Culture Conditions Optimization And Control For N-acetylglucosamine Production By Bacillus Subtilis

Glucosamine（GlcN） and N-acetylglucosamine（GlcNAc） are not only used in nutritional chemicals and pharmaceuticals, but also have been widely applied in nutraceuticals, cosmetics and pharmaceutical industries. GlcN and GlcNAc are currently produced by acid or enzymatic hydrolysis of chitin extracted from shrimp shells and crab. The B.subtilis was used as the starting strain. A recombinant bacteria was obtained by pathway engineering, which has the ability to product GlcNAc. The optimal fermentation medium was obtained by medium optimization. Yeast has the highest price in the optimal fermentation medium, which hinder the amplification of industrialization production. The high-throughput screening of Bacillus subtilis for N-acetylglucosamine production by ARTP mutation technolog. The optimal inorganic nitrogen source was optimal inorganic nitrogen source based on inorganic nitrogen source optimization.（1） Firstly, a novel atmospheric and room temperature plasma（ARTP） mutagenesis system was used by combining with the high throughput screening of 96-well microplates and chromogenic reaction to obtain high-yielding mutants. The best condition for chromogenic reaction is as follows: otassium tetraborate solution 1 μL, PDABA 125 μL, reaction time 5 min, temperature 96℃. Through several rounds of ARTP mutation and high-throughput screening, a mutant（4A12） was obtained. In shake flask fermentation, the maximum GlcNAc production reached 9.1 g·L^-1, which was improved 19.7% compared with the control strain BSGN6.In 3 L fermentor, the maximum GlcNAc production reached 36.1 g·L^-1, which was improved 64.8% compared with the control strain BSGN6. The results of subculture test show that the mutant strain had stable hereditary characteristics after 50 generations. ARTP mutagenesis techniques as an effective way to obtain high-yield GlcNAc strains was compared with molecular biology methods.（2） Next, nitrogen source composition in the culture medium is too high in composite organic: yeast 20 g·L^-1, corn starch 20 g·L^-1. Yeast has the highest price in the optimal fermentation medium. Ammonium sulfate is optimal inorganic nitrogen source based on inorganic nitrogen source optimization. The optimal semi-synthetic medium optimization was obtained, which containing(g·L^-1): yeast extract 6,（NH₄）₂SO₄ 8.49, glucose 60, K2HPO4 12.5, KH2PO4 2.5, MgSO4·7H2O 3, CaCO3 5, trace element solution（separating the sterilization）. In 3 L fermentor, the maximum GlcNAc production of the mutant（4A12） reached 41.3 g·L^-1 in the optimal synthetic medium, which was improved by 13.8% compared with the optimal composite medium.（3） Finally, the biomass of the mutant（4A12） and the recombinant bacteria（4A12-△tnrA） is lower the biomass the starting strain BSGN6. The role of the global regulatory factors tnrA in the metabolism of nitrogen source. TnrA suppressed the deactivation in the optimal composite medium and it has no effect on the growth of bacteria and GlcNAC biological production. The fermentation results of the mutant（4A12） and the recombinant bacteria（4A12-△tnrA） has obvious difference in the inorganic salt medium. TnrA is activated in the inorganic salt medium, and it can activate nrgAB, nasBC and nasDEF operon expression, the mutant（4A12） can grow by using the nitrogen source in the environment. The recombinant bacteria（4A12-△tnrA） lose △tnrA, it can not activate nrgAB, nasBC and nasDEF operon expression, which affect the growth of the recombinant bacteria（4A12-△tnrA）. TnrA is activated in the optimal composite medium, and it can activate nrgAB, nasBC and nasDEF operon expression, which is way to solve the growth of bacter.