Construction Of The Gene Deletion And Expression System In Corynebacterium Glutamicum And Their Application

Corynebacteria are gram-positive bacterias with moderately high G+C content, andhave been widely used in the industrial production of amino acids and vitamins.Corynebacterium glutamicum is one of the most widely used Corynebacteria in industrialfermentation.Promoted by genomic sequencing of C. glutamicum strains ATCC13032and ATCC14067, metabolic engineering in C. glutamicum has been used to construct high amino acidproducing strains. However, expression vectors for massive overexpressions and effectivegene deletion systems for chromosomal manipulations in C. glutamicum are still lacking. Inthis work, we have developed a constitutive expression system to facilitate both lab researchand industrialized fermentation, and a gene deletion system generally applied in C.glutamicum strains. Both systems have been used in engineering C. glutamicum ATCC14067’s metabolics towards highly efficient L-valine accumulation. The main results arelisted below:(1) Constitutive E. coli-C. glutamicum shuttle vector pJYW-4and pJYW-5wereconstructed. Both of the two vectors harbor two selective markers: one is the kanamycin-resistant gene kan; the other marker is based on plasmid-borne auxotrophic complementarysystem. Both vectors harbour a large MCS containing11restriction sites. pJYW-4carries amoderate tac promoter, while pJYW-5carries a stronger tacM promoter; the differencebetween the intensity of the two promoters facilitates expressions of different genes. Thereplicon of the vectors differs from that of pDXW-8, providing an alternative choice underspecific circumstances.(2) Construction of a new gene deletion system that can be applied in multiple-gene-deletions in C. glutamicum. The system is based on the homologous recombination andCre/lox site-specific recombination. It consists of three plasmids, namely pDTW109,pDTW102and pDTW202. pDTW109is a temperature-sensitive vector which harbours a catunder the tacM promoter, a cre gene under the tac promoter. pDTW201and pDTW202carries a loxp-flanked or mutant lox-flanked kan, respectively.(3) Chromosomal manipulations in ATCC14067have been done with the new deletionsystem, resulting in a series of deficient strains. Aimed at concentration of the strain’smetabolism towards L-valine synthesis, aceE, ilvA, leuA, alr were deleted in a sequentialway, either to focus metabolic flux or to facilitate subsequent overexpression. The final strainwas named YTW-104.(4) pJYW-4-ilvBNCE-brnFE1was built to intensify the in vivo carbon flux on L-valinesynthesis and its extracellular transportation. The final strain, YTW-104/pJYW-4-ilvBNCE-brnFE1, was tested for its L-valine production. pJYW-4-ilvBNCE-brnFE1can exist in cellswithout the need of antibiotics. YTW-104/pJYW-4-ilvBNCE-brnFE1could produce7.5mM g-1L-valine production. which is200%increase, compared with that of the controlstrain YTW-104and a finial production of12.8mM g-1L-valine in complex media.