 M respectively.The K_D values of CYP153A33/M228L and trans-2-decenoic acid were one order of magnitude smaller than those of CYP153A33 and trans-2-decenoic acid.Through the above optimization and modification of the key components in the synthesis route,0.5 g/L decanoic acid can produce 0.275 g/L 10-Hydroxy-2-Decenoic acid through two-step catalysis,and the conversion rate is 55%.This paper provides a two-step whole cell catalytic method for the biosynthesis of decanoic acid and 10-HDA using decanoic acid as substrate.The results of this study provide new ideas and approaches for the biosynthesis of 10-HDA and other medium chain hydroxy fatty acids and unsaturated fatty acids.