Construction Of Engineered Strains For Biosynthesis Of 1,2,4-butanetriol

The 1,2,4-Butanetriol is a valuable energy material precursors of 1,2,4-trinitrate,which is widely used in numbers of fields including polymer materials,agriculture,tobacco,military,and medicine.1,2,4-trinitrate alternative nitroglycerin becomes an ideal material due to its excellent thermal stability and many other advantages.Conventional chemical synthesis method is featured by high cost,harsh reaction conditions and many byproducts.Thus,it has become a hot research field of useing lignocellulosic raw materials to produce 1,2,4-Butanetriol resource development to produce 1,2,4-butanetriol by biosynthetic methods.The paper was amptty to obtain engineering Escherichia coli which can produce 1,2,4-butanetriol from xylose,which made full use of renewable resource lignocellulose and obtained nunmerous;engineering strajins producing butanetriol.It provided theoretical and practical significance.In this paper,the gene of the benzoylformate decarboxylase from P.putida CICC21884 and xylose dehydrogenase from B.subtilis168 were amplified by PCR,The results showed that the length of mdlC was 1599 bp,the length of gdh was 786bp.And the sequence was submitted into NCBI;and transfer the pRSFDuet-1 vector connected with the two target genes into the JM109(DE3).Preliminary fermentation by shake,the production of butanetriol is about 0.42g/L by HPLC test.As a result,this paper successfully constructed recombinant E.coli containing the activety of benzoylformate decarboxylase xylose dehydrogenase,which can biosynthesis butanetriol.The benzoylformate decarboxylase and xylose dehydrogenase activity was tested a high activities.The enzymes induced condition was optimized,and the best conditions 0.5mmol/L IPTG,8h and 37? culturing was achieved.SDS-PAGE clearly showed that the mdlC and gdh recombinants protein products band 55kD and 34kD respectively.The paper first transform glucose dehydrogenase and benzoylformate decarboxylase recombinate plasmids to E.coli JM109(DE3).Through the introduction of two gene,a 1,2,4-butanetriol producing E.coli was constructed.The native pathway for D-xylose catabolism in E.coliJM109(DE3)was also blocked by disrupting two genes(xylAB)both encoding xylose isomerase and xylulose kinase genes by means of Red recombinase.The mutant strains of xylAB deletion were constructed.After transferring the engneering expressing plasmid pSLQMG into the mutant strains.Through the HPLC test,1,2,4-butanetriol production of mutant strains was 0.58g/L,38%higher than that of the original strain which was 0.42g/L.These results suggest that the engineered E.coli has a promising application for the industrial-scale production of 1,2,4-butanetriol.This research has been a great significance to the construction a high-yield gene engineering bacteria producing 1,2,4-butanetriol,and provided a new successful example to transform xylose metabolic ways of Escherichia coli and laid a theoretical and practical foundation for the industrial production of 1,2,4-butanetriol in a' biblogical way.