Breeding OF L-Isoleucine Producing Strain And Cloning And Expression OF Threonine Dehydratase Gene

L-isoleucine is one of the eight essential amino acids in human body, and which together with L-valine and L-leucine are referred to as the branched chain amino acid. Due to its special structure and function, L-isoleucine play important role in the metabolism of human life, In order to get an L-isoleucine overproducing strain for large scale production, we modify a Brevibacterium flavum Bf420by means of mutagensis breeding and gene cloning. The.ain contents and results are as follows:1、L-isoleucine concentration in fermentation broth was quantitatively determined with methods of paper chromatograghy(PC), paper chromatograghy-mottle elution chromometry (PC-MEC) and high pressure liquid chromatography-orthophthalaldehyde (HPLC-OPA). The quantitative determination condition and computing method were decided. By comparing the Rf values of different amino acids, paper chromatograghy is a simple, rapid, convenient qualitative measurement of L-soleucine in fermentation samples. Compared the results from PC-MEC, the determination value of L-isoleucine concentration with HPLC-OPA is more accurate. But the former is a more practical and cheaper way especially for large numbers of samples.2、The starting strain Brevibacterium flavum Bf420was treated both by ultraviolet (UV) and nitrosoguanidine (NTG), and a L-isoleucine high-producing mutant strain BM2610, which showed the double genetic markers of methione defect (Met") and alpha aminabutyric acid resistance (α-ABr), was obtained after series of primary screening and shaking flask screening. The genetic markers of BM2610was stable and the L-Isoleucine production of mutant BM2610reached to7.12g/L after96h fermentation under a fermentation condition without being optimized, which was increased122.5%compared to its parental strain Bf420.3、The mutant strain BM2610was further modified by molecular way. The ilvA gene, which codes the key enzyme-threonine dehydratase in L-isoleucine metabolism, was amplified from E.coli JM109by PCR. And the recombinant expression vector of pET-28a-ilvA was constructed and transformed into Brevibacterium flavum BM2610by electroporation way. The recombinant enzyme was functional expressed after inducing by IPTG. A recombinant fusion protein with a size of4.6kDa was detected by SDS-PAGE analysis. The threonine dehydratase activity of the crude enzyme from the cells containing the recombinant plasmid was increased36%compared to that of the control group.