| ?-Poly-L-lysine(?-PL)is a natural homopolymer consists of 25?35 L-lysine residues linked by dehydration condensation of?-COOH and?-NH2.Because of its good solubility in water,biodegradability,broad spectrum of inhibition capability,nontoxicity,?-PL is considered to be a natural food preservative.However,wild?-PL-producing strains have low?-PL biosynthesis capability.Therefore,it is significant to improve the yield of?-PL by mutation breeding and the efficiency during fermentation process.In the study,Atmospheric and Room Temperature Plasma(ARTP)mutagenesis technology was conducted on screening the mutant strain of S.albulus for high?-PL yield,and metabonomics and genomics were used to explore the preliminary mutagenesis mechanism.The ultrasound-assisted the mutant S.albulus strains fermentation technology was established for?-PL yield promotion.Kinetics,cell respiratory activity,respiration activity,intracellular ATP,key enzymes and the transcription levels of related gene in the synthesis pathway of?-PL were conducted to explore the effect mechanism of ultrasonic treatment on the fermentation of S.albulus.The main research contents and results of this research are as follows:(1)A higher?-polylysine mutant strain of S.albulus was obtained by ARTP mutagenesis,taking S.albulus(CICC 11022)as the original strain.The results showed that the lethality rate of ARTP mutagenesis could reach 88.78%under the conditions of ventilation rate of 10 SLM(Standard Litre per Minute),power of 120 W and mutagenesis time of 120 s.Under these conditions,a mutant strain named SAR14-116 with higher?-PL activity and stable inheritance was screened from a total of300 single colonies by using?-PL+Gly resistance plate and antibacterial circle measurement methods.The yield of SAR 14-116(1.04 mg/mL)increased by 18.46%compared with original strain SAB.It was observed that the mutant strain SAR 14-116 cultured in seed liquid had a larger proportion of spores with"burr-like"protrusions compared with the original strain.It was found that the aerial mycelia of the mutant strain cultured in fermentation broth were thicker and more branching.In addition,the increase of intracellular calcium ion concentration of SAR 14-116 indicating that the ARTP mutagenesis changed the permeability of cell membrane.(3)Effects of ARTP mutagenesis on S.albulus were investigated at molecular levels to elucidate the mechanism of?-polylysine(?-PL)yield enhancement by using genome resequencing analysis.Compared with the original strain SAB,the result showed that 88 SNPs and 57 In Dels involving 65 genes occurred in SAR 14-116.The variations of the genes indicated that the ARTP possibly changed cell growth and secondary metabolism,amino acid,lipid,carbohydrate transport and metabolism,cell membrane,and so on,therefore improved the?-PL yield.(4)Effects of ARTP mutagenesis on S.albulus were investigated at metabolism levels to elucidate the mechanism of?-PL yield enhancement by using untargeted metabolomics analysis.According to the results,the metabolic pathway enrichment involved a total of 64 complex metabolic processes with 851 differential metabolites based on KEGG database.The results also showed that the?-PL yield increase in SAR 14-116 might have a relationship with the up-regulated metabolites in the L-lysine synthesis and degradation pathways,such as N-acetyllysine,L-2,3,4,5-Tetrahydrodipicolinate and D-pipecolic acid.The content of some intermediate metabolites in the metabolic pathways related to?-PL synthesis was changed significantly.Compared to the original strain,the content of basic amino acids including arginine,lysine and histidine increased.The carbon metabolism involved in the synthesis of?-polylysine in the mutant strain was affected by the up-regulation of erythrose-4P and 5-methyl-THF.The significant decrease in the content of L-glutamate and L-glutamine indicated the flux of nitrogen metabolism might have changed in the mutant strain.Changes in the TCA cycle and glycolysis bypass which was caused by the decrease of acetyl tributyl citrate and S-D-lactoylglutathione affected energy metabolism.In addition,according to the results of lipid metabolism,it was found that the materials related to membrane system in S.albulus could be changed by ARTP treatment of appropriate intensity.(5)Ultrasound-assisted fermentation technique of SAR 14-116 for?-PL,production was established.The optimum ultrasonic treatment conditions of SAR14-116 included 28 kHz ultrasound frequency with power density 280 W/L,SAR14-116 cultured at mid logarithmic phase,and were treated for 1 h.The biomass of SAR 14-116 and the concentration of?-PL increased by 14.92%and 28.45%under these conditions.The fermentation kinetics study showed that the product constant associated with SAR 14-116 cell growth of the ultrasonic group model increased by2.88 times compare with the control.Analysis of key enzymes activities showed that pyruvate kinase(PK)and aspartate aminotransferase(AAT)activities increased by 249%and 81.15%at 3 h and6 h after ultrasound stimulation,respectively,which could cause more precursor lysine production.Activities of HK increased by 4.61-fold after ultrasonic treatment,suggesting that the Embden-Meyerhof-Parnass(EMP)pathway was more active after ultrasound treatment.Activities of Glucose 6-Phosphatedehydrogenase(G6PDH)decreased after ultrasound treatment,which may change the carbon flux distribution of the PPP pathway.In addition,ultrasonic treatment also increased the intracellular ATP content of the SAR 14-116 by 90.63%.Analysis of genes involved in?-PL biosynthesis pathways including pk,cs and aat in the the ultrasonic group were significantly overexpressed with 1.69,1.54 and 1.34-fold higher expression than the non-ultrasonic group(p<0.05).The metabolic intensity of glycolysis pathway,TCA cycle and DAP pathway increased after ultrasound treatment,which might promote the synthesis of the precursor substance consumed in L-Lys biosynthesis.The expression level of pls encoding?-PL synthase was significantly 4.58-fold higher after ultrasound treatment(p<0.05),which might directly improves the synthesis ability of?-PL in cells. |
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