Breeding Of L-valine Producer And Study On Its Unique Natures

L-Valine is not only one of three branched-chain amino acids but also a member of eight essential amino acids which can’t be synthesized by humans themselves. It plays an important role in many kinds of physiological functions in vertebrates’bodies. The applications of L-valine are very broad: precursor of pharmaceuticals, additives to food or feedstuff for livestocks, material for pesticides, nutrition infusion for patients, health care product, and condiment and so on. Nowadays, scales of the industrial plants for L-valine in China are small, and the actual production level of L-valine prodution isn’t high. The annual production amount of L-valine can’t meet the growing demand of domestic market. In order to solve problems mentioned above, it is necessary to breed highly efficient L-valine-producing strains based on metabolic engineering principles.（1） The origin strain is strain ZGH6128 （Brevibacterium flavum） which was treated with DES and NTG in the screening process. Strain NVT1103 (Leu^l,α-AB^hr, 2-TA^hr,SG^r) and strain JVHK597（Leu^-, Ile^-, Met^l,α-AB^r, 2-TA^r） were obtained in the early stages. Under unoptimized conditions, the L-valine productions in flasks of the two strains were 37.6g/L and 41.2g/L respectively.（2） Scientists at home and abroad were drawn attention to the application of protoplast fusion technology to screen L type amino acids producing strains. Strain NV1103 and strain JVHK597 was used as parent strains during the process to beed L-valine hyper-producer by protoplast fusion technology in this thesis. Parent strains （NV1103 and JVHK597） were pretreated for 2.5 hours and 3 hours with 0.5 U/mL and 1U/mL penicillin sodium salt solution. The two parent strains were treated by lysozyme in the optimal concentration of 1g/L and 2g/L for 9 hours and 11hours repectively. The optimal concentration of osmotic pressure stabilizer in the regeneration medium is 0.6 mol/L. A good fusion rate was got when the two parent stains was mediated by PEG for 25 minutes in pH 10 buffer system at 34°C. Eventually, L-valine hyper-producer NJv61 was obtained, and genetic markers of strain NJv61 were (Leu^-, Ile^-, Met^l, 2-TA^hr, SG^r, Glc^hr,α-AB^hr). Under unoptimized conditions, the L–valine production in flasks of the strain was 45.6 g/L.（3） The composition of seed medium of strain NJv61 was fixed through orthogonal table experiment design: Glucose 24g/L, （NH₄）₂SO₄ 5g/L, KH₂PO₄ 0.6 g/L, MgSO₄·7H₂O 0.5g/L, corn-steep 35g/L, CaCO₃ 10g/L. Single-factor experiments were conducted, including carbon or nitrogen source, inorganic salts, growth factors and other necessary factors. The optimal composition of the fermentation medium was fixed by response surface methodology. The culture medium used for the fermentation of NJv61 contained: Gulcose 149g/L, （NH₄）₂SO₄ 48.4g/L, KH₂PO₄ 1.5g/L, MgSO₄·7H₂O 0.6g/L, VB1 150μg/L, biotin 60μg/L, corn-steep 19.4g/L. The L-valine production of strain NJv61 in 7L fermentor was 51.8 g/L under unoptimized conditions after 72 hours fermentation.（4） Representative strains （ZGH6128, NVT1103, JVHK597 and NJv61） were obtained in the screening process. And broth samlpes of the representative strains were analysed when these strains accumulated in the 7 fermentor during the late metaphase of the fermentation process. Metabolic flux distribution network diagram from glucose to L-valine was figured out based on results of analysis of the samples. Diagrams of frames of carbon distribution in metabolic network were compared between the initial strain ZGH6128 and the target strain NJv61. In the screened strain NJv61, Carbon flows into the EMP pathway was enhanced at the G6P node, and there was still sufficient metabolic fluxes in the HMP pathway. Compared with the original strain, metabolic fluxes into the TCA cycle were decreasing sharply at pyruvate node in order that more carbon frame fluxes flowed into the L-valine biosynthetic pathway. Compared with the starting stain, Carbon frame fluxes in the L-alanine biosynthetic pathway didn’t decline markedly in the strain NJv61.（5） Target strain NJv61 could resist the high concentration ofα-AB and 2-TA because of the mutations in acetohydroxyacid synthase gene. In cells of strain NJv61, L-valine feedback inhibition and repression effect were released drastically. Meanwhile, relative activity of the enzyme was changed. Strain NJv61’s AHAS gene was cloned and sequenced in the thesis, and the result was analysed with biological softwares. There are nearly 10% of the total uumber mutations in the whole gene distributed in the section encoding regulatory subunit; while there are 90% of mutations in the entire gene distributed in the AHAS gene segment encoding the catalytic subunit. Finally, the NJv61’s AHAS gene was connected with a given expression vector and exported into a mode strain. Induced by IPTG, the enzymatic activity of enzyme protein molecules expressed by the mode strain was unstable at 4℃, and the relative activity of the enzyme decreased rapidly in the first 22 hours, just keeping about 20% of relative activity only.