Study On Cloning Of Key Enzyme Molecules And Construction Of Engineering Strain For Biosynthesis Of 10-HDA

10-hydroxy-2-decenoic acid?10-HDA?is the derivation of medium-chain fatty acids with two special functional groups,double bond and terminal hydroxyl group.Scientific research has shown that 10-HDA has many important physiological functions,such as antibacterial,immunomodulatory,neuroregulatory,anticancer,antineoplastic,anti-inflammatory,and so on.It can also be used as a health product,and can be used as a synergist to increase the effect of the cosmetic.Conventionally,the ways of obtaining 10-HDA include direct extraction and chemical synthesis at present.Direct extraction method is difficult to meet the market demand.Chemical synthesis method has inevitable shortcomings.In addition,chemical processes which steps are very complicated and the chemical reagent is toxic to a certain extent.So it has great scientific significance to search a new method.The analysis of its structure shows that hydroxyl group and double bond are two special groups in 10-HDA.So,clarifying the formation process of these two groups is important to the synthesis of10-HDA.In this paper,the idea of constructing a pathway for 10-HDA biosynthesis is presented.Decanoic acid was used as substrate to produce 10-hydroxy decanoic acid.This?functionalization can be achieved through the use of the P450 enzyme.Then through?-oxidation cycle requires the use of two corer BOX enzymes along with thioesterase able to convert 10-hydroxy decanoic acid-CoA to the corresponding production.The 10-hydroxy decanoic acid is catalyzed by thiokinase?acyl-CoA dehydrogenase and two hydrogen atoms are removed at the C?and C?,a trans double bond was formed.Finally,coenzyme A was removed under the catalyzed of thioesterase,and the 10-HDA was formed.?1?In this study,four cytochrome P450 enzymes were cloned:Cytochrome P450enzyme P450_BM3 from Bacillus megaterium,CYP102A1 from Bacillus subtilis,Cytochrome P450 enzyme CYP52B1 and its reductase from yeast,CYP153A from Marinobacter aquaeloei and the reductase of P450_BM3 from Bacillus megaterium.And four engineering bacteria were constructed to express cytochrome P450 enzymes:E.coli(pET-21b-CYP153A-P450_BM3 CPR)?E.coli?pET-28a-CYP102A1??E.coli(pET-28a-P450_BM3)?E.coli?pET-28a-CYP52B1-CPR?.?2?The engineering strain E.coli?pET-SUMO-ydiI?was constructed by using a vector which was helpful to the expression of small protein.?3?The bioactivity of cytochrome P450 was tested by shaking flask fermentation.Resting cell was used in the study.Finally,only E.coli(pET-21b-CYP153A-P450_BM3CPR)had the ability of decanoic acid terminal hydroxylation.The results of shaking flask fermentation of thioesterase engineering bacteria showed that the engineering bacteria could convert decane to trans-2-decenoic acid,and could convert10-hydroxydecanoic acid to 10-hydroxy-2-decanoic acid.In this experiment,two biocatalytic elements which can form the key functional groups of 10-HDA,were obtained by screening and cloning.And their catalytic characteristics were studied.This is of great theoretical significance to the further construction of 10-HDA synthesis pathway using synthetic biology,and provides an important direction for the industrial production of 10-HDA in the future.