Improving Biosynthesis Of Poly-γ-glutamic Acid And Regulating Its Stereochemical Configuaration

γ-PGA is a polymer polymerized from L-glutamic acid or/and D-glutamic acid monomer,which can be divided intoγ-L-PGA,γ-D-PGA,γ-L/D-PGA.γ-PGA has a variety of important physiological functions,such as moisture retention,biocompatibility,and complete biodegradability.It has very important application prospects in the fields of food,cosmetics,biomedicine and biomaterials,and has a large market demand.At present,the main strain for industrial production ofγ-PGA is Bacillus,and the fermentation medium needs to be added with expensive raw materials such as L-glutamic acid,so the production cost is high.In this study,a strain of Corynebacterium glutamicum that could directly synthesizeγ-PGA from glucose,which was constructed in the early stage of the research group,was optimized for fermentation to increase the yield ofγ-PGA.On this basis,through exogenous addition of D-glutamic acid and endogenous expression and regulation of the glutamate racemase gene rac E from Bacillus subtilis,γ-PGA with different D/L monomer ratios was synthesized,and the fermentation conditions of the strain were optimized.Improved the yield ofγ-PGA with a specific D/L monomer ratio.The main results of this paper are as follows:（1）In order to increase the production ofγ-PGA in shake flask of engineered strain,this paper explored the effects of biotin concentration,inoculum amount,initial glucose concentration,and induction time onγ-PGA fermentation.On the basis of the above research,three factors that had a greater impact on the yield ofγ-PGA were selected:biotin concentration,inoculation amount,and induction time,and L₉（3³）orthogonal test was performed.The results showed that when the biotin concentration was 10μg·L^-1,the inoculation amount was 6%,and the induction time was 2 h,the highest yield ofγ-PGA reached 10.12 g·L^-1,the conversion rate Yp/s was 0.20 g·g^-1,and the productivity was 0.28g·（L·h）^-1.On this basis,by screening different types of oxygen vectors,the best oxygen vector was determined to be Tween 80.At the same time,the concentration and time of addition of Tween 80 were optimized to be 0.3%and 0 h,respectively.The maximum yield ofγ-PGA reached 13.76 g·L^-1.The conversion rate Yp/s was 0.26 g·g^-1,and the productivity was 0.38g·（L·h）^-1.（2）In order to improve theγ-PGA production of engineered strain in fermenter,this paper explored the influence of different stirring paddle speeds and inoculation amounts on the production ofγ-PGA based on the optimization of the shake flask.The results show that when the stirring speed is 700 r·min^-1,the yield ofγ-PGA was 38.26 g·L^-1,the conversion rate g_γ-PGA/g _glucose was 0.48 g·g^-1,and the productivity is 1.06 g·（L·h）^-1;when the inoculation amount is 15%,the yield ofγ-PGA was 40.10 g·L^-1,the conversion rate Yp/s was 0.50 g·g^-1,and the productivity was 1.11 g·（L·h）^-1.On this basis,by adding glucose during the fermentation process,the residual glucose level is maintained at 15～25 g·L^-1,the maximum yield ofγ-PGA is 51.98 g·L^-1,the conversion rate Yp/s was 0.56 g·g^-1,and the productivity was 1.44 g·（L·h）^-1,respectively 3.78 times,2.15 times,and 3.79 times of the shake flask.（3）In order to synthesizeγ-PGA with different D/L monomer ratios,this paper determined the stereochemical configuration ofγ-PGA synthesized by the engineered strain,and L-glutamic acid accounting for 97.10%.On this basis,firstly by adding D-glutamic acid at different concentrations exogenously,γ-PGA with D-glutamic acid accounting for15.71～33.52%was synthesized.Then,by expressing the glutamate racemase from B.subtilis in the engineered strain,and using three different strength RBS and four different strength promoters to regulate the expression level of glutamate racemase,further improving the D/L monomer ratio control range,andγ-PGA with D-glutamic acid accounting for 30.82～52.53%was synthesized.Finally,the fermentation conditions of one of the engineered strain were optimized,which greatly increased the yield ofγ-PGA with D-glutamic acid accounting for33.82%,from 3.92±0.46 g·L^-1in shake flask to 26.70 g·L^-1 in fermenter.The yield ofγ-PGA was 9.14 times of the original.