Metabolic Balancing On The Microbial Lipids Production By Corynebacterium Glutamicum

Due to its short life cycle,continuous production,broad natural substrates and lipid composition can be easily modified by genetic engineering,microbial lipid becomes the important raw source of the third-generation biodiesel.A metabolic engineered Corynebacterium glutamicum CT14 has been construed to produce triacylglycerols(TAG)in previous work,based on which further studies have been applied to regulate the TAG production in this research.First,enhance the supplement of the precursor 3-phosphate glycerol.CT14 enhanced the supplement of one of the precursors,fatty acid,which led to the waste of the extracellular fatty acid.In this research,the 3-phosphate glycerol path has been enhanced to balance the supplement of both two precursors and to eliminate the extracellular fatty acids.Optimal combination of gene regulation strategy has been achieved through the analysis of the effects on extracellular fatty acids and TAGs production by single gene engineering.Overexpression of gpsA plus knocking out of glpD led to an achievement of zero extracellular free fatty acid,1.8 g/L total fatty acid and an increase of intracellular triacylglycerols by 5-10%.Second,improve the step-limited path of GPAT.Documents show that there is no obvious GPAT gene in the genome of C.glutamicum.The functional gene of GPAT and its effects on TAG production in C.glutamicum have been identified and studied in this research.Aided by bioinformatics technology,several candidate genes(cg0896,cg2095,cg2771,cg2096 and cg3254)encoding GPAT in C.glutamicum ATCC 13032 have been predicted out.Cg2777,which is a putative membrane protein gene,has been selected for subsequent analysis and verification.Knockout of cg2777 gene inhibited the growth of C.glutamicum and TAG production.Over expression of cg2777 had no significant improvement of fatty acids,which implied that cg2777 performs part of GPAT function with relative low activity.The heterologous expression of plsBl from Rhodococcus opacus PD630 reduced the amount of extracellular free fatty acids,the total fatty acids was 1.44 g/L and the fatty acids content was 13.2%,which increased by 18.9%and 32%compared with CT14.Final,construction of resistance-free recombinant strain.The TAG production plasmid,pZ8-TAG4,is too huge for staying stably during the fermentation.A homologous integration vector pk18mobsacb-TAG4 carrying all the TAG synthesis genes(atfl and atf2 for encoding diacylglycerol acyltransferase DGAT,pgpB for encoding phosphatidic acid phosphatase PAP,and tadA for the accumulation of TAG)has been successfully constructed,which is designed to be integrated into the chromosome of C.glutamicum via transposon ISCg3a.The recombinant is about to be subjected to further verifications such as sequencing and phenotypic test.Stable and resistance-free gene expression in C.glutamicum would lay a solid foundation for the further metabolic engineering of C.glutamicum for higher capacity of TAG production.