| Poly-?-glutamic acid is a multifunctional biodegradable polymer that has been widely used in medicine,agric?Lture,food,environmental protection and light chemicals.Currently,the commercial production of poly-?-glutamic acid mainly depends on the microbial fermentation method.The conversion of glucose to poly-?-glutamic acid is low.because the need to add poly-?-glutamic acid synthesis precursors and excessive fermentation by-products.This study intends to reduce the synthesis of by-products and improve the conversion of glucose to poly-?-glutamic acid by central carbon metabolism modification and coenzyme balance modification of Bacillus licheniformis.The C4 compound oxaloacetate plays a leading role in the synthesis of tricarboxylic acidderived amino acids,affecting cell growth,and its intracell?Lar accumulation affects the speed of the tricarboxylic acid cycle.In this study,we verified the effect of the C4 replenishment pathway on the synthesis of poly-?-glutamic acid by exogenous expression phosphoenolpyruvate carboxylase gene pp C of Escherichia coli and overexpression pyruvate carboxylase gene pyc A of B.licheniformis WX-02.And the poly-?-glumatic acid yield increased 65.59% by the overexpression of the pyruvate carboxylase gene pyc A.The glyoxylate cycle is a branch of the TCA cycle.Isocitrate produces glyoxylic acid by isocitrate lyase;glyoxylic acid forms succinic acid and malic acid.In order to ensure the carbon flux from oxaloacetic acid,citric acid to ?-ketoglutaric acid and then to glutamic acid,this study deleted the isocitrate lyase gene ace A and blocked the glyoxylate cycle.Subsequently,the strong promoter PRBS6 was used to replace pyruvate carboxylase gene pyc A,citrate synthase gene cit Z,succinate dehydrogenase gene sdh B promoter based on B.licheniformis WX-02?ace A to enhance the operation of TCA cycle,respectively.They named WX-02?ace A-RBS6-pyc A,WX-02?ace A-RBS6-cit Z,WX-02?ace A-RBS6-sdh B and the yield of poly-?-glutamic acid was increased by 57.91%,61.28%,20.03% compared with the control strain,respectively.Moreover,the total amount of by-products such as acetoin,2,3-butanediol and acetic acid also decreased to some extent in the corresponding strains;however,the accum?Lation of byproducts was still excessive.Acetyl and 2,3-butanediol are the main by-products of the WX-02 poly-gamma-glutamic acid fermentation process,and the synthesis of the two consumes a large amount of NADH.In this study,the synthesis of the two was blocked by the deletion of the acetolactate dehydrogenase gene als D,but the loss of als D caused acidification and coenzyme imbalance,which significantly reduced the growth ability of the strain,thus affecting the synthesis of poly-?-glutamic acid.NADPH as a reducing power was used to synthesize glutamic acid by glutamate dehydrogenase of WX-02.Firstly,We screened glutamate dehydrogenase from Bacillus licheniformis WX-02,Corynebacterium glutamicum ATCC 13032,Bacillus subtilis168,and Escherichia coli BL21,and enhanced expression of the glutamate dehydrogenase gene roc G of B.licheniformis WX-02 is most favorable for the synthesis of poly-?-glutamic acid.This study used a saturated mutation technique to convert the coenzyme specificity of glutamate dehydrogenase from NADPH to NADH.Subsequently,the mutated glutamate dehydrogenase was expressed in WX-02?als D,and it was found that the growth and the ability to synthesize poly-?-glutamic acid of two engineered bacteria were significantly improved.The yield of poly-?-glutamic acid in the mutant strain roc G2 was increased by 101.82%,and the biomass was increased by 71.04%.Increasing the ATP supply level is an effective means for high-yield production of microbial fermentation.Oxidative phosphorylation is the main energy supply pathway under aerobic conditions.This study verified that overexpression of the electron transport chain pathway gene can promote the synthesis of poly-gamma-glutamic acid.At the same time,we localize Vitreoscilla hemoglobin VHb in the periplasmic space and cell wall by Tat-type signal peptide.It was found that VHb localized in periplasmic space was superior to VHb expressed in cell wall and VHb expression in periplasmic space.The strain's poly-?-glutamic acid production increased by 26.13%,and the ATP concentration increased by 76.37% in logarithmic growth phase.Finally,a superimposed engineering strain WX-02?ace A-RBS6-cit Z-pyc A/300-roc G2 was constructed,and its poly-?-glutamic acid yield was 18.32 g/L,which was 2.41 times higher than that of the starting strain WX-02.In summary,the reaction of pyruvate to oxaloacetate may be the first metabolic bottleneck for the synthesis of poly-?-glutamic acid by WX-02.Increasing the metabolic flux of the TCA cycle enhances the synthesis of intracell?Lar glutamate.At the same time,reducing by-product production and rational use of coenzyme can enhance the ability of B.licheniformis to utilize glucose to transform into poly-?-glutamic acid. |
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