Construction Of Predominant Strain For Polyglutamic Acid Synthesis And Optimization Of Fermentation Process

γ-Polyglutamic acid（γ-PGA）,as a natural harmless biopolymer,is a water-soluble substance widely used in daily life,and can be used in food industry,medical materials,environmental protection and other fields.At present,the application market ofγ-PGA is very broad,coupled with the high market demand,it is a high-value macromolecular polymer.Bacillus subtilis（B.subtilis）is considered to be a probiotic approved by FDA in the United States,with reliable safety,and is the most commonly used strain in industrial production ofγ-PGA.Some studies have shown that the production ofγ-PGA by Bacillus fermentation has a good prospect,and the application ofγ-PGA is also popular in some international journals in recent years.In this paper,the dominant mutant strains with high yield ofγ-PGA were screened first,then the fermentation conditions were optimized,and the production ofγ-PGA was affected by the addition of Fe³⁺.Finally,the gene expression level andγ-PGA configuration were analyzed,which laid a theoretical foundation for further research on improvingγ-PGA in the later stage.The main research contents and results are as follows:（1）The genes related to two-component regulatory system inγ-PGA synthesis pathway were knocked out or overexpressed,and a new mutant strain was constructed by molecular biological means.It was found that the changes of different genes had significant effects onγ-PGA and other phenotypes of B.subtilis.The results showed that in theγ-PGA synthesis pathway,the excessive expression of key genes which are synthetic precursors can improve the accumulation ofγ-PGA to a certain extent.Finally,the dominant strain for the synthesis ofγ-PGA was screened and named as BS168-roc A.（2）The effect of metal ions on the synthesis ofγ-PGA by BS168-roc A was investigated.The accumulation ofγ-PGA was increased by adding metal ions in 250 m L shaking flask fermentation broth.By analyzing the difference in the yield ofγ-PGA under the condition of adding different metal ions,the species and concentration of metal ions that could improve the accumulation ofγ-PGA most significantly were selected.BS168-roc A was cultured in the fermentation medium with the addition of Mg²⁺,Mn²⁺,Fe³⁺,Ca²⁺,Zn²⁺,Al³⁺,and Na⁺,respectively.Compared with other kinds of metal ions,the addition of Fe³⁺could significantly improve the accumulation ofγ-PGA.It was concluded that the addition of an appropriate amount of Fe³⁺in the fermentation medium could indeed improve the ability of BS168-roc A to synthesizeγ-PGA.The yield ofγ-PGA of the BS168-roc A strain optimized by fermentation conditions is greatly improved compared with that of the original strain.（3）The real-time fluorescent quantitative technique was used to analyze the gene transcription level of B.subtilis mutant.After obtaining the dominant producer,the relevant key genes in the biosynthesis pathway ofγ-PGA in bacteria were further analyzed.We mainly studied whether the addition of Fe³⁺was related to the transcriptional expression level ofγ-PGA synthetase or hydrolase genes.Quantitative real-time PCR（qRT-PCR）results showed that the transcription ofγ-PGA synthetase and hydrolase genes in strain BS168-roc A was significantly changed when Fe³⁺was added for fermentation.At the same time,the transcription level of the gene would affect the molecular weight of the generatedγ-PGA,the ratio of D/L-glutamic acid,and so on.These results provide an important theoretical value for the future modification of key genes in theγ-PGA metabolic pathway by B.subtilis.