Reseach On The Construction Of L-glutamine Genetically Engineered Bacteria And Its Fermentation Process Optimization

L-glutamine is a neutral amino acid ofγ-carboxyl amidation of L-glutamic acid,and it is one of the 20 basic amino acids that make up the protein of organism.L-glutamine plays an important role in maintaining human function and life activities.the content of L-glutamine in human body is very high,accounting for 61%of human free amino acids.In recent years,with the in-depth study of L-glutamine,L-glutamine is widely used in medicine,health food,feed and other fields.As a potential new drug,L-glutamine is mainly used in clinical treatment of gastrointestinal ulcers,relief of exercise fatigue,improvement of brain nerve function and so on.The key problems in the construction of L-glutamine genetically engineered bacteria and the optimization of its fermentation process include:high content of by-product glutamate,low yield of glutamine,weak activity of glutamine synthetase and low conversion rate of sugar and acid.In this paper,the above problems are solved by rational metabolic engineering transformation and fermentation process optimization.Using the glutamine-producing bacterium,GM34,as the starting strain.The GS-GOGAT pathway of Corynebacterium glutamicum was optimized by knocking out glutamate synthase genes NCgl0181,NCgl0182and glutaminase genes NCgl2395 and NCgl2500.One copy of glutamine synthase gene gln A_bsufrom Bacillus subtilis and one copy of glutamine synthase gene gln A_lcbfrom Lactobacillus acidophilus were integrated at genomic NCgl0182 and NCgl2500 sites,respectively.Furthermore,glutamine synthetase genes gln A_bsuand gln A_lcbwere overexpressed by p XT01plasmid with strong tuf promoter.The glutamine yield of the constructed strain G-6 increased by 41.9%compared with the original strain GM34,reaching 68.4 g/L,the content of glutamic acid decreased by 56.9%,reached13.1 g/L,and the sugar-acid conversion rate increased by 6.6%to 37.1%.Using strain G-6 as the starter strain,we optimized the nitrogen regulation system in Corynebacterium glutamicum,knocked down the nitrogen-regulated global repressor amtr,glutamine synthetase adenotransferase gene gln E,and strengthened the two ammonium transporter genes amt and amtb in Corynebacterium glutamicum.Construction strain G-11,glutamine production reached 80.3 g/L,glutamate content reached 10.6 g/L,and the sugar-acid conversion rate reached 38.3%.Strain G-11 is the most potent glutamine production strain constructed in this subject with the potential for industrialization.The uncertainty of biotin content in corn pulp used in the traditional biotin sub-moderate process will lead to the instability of fermentation results in each batch.In order to solve this problem,the effect of biotin addition on glutamine fermentation was studied.By adding 40 m L/L soybean concentrate and 25μg/L V_Hto the seed medium,and adding 50 m L/L soybean concentrate and 4μg/L V_Hto the fermentation medium instead of the corn pulp components of the traditional biotin sub-moderate process,a new glutamine fermentation medium was obtained,which increased the glutamine yield to 48.2 g/L,reduced the glutamate content to 30.4 g/L,and increased the sugar-acid conversion rate to 32.5%.NH₄⁺is a key factor in glutamine fermentation.NH₄⁺,is needed to catalyze the production of glutamine by glutamine synthetase,but high concentration of NH₄⁺will affect cell growth and reduce the activity of glutamine synthetase.Under the condition of low concentration of NH₄⁺,the activity of glutamine synthetase was at a high level,but too low concentration of NH₄⁺would reduce the production of glutamine.In order to solve the contradiction of the amount of NH₄⁺in the fermentation process and improve the activity of glutamine synthetase,a NH₄⁺limited fermentation process was designed to explore the effect of NH₄⁺concentration on glutamine fermentation and regulate the NH₄⁺concentration.Urea was used to regulate the p H of seed culture and early fermentation.By adding urea of 3 g/L at the beginning of seed culture,p H naturally decreased to 7 after 6 h of seed culture,that is,urea was exhausted,and the OD₆₀₀of seed culture about 6 h just reached 40,that is,the inoculation condition was reached.2 g/L urea was added in the early stage of fermentation,and when the urea was exhausted,p H fell back to neutral in about 3hours,began to use 25%ammonia to regulate p H,and began to（NH₄）₂SO₄to the end of fermentation at the same time.It effectively increased the activity of glutamine synthetase and increased the yield of glutamine by 12.8%to 90.6 g/L,the content of glutamic acid decreased by 32.3%to 6.7 g/L,and the sugar-acid conversion rate increased by 3.8%to 40.9%.In view of the decrease of acid production efficiency and aging in late fermentation,a total nutrient feed fermentation process was designed,and the effects of betaine and choline chloride on glutamine fermentation were studied.The fermentation medium was divided into initial fermentation medium and fermentation feed medium at 4:6.The fermentation medium was fed evenly 4 hours after the beginning of fermentation to 2 hours before the end of fermentation,and 3 g/L betaine and 2 g/L choline chloride were added to the glucose sugar solution with the feeding of glucose.Using the best feeding strategy of total nutrient feed and the feeding strategy of betaine and choline chloride,glutamine production increased by 2.9%to96.3 g/L,glutamate content by 22.4%to 5.2 g/L,and the sugar-acid conversion rate by 0.7%to 41.6%.In this project,the glutamine production bacterium GM34 was used as the starting strain to construct a strain G-11 with the strongest glutamine production capacity,and the glutamine fermentation process was optimized.The final glutamine yield reached 96.3 g/L,glutamate content 5.2 g/L,and the sugar-acid conversion rate 41.6%.