Improvement Of Fermentation Of Bacillus Licheniformis With Poly-γ-glutamic Acid Hydrolase Deletion By Using L-glutamine As Precursor

Poly-γ-glutamic acid(γ-PGA)is a natural multifunctional biopolymer,which is biodegradable,edible,and harmless to human beings and the environment.It has a wide application prospect in the fields of food,agriculture,medicine,cosmetics,and sewage treatment,etc.At present,studies on increasing the yield of γ-PGA by metabolic engineering mainly focus on regulating gene expression in metabolism related to γ-PGA synthesis.By contrast,few studies have focused on genes related to γ-PGA catabolism.In this study,we investigated the effects of genes related to the hydrolytic metabolism ofγ-PGA in Bacillus licheniformis on the synthesis of γ-PGA.The relevant experiments were conducted and the following research results were obtained:The D,L-endopeptidase cwlO deletion strain WX-02-ΔcwlO was constructed by double exchange homologous recombination,and cultured in a 3 L fermentor with L-glutamine as the precursor,and the yield of γ-PGA reached 29.2 g/L,which was 57.8%higher than that of wild strain(18.5 g/L).Furthermore,under the culture condition using L-glutamine and L-sodium glutamate as mixed precursors,the yield of γ-PGA of WX-02-ΔcwlO reached 36.3 g/L,which was 48.8% higher than that of the wild-type(24.4g/L).The experimental results of real-time fluorescent quantitative PCR(q RT-PCR)for determination of gene relative transcription levels showed that the transcription levels of key genes in the γ-PGA synthesis pathway and respiratory chain were up-regulated when L-glutamine was used,compared with the wild-type.WX-02-ΔcwlO cells became shorter and rounder when observed under the fluorescence inverted microscope.Further,measurement of cell wall peptidoglycan content and composition revealed that cell wall peptidoglycan content in WX-02-ΔcwlO was decreased,and the proportion of protein content in peptidoglycan was also decreased.In summary,the absence of cwlO in B.licheniformis caused the decrease of cell wall peptidoglycan content,which resulted in promoting the utilization of L-glutamine and enhancing the synthesis of γ-PGA.The γ-DL-glutamyl hydrolase pgdS deletion strain WX-02-ΔpgdS was also constructed by double exchange homologous recombination.The recombinant strain WX-02-ΔpgdS was cultured in a 3 L fermentor with L-sodium glutamate as the precursor,the results showed that the strain could not grow normally and the yield ofγ-PGA was only 3.9 g/L.Subsequently,shaking flask cultures of WX-02-ΔpgdS with different nitrogen sources and precursors were conducted,and it was found that the recombinant grew normally only when L-glutamine was added.Furthermore,L-glutamine was used to partially replace(20 g/L L-glutamine and 20 g/L L-sodium glutamate)or completely replace(40 g/L-glutamine)L-sodium glutamate for the fermentation of WX-02-ΔpgdS,and the yield of γ-PGA reached 17.2 g/L and 19.7 g/L respectively,26.5%and 44.9% higher than that cultured in L-sodium glutamate medium.The fluorescence inverted microscope observation showed that the cell adhesion phenomenon appeared when the recombinant was cultured in the L-sodium glutamate medium,while the cells were relatively dispersed in the L-glutamine medium.q RT-PCR results showed that the transcriptional levels of key genes related to glutamic acid synthesis,nitrate metabolism,respiratory metabolism,and γ-PGA synthesis,were all up-regulated in the recombinant when L-glutamine was used.The analysis of fermentation product characteristics and components showed that extracellular polysaccharides yield produced by WX-02-ΔpgdS was significantly higher than that of the wild-type.However,the substitution of L-glutamine for L-sodium glutamate could reduce extracellular polysaccharides yield,especially in the cell growth phase.Based on the above results,it could be concluded that the increase in extracellular polysaccharides yield caused by deletion of the pgdS gene would make cells adhere together.Using L-glutamine as a precursor could effectively reduce the yield of extracellular polysaccharides of the recombinant strain,alleviate the phenomenon of cell adhesion,and promote the synthesis of γ-PGA.This study explored the effects ofcwlO and pgdS gene deletion on the physiology and metabolism of B.licheniformis,revealed the deep mechanism of L-glutamine promoting the synthesis of recombinant strain γ-PGA,and provided a new idea and research basis for further understanding the functions of genes related to the catabolism of B.licheniformisγ-PGA and enhancing the synthesis of γ-PGA by the strain.