Selection Of Bacillus Licheniformis With High Production Of Extracellular Polymer Based On ARTP And Analysis Of Its Metabolic Mechanism

Extracellular polymeric substances(EPS)have been widely used in the fields of food,cosmetics,agriculture,medicine and sewage treatment due to their adsorption,flocculation and water retention capabilities,as well as their safety,non-toxicity and environmental degradation.In this study,the EPS-producing strain Bacillus licheniformis CGMCC 2876 which was independently screened in the laboratory,was selected as the research object.The high-yield of EPS strain was obtained by ARTP mutagenesis technology,and then the changes of EPS metabolic pathway before and after strain mutagenesis were studied,and the optimization strategy of EPS synthesis process was obtained.First of all,Bacillus licheniformis M32,a strain with high EPS yield was treated by ARTP mutagenesis technology and screened out,and its genetic properties were stable.The total yield of EPS in the mutant strain was 13.6 g/L,which was 35%higher than that of the original strain.Among them,the yield of γ-PGA was 6.68 g/L,which was 11%higher than that of the original strain.The contents of polysaccharide and protein in EPS did not change significantly.The molecular weight of the EPS was about 6.69-8.57×107 Da,and there was no significant difference between the original strain and the mutant strain.The EPS of the mutant strain contained more substances with high molecular weight.Secondly,the gene information of the mutant strain was studied by gene sequencing,and molecular docking method was used to explore the molecular mechanism of increasing γ-PGA yield.A total of 54 single nucleotide polymorphisms(SNPs)and 7 insertion/deletion variations(Indels)were detected in the genome of mutant Bacillus licheniformis M32.Among them,five non-synonymous SNPs were detected in the gene ppsc encoding lipopeptide Plipastatin synthase subunit C(PpsC)in the mutant strain.Through molecular docking simulation,it was found that after mutation,PpsC had more binding sites with glutamic acid and binding energy was enhanced,while PpsC had fewer binding sites with valine and alanine,and binding energy was reduced.This change may promote the activation and polymerization of glutamic acid,and then promote the metabolic flux of γ-PGA synthesis pathway.The distribution of metabolic flux was analyzed by metabonomics and qRT-PCR.For the synthesis pathway of γ-PGA,capB,capC and capA were upregulated in the mutagenic strain,leading to the increase of γ-PGA production.The intracellular metabolite alanine accumulated due to the decrease of its binding ability to PpsC,thus stimulating the TCA cycle and facilitating the synthesis of glutamate.At the same time,the up-regulation of alanine promoted the mutual transformation of L-glutamic acid and D-glutamic acid,enhanced the synthesis of glutamic acid and resulted in the upregulation of γ-PGA production.For the glycolysis pathway,crr,pyk,pdh,pgi and glpk were significantly up-regulated in the mutant strain,indicating that the strain could better utilize glucose.At the same time,the up-regulated metabolic pathway of glycolysis may provide more energy for the EPS.The change of pyruvate kinase activity induced by serine upregulation may be the cause of the upregulation of glycolysis pathway.For polysaccharide metabolism pathway,gene and metabolite on polysaccharide metabolism pathway were upregulated in mutant strain,but the yield did not change significantly.The extracellular polymer synthesis process of Bacillus licheniformis was optimized.Adding sodium glutamate,glucose and NaNO3 significantly increased the extracellular polymeric substance yield,and the maximum total yield of EPS was 47.54 g/L,which was 17.44%higher than the original strain,and the γ-PGA yield was 28.43 g/L.