Construction Of High-biomass Lactococcus Lactis Strain Based On Carbon Redistribution And Its Production Applications

Lactococcus lactis is a kind of Gram-positive bacteria whose major metabolite from fermentable sugars is lactate when grown anaerobically.Not only is it widely used in food industry,L.lactis also has great potential as a cell factory for large-scale production of bio-based chemicals.However,like many anaerobic bacteria,glycolysis is the sole pathway to generat energy for the growth and production of L.lactis,resulting in its incompetent biosynthetic capacity.Another critical issue is that pyruvate is mainly converted to lactate,the lactate accumulation of which leads to feedback inhibition and environmental acidification that significantly influence the growth performance and make it difficult for strains to achieve high-biomass cultivation.In this study,to construct high-biomass strain,we relieved the inhibition of metabolite accumulation by re-routing carbon metabolism,and researched on the production capacity of the high-biomass strain.Several strategies were adopted to realize carbon redistribution on L.lactis F44,including the introduction of heterogenous pathways,enhancement of endogenous pathways,adjustment of redox status and weakening of lactate synthetic pathways.The results indicated that expression of pck A,overexpression of nox E and knockout of ldh respectively increased the biomass by 4.4%,8.6%,and 18.4%.Secondly,based on L.lactis ?LDH,ldh B was deleted to further inhibit biosynthesis of lactate.The biomass of the strain was enhanced to 7.32 by knocking out ldh and ldh B gene,and the nisin yield increased to 4209 IU/m L,which were respectively 46.1% and 117.5% higer than the original strain.Additionally,the lactate detected in the fermentation broth was significantly decreased from 22.83 g/L to 2.48g/L,while the concentrations of formate,acetate,acetoin,and ethanol were increased.In order to alleviate the toxic effect of formate accumulation caused by the deletion of ldh and ldh B genes,two strategies were applied to reduce formate level including knockout of pfl and expression of fdh.The knockout of pfl limited the growth of the strain and sharply decreased the biomass,presumably due to the decreased biosynthetic capacity of acetyl Co A.The fdh gene from Candida boidinii was expressed in L.lactis?LDH?LDHB,which generated the increasement of the biomass and nisin yield to7.64 and 4763 IU/m L with 6.56% and 13.16% improvement respectively compared with controlled strain.Finally,we studied the production capacity of high-biomass strain by introduction the biosynthesis pathway of phenyllactic acid.Phenyllactic acid(PLA)is a natural organic acid with significant bacteriostasis on a range of Gram-positive foodborne bacteria,Gram-negative bacteria pathogens,yeast and mycete,which is a potential substitute for chemically preservatives.The synergistic effect of the combined use of nisin and PLA had been proved by bacteriostatic experiments and food preservation test.To improve the effectiveness of the fermentation product of L.lactis in food preservation,a fermentation system of co-production of nisin and PLA was developed in high-biomass L.lactis.The engineered strain yielded up to 1.67 g/L PLA with the55.7% conversion rate of phenylpyruvate.We envision that the high-biomass strain constructed in this study has the potential to be extended to the production of other high-value biobased products,such as heterologous proteins,vitamins,vaccines and fermetation agents.