Breeding Of L-threonine Producing Corynebacterium Glutamicum And Construction Of Engineering Bacteria

Since the isolation of non-pathogenic Corynebacterium glutamicum producing L-glutamic acid from soil for the first time,its excellent amino acid biosynthesis ability and biological safety that make it become one of the most important platform microorganisms in amino acid industry production.In this study,we isolated Corynebacterium glutamicum from soil that it can secrete L-threonine from soil,and make it as an basal cells.Combining mutagenesis breeding and metabolic engineering to structure a engineering strain with high yield of L-threonine.The main contents of the study are as follows:(1)According to the biosynthesis of threonine and the growth characteristics of Corynebacterium glutamicum,we used a plate that contained low concentration of L-threonine structure analogue to filter high produce L-threonine bacterial directional.By paper chromatography and high throughput screening method to preliminary screening,on the basis of amino acids corresponding to Rf value and half-quantity of color spots.The wild strains were obtained by shaking flask re-screening with the ability to synthesize and accumulate L-threonine.One of strains named MD4027 can accumulation of L-threonine 0.23 g/L.Throgh morphological characteristics and 16S rRNA gene molecular identification,MD4027 was closest genetic relationship with Corynebacterium glutamicum,so we successfully obtained a new L-threonine-producing strain that belong to Corynebacterium glutamicum initially.(2)Using C.glutmicum MD4027 as the research object,we optimized the processing conditions of ARTP.Then according Penicillium method and spot planting control method to screened the nutrient deficiency type.Through 96 microporous plate high throughput screening and shaking flask double screening,one stable L-threonine-producing strain with double-defect of methionine and isoleucine(Met~-/Ile~-)that named C.glutmicum MT-04 was obtained.By shake flask,it production of L-threonine up to 2.65 g/L,which was 10.5 times higher than that of C.glutmicum MD4027.(3)Weakening the intracellular degradation of L-threonine.The yield of threonine can be increased by using a no-mark knockout vector pK18mobsacB and a modified gene glyR,the strain MT-4-(35)glyR was constructed.it was named C.glutmicum T11 whose shake flask L-threonine production reached 3.16 g/L,and it was 19.2%higher than the control strain C.glutmicum MT-4.(4)For further modified the strain C.glutmicum T11.Using strong promoter Ptac as the regulatory element to overexpression the key enzyme genes lysC,asdA,hom,thrB in the L-threonine pathway were efficiently or expressing in tandem with one or more the key genes,and the carbon flux of L-threonine synthesis was further increased.Analyzing the function of the key enzyme gene lysC and site-directing mutagenesis(Ala 279 Thr)can performed to relieve the feedback inhibition of L-threonine.The highest level of L-threonine production was found in the C.glutmicum T11/pZ-lys~rasd strain(4.8 g/L),which was 51.9%higher than that of strain C.glutmicum T11.(5)On the basis of the original medium,fermentation conditions of C.glutmicum T11/pZ-lys~rasd engineering strain and optimization experiment of shake flask fermentation medium.We obtained its optimum L-threonine fermentation medium formula,the result showed that the acid value of the fermentation medium reached 5.7g/L and the original formulation increased by 18.8%.On the basis of the optimization of the medium formula,we batch fermentation of C.glutmicum T11/pZ-lys~rasd by feeding on 30 L Tank,through existing fermentation technology,the concentration of L-threonine in the fermentation broth of the modified strain C.glutmicum T11/pZ-lys~rasd can reach up to 54.6 g/L,it was 32.5%more than the original formula.