Construction Of Vector Systems For Brevibacterium Flavum And Its L-valine Production Metabolic Engineering

Since C. glutamicum was first isolated to produce L-glutamate, three major representatives of the non-pathogenic soil corynebacteria, Corynebacterium glutamicum, Brevibacterium flavum and Brevibacterium lactofermentum, have been widely used for industrial production of amino acids. At present, metabolic engineering breeding has become main strategy for strain development in corynebacteria. Vector systems are the basis of conynebacteria molecular biology and metabolic engineering breeding study. Due to their shortcomings, most currently available vectors are not suitable for conynebacteria metabolic engineering. In this dissertation, a few new B. flavum vector systems have been constructed, and their application in B. flavum was studied. The main results are listed below:(1) A novel induced E. coli–B. flavum shuttle expression vector pDXW-8 has been constructed. The vector pDXW-8 habors oriE for replication in E. coli, dso and sso for replication in corynebacteria, an ampicilin resistance gene amp and a kanamycin resistance gene kan, a tac promoter, a lacIPF104 segment and a large MCS sequence. In comparison to the available expression vectors used in corynebacteria, the major advantage of pDXW-8 is that it contains a large MCS and a lacIPF104 segment. The large MCS containing up to 11 single-cutting restriction enzyme sites is very convenient for cloning multiple genes in a metabolic pathway or a DNA fragment harboring multiple restriction enzyme sites. The lacIPF104 fragment can stringently control the gene expression in corynebacteria, therefore pDXW-8 can be used to clone heterogenous genes encoding proteins poisonous to host cells. The applicability of pDXW-8 was confirmed by the expression of the vhb gene in E. coli, B. flavum and C. glutamicum. The vector pDXW-8 will be very useful for research on metabolic engineering in corynebacteria.(2) A novel E. coli-B. flavum shuttle promoter-probe vector pDXW-11 has been constructed. The vector pDXW-11 habors oriE for replication in E. coli, dso and sso for replication in corynebacteria, and kan used as selected marker. The region involved in promoter-probe has the following characteristics: rrnBT1T2 terminator closely located in the upstream of MCS, which prevents transcriptional readthrough from potential promoters; the MCS consisting of seven unique restriction sites is located in the upstream of the cat reporter gene; the region between MCS and the cat gene contains three tandem translational stop codons, preventing potential translational readthrough from the inserted fragment. Six promoters were evaluated by pDXW-11 in B. flavum. According to their activities, the six promoters can be listed in order from the strongest to the weakest as the following: tac-M, Pkan, tac-M1, tac, PF104, and PilvA. These promoters, especially tac-M, would be very useful for research on metabolic engineering in B. flavum.(3) A novel constitutive E. coli-B. flavum shuttle vector pDXW-10 has been constructed. The vector pDXW-10 harbors an origin oriE for replicating in E. coli, the dso and sso origins for replicating in B. flavum, selected marker kan, a large MCS, and a tac-M promoter. In comparison to the available expression vectors used in corynebacteria, the shuttle vector pDXW-10 has the following advantages. The first is the large MCS consisting of 10 single-cutting restriction enzyme sites, which is very convenient for cloning big DNA fragments. The second is the tac-M promoter. Under the control of tac-M promoter, the inserted genes can be moderately expressed in B. flavum, and moderate enzyme quantity is suitable for cell metabolites production. The third is that the vector performs constitutive gene expression in B. flavum, which make the engineered strains more convenient for large-scale industrial production. Finally, gene expression in E.coli needs to be induced when using pDXW-10, which would benefit the engineering of B. flavum genes that are poisonous to E. coli. The applicability of pDXW-10 was confirmed by subcloning the cat gene into the vector and measuring the CAT protein production in E. coli and B. flavum. High-level CAT production in E. coli and moderate-level CAT production in B. flavum were observed. The vector pDXW-10 would be an ideal vector used for research on metabolic engineering in B. flavum.(4) A novel intergrative vector pDXW-3 used for unmarkless gene knockout and replacement in B. flavum has been constructed. We found that transformation efficiency of the intergrative vector pK18mobsacB is very low in B. flavum ATCC14067. pDXW-3 can be high-efficiently transformed to B. flavum ATCC14067, and uses kan and the sacB gene using lac-M promoter as selection markers, which make it a good intergrative vector used for unmarkless gene knockout and replacement in B. flavum.(5) A genetic engineering Brevibacterium flavum strain,which can over-produce L-valine, has been constructed. Gene ilvBN, encoding Acetyl-carboxylic acid synthase (AHAS) that is the rate-limiting enzyme of L-valine biosynthesis, was amplified by PCR from Corynebacterium glutamicum ATCC13032, Anti-feedback inhibition form of ilvBN, ilvBNr was obtained by site-directed mutagenesis. The ilvBNrgene was inserted in E. coli-Brevibacterium flavum shuttle expression vector pDXW-10, resulting the recombinant plasmid pDXW-10-ilvBNr. pDXW-10-ilvBNr was transformed to B. flavum ATCC14067, resulting engeering strain ATCC14067/pDXW-10-ilvBNr. 3L fermentor experiments showed that ATCC14067/pDXW-10-ilvBNr could produce 5.0 g/L of L-valine, while the control strain ATCC14067 could not accumulate L-valine.