Breeding L-lysine Hyper Producer By Corynebacterium Glutamicum Based On Metabolic Engineering

Corynebacterium glutamicum, a non-pathogenic Corynebacterium, has been appliedworldwide for producing amino acids, such as L-lysine. L-lysine is one of the eight essentialamino acids for humans and animals, and has been used in many fields. In this dissertation,an L-lysine high-producing strain had been constructed by genetic modification based onmetabolic engineering. Moreover, in order to overcome the defects of plasmid-mediated geneamplification, a method for the replacement of a given gene by a target gene, leaving nogenetic markers, had been developed. Furthermore, the L-lysine production of targeted-strainwas further improved with the helps of optimizing medium and fermentation parameters,which can provide a theoretical basis and technical support for producing L-lysine in industrywith genetically modified bacteria. The main results are as follows:1. A method for genetically modifying C. glutamicum genome has been developed. Themethod is based on insertional inactivation and double-crossover homologous recombination,and can be used for the simultaneous replacement of a given gene by a target gene, leaving nogenetic markers. The integrated cassettes in host are successfully expressed and maintained asstable chromosomal insertions in C. glutamicum. With this method, the L-lysine-producingstrain C. glutamicum lysCfbrwere genetically modified, and the targeted-transformant C.glutamicum pck::lysCfbr alaT::fbp avtA::ddh showed better L-lysine producing capacitythan original strain (37.3vs.0mmol·L-1). Experiments showed that this method can be usedto construct amino acid high-producing strains with unmarked gene amplification andsimultaneous deletion in genome.2. The L-lysine production is improved because of improving utilization of molasses byheterologus expression of E. coli fbp and C. acetobutylicum scrK. PPP is beneficial toL-lysine production, and FBPase (fbp gene product) and ScrK (scrK gene product) haveimportant roles in regenerating glucose-6-phosphate in the PPP. C. glutamicum lysCfbrshowed bad cell growth, sugar consumption and L-lysine production (only25.0±0.85mmol·L-1) during growth on molasses. Heterologous expression of fbp or scrK not onlyincreased the utilization of molasses, but also improved cell growth and L-lysine productionduring growth on molasses. The targeted-transformant C. glutamicum lysCfbr/pDXW-8-fbp-scrK accumulated47.1±2.36mmol·L-1L-lysine after48h fermentation in shake flask,88.4%higher than original strain. Moreover, the heterologous FBPase in C. glutamicum wasinsensitive to catabolites.3. The by-products accumulation has been decreased, but the supplies of precursor andNADPH have beed improved via gene site-dirested mutation and gene knock-out. The detailresults of this experiment were:①pyruvate accumulation had been increased because of aceE deletion;②L-alanine biosynthesis had been blocked because of alaT and avtA deletion;③lactate or succinate accumulation had been decreased because of ldhA or mdh deletion;④L-valine accumulation had been decreased because of ilvNC-Tdeletion;⑤L-methionine andL-threonine accumulation had been decreased because of hom mutation. Heterologousexpression of E. coli fbp or replacement of NAD-dependent GADPH with C. acetobutylicumNADP-dependent GADPH not only released the inhibition of GADPH by NADH, but alsoimproved glucose-uptakeing rate and the supply of NADPH. The targeted-transformant C.glutamicum Lys5-3accumulated59.1±5.73mmol·L-1L-lysine and3.5±0.37mmol·L-1L-valine after48h fermentation in shake flask.(4) L-lysine high-producing strain C. glutamicum Lys5-10has been constructed viaoverexpression of genes (i.e., lysC, asd, dapA, dapB, ddh and lysA) in L-lysine biosyntheticpathway. In fed-batch fermentation, Lys5-10began to produce L-lysine at phase of glucosefeeding and continuously increased to a final titer of853±27.8mmol·L-1, and the conversionof glucose arrived to47.2%after48h. In addition, the by-products (i.e., L-threonine,L-methionine and L-valine) were significantly decreased, and L-alanine, lactate and acetatesynthesis were not detected.(5) A sequential optimization strategy was used to enhance the L-lysine production of C.glutamicum Lys5-10. The medium components influenced significantly L-lysine productionwere determined by Plackett-Burman design, which are (NH4)2SO4, NaAc and L-alanine. Theoptimal value of medium was determined by RSM based on the CCD, which is (g·L-1):glucose100, beet molasses20mL, corn steep liquor30mL,(NH4)2SO447.3, NaAc31.7,urea5, L-alanine1.34, KH2PO42, MgSO4·7H2O1.35, FeSO4·7H2O0.02, MnSO4·H2O0.04,glycine betaine0.05, nicotinamide0.008, biotin0.001, thiamine·HCl0.0006. Moreover, theoptimal value of fermentation parameters were determined, which are pH7.0, inoculum size8%, agitation speed640r·min-1, temperature34°C and ventilatory capacity2.69L·min-1.Under this optimized condition in the fed-batch culture, the L-lysine with the concentration of976±70.1mmol·L-1was obtained after48h, and the conversion of glucose arrived to56.8%.