The Optimization Of Metabolic System And Fermentation Process For 1,2,4-butanetriol Biosynthesis

D-1,2,4-butanetriol is an important fine chemical.It can be used in military,medicine,polymer materials and other fields.At present,many domestic and foreign laboratories have preliminarily constructed a biosynthetic route from D-xylose to D-1,2,4-butanetriol,but both the yield of the target product and the substrate conversion rate are low.And no scholars have carried out small-scale fermentation experiments on D-1,2,4-butanetriol.This study has carried on the thorough research aim at the problems above.First of all,we established a heat map of 45 efflux pumps in E.coli and 13 kinds of alcohols.Through this map,we can directly select the correct efflux pumps that can transport the 13 kinds of alcohols including D-1,2,4-butanetriol.This study screened two D-1,2,4-butanetriol-related transporters Mdt H and Yde A.The overexpression of them effectively increased the tolerance of the strain to D-1,2,4-butanetriol and the yield of engineering bacteria that can biosynthetic D-1,2,4-butanetriol.In order to optimize the gene expression level,IPTG inducible promoter was identified as the optimal promoter for the biosynthesis of butanetriol.Compared with two constitutive promoters with different expression intensities,IPTG inducible promoter showed better induction effect.The yield of butanetriol was significantly increased after increasing the expression level of mdl C which is an important gene in the butanetriol synthesis pathway than the control strain.The mdl C gene and the efflux pump gene mdt H were overexpressed in low copy number plasmid p BBR1 MCS to construct the optimal synthesis of butanetriol MJ138K-10,whose butanetriol yield was 4.8 g/L.In shake flask fermentation,the optimal initial glucose concentration was determined to be 5 g/L and the best fermentation liquid volume is 50 ml.For small-scale fermentation experiments,the D-1,2,4-butanetriol biosynthesis yield has been great improved increased to 18.8 g/L by the fed-batch cultivation and the rational controlling the parameters of fermentation process such as p H and dissolved oxygen.