The Cloning Of Key Genes And The Recombinant Construction Involved In The Biosynthesis Of D-1,2,4-butanetriol

D-1,2,4-butanetriol is a kind of four carbon polyol, and it has many important applicationvalues, particularly in the synthesis of drug and solid propellant. D-1,2,4-butanetriol belongsto non-natural products, which couldn’t be detected in any microorangnisms. In this paper,according to the principle of synthetic biology, the recombinant E. coli W3110wasconstructed to convert D-xylose to D-1,2,4-butanetriol through four catalysis steps.Toestablish the suitable conditions for the synthesis of D-1,2,4-butanetriol, the fermentationconditions were optimized preliminarily.In order to obtain D-xylose dehydrogenase gene xylB and benzolyformate decarboxylasegene mdlC, Caulobacter sp S1304and Pseudomonas putida S1211, confirmed byphysiological-biochemical indentification and molecular indentification, were isolated in themedium with D-xylose and mandelic acid as the sole carbon source. According to the knownsequences, xylB gene and mdlC gene were acquired by PCR from strain S1304and S1211respectively, and the bioinformatic analysis was as follows: in the comparison of amino acidssequence, xylB gene achieved96%similarity with Caulobacter segnis ATCC217560D-xylose dehydrogenase, and mdlC gene achived99%similarity with Pseudomonas putidaS12benzolyformate decarboxylase.To testify the method for the biosynthetic D-1,2,4-butanetriol, a segmented systemconsisted of oxidation, dehydration, decarboxylation and reduction, was in need to build. Therecombinant E. coli W3110(pEtac-xylB) and E. coli W3110(pEtac-mdlC) were acquired bythe way of molecular biology. The results were as follows: xylB gene and mdlC gene gainedefficient expression in E. coli W3110, and then the D-xylonic acid was detected in thesupernant broth, when E. coli W3110(pEtac-xylB) was cultivated for48h in LB mediumsupplemented with D-xylose, while D-1,2,4-butanetriol was also found in the supernantderived from E. coli W3110(pEtac-mdlC) fermentation broth using D-xylonic acid as rawmaterial. D-1,2,4-butanetriol was confirmed by GC-MS method, and it indicated the validityof designed pathway for the D-1,2,4-butanetriol production from D-xylose.The fragment tac-xylB was amplified from the plasmid pEtac-xylB and then ligated intopEtac-mdlC to produce the plasmid pEtac-mdlC-tac-xylB.Next, the recombinant E. coliW3110(pEtac-mdlC-tac-xylB) was constructed. The recombinant had the ability to expressefficiently in E. coli W3110, and its fermentation property was as follows: D-1,2,4-butanetriolwas produced when E. coli W3110(pEtac-mdlC-tac-xylB) grew in the LB medium containingD-xylose, so it indicaed the achievement of one-step fermentation method for the productonof D-1,2,4-butanetriol using D-xylose as the start material. The optium fermentation conditionwas established as follows: LB medium containing8g L-1peptone, induction temperature37℃, IPTG concentration1.0mmol L-1, D-xylose concentration30g L-1. Therefore, underthese conditions, the titre of D-1,2,4-butanetriol could reach945mg L-1at48h.It provided akind of efficient approach for biosynthetic D-1,2,4-butanetriol.