Process Development And High-yield Mechanism Analysis For ε-poly-L-lysine Production Based On L-lysine Feeding Strategy

ε-Poly-L-lysine（ε-PL）is a homo-amino acid polymer formed by the dehydration condensation of 25-35 L-lysine monomers betweenα-COOH andε-NH₂,which has currently been approved as an excellent natural food preservative in many countries for its broad antibacterial activity.In our previous study,a high-yield mutant S.albulus GS114 was obtained by engineering,and itsε-PL production has achieved 60.2 g·L^-1 in a 5 L fermenter.To further improveε-PL production,a fermentation process based on a L-lysine feeding strategy was developed,and the main results are as following:（1）The optimal organic nitrogen source for S.albulus GS114 was determined by systematic optimization of organic nitrogen source types and addition amount.Based on the principle of the equal total organic nitrogen,the effects of 12 organic nitrogen sources and their concentrations onε-PL synthesis and cell growth during natural fermentation,batch fermentation,fed-batch fermentation were systematically evaluated,and the best organic nitrogen source for S.albulus GS114 was the FM760 with the optimal concentration of 9.27g·L^-1.In the shake flask fermentation,ε-PL production reached by 2.60±0.02 g·L^-1,which was 22.07%higher than the pre-optimized organic nitrogen source.In a p H-controlled batch fermentation,ε-PL production reached by 6.41±0.23 g·L^-1,which was 42.64%higher than the control.In a fed-batch fermentation,ε-PL production reached 62.38 g·L^-1 with the increasement of 18.80%inε-PL titer and 25.77%in glucose conversion ratio than the control.The analysis of amino acid composition of organic nitrogen source initially revealed that the promotion ofε-PL synthesis by FM760 may be related to the concentrations of isoleucine,leucine and alanine.（2）A novel fermentation process based on a L-lysine feeding strategy for higherε-PL production was developed,which is overcoming the problems of complex biosynthetic pathways,low substrate conversion ratio and low productivity.The optimal fermentation process by a L-lysine feeding strategy were determined by systematically optimization of the concentration,addition manner and time of precursor L-lysine.Finally,the highestε-PL production was achieved by maintaining the L-lysine concentration at 1～2 g·L^-1 from 96 h.Under the optimal fermentation conditions,theε-PL production and DCW in a 5 L fermenter was achieved by 73.1 g·L^-1 and 62.6 g·L^-1 within 150 h,which was 35.4%and44.2%higher compared to that of the control.Meanwhile,theε-PL productivity was also increased by 78.20%.Importantly,Scaled-up the developed strategy in a 50 L fermenter,theε-PL production was increased from 53.78 g·L^-1 to 69.80 g·L^-1,demonstrating the potential of this novel strategy in industrial production ofε-PL.（3）The physiological mechanism for highε-PL production by exogenous addition of L-lysine was elucidated based on a comparative transcriptomic analysis.It was found that the addition of exogenous L-lysine resulted in the enhancement of the TCA cycle and the lysine synthesis pathway,providing more ATP,intermediate metabolites,and cofactors forε-PL synthesis.In addition,oxidative phosphorylation pathway was substantially up-regulated,generating a large amount of ATP to meet the energy demand duringε-PL biosynthesis,which may be the reason for the decreased dissolved oxygen after the addition of L-lysine.In addition,L-lysine adding lead to oxidative damage of the S.albulus,which may explain the increase of dissolved oxygen in the late stage of fermentation.Moreover,the synthesis pathways of many other amino acids were weakened,introducing more carbon flux into the L-lysine synthesis and the TCA cycle,and consequently providing more NADH for oxidative phosphorylation.The decrease in fatty acid synthesis not only saved more NADPH for L-lysine synthesis,but also might improve cell membrane permeability,thus promoting the uptake of the exogenous L-lysine.