Construction Of Nisin High-yield Lactococcus Lactis Strain And Development Of Novel Nisin Production System

Nisin,an antimicrobial peptide,is produced by certain Lactococcus lactis strains.It exhibits broad-spectrum antimicrobial activity against a majority of Gram-positive foodborne bacteria and some Gram-negative pathogens.It has been approved by the Food and Agriculture Organization of the United Nations and World Health Organization for utilization as a safe and non-toxic food preservative.In recent years,nisin is considered an excellent antibiotic candidate due to the increasingly serious threat of antibiotics-resistance.It has shown efficacy in various clinical applications such as treatment of skin and soft tissue infections,Clostridium difficile-associated diarrhea,and even cancer therapy.However,the low yields and high production cost of yield severely restrict its wider applications.Therefore,this research focuses on three aspects including reconstruction of nisin high-yield strain,development of low-cost nisin production system and improvement of application efficacy of fermentation products.Below are the main results.Like many anaerobic bacteria,L.lactis uses glycolysis as its sole energy generating pathway to support growth.However,pyruvate is mainly converted to lactic acid,the accumulation of which leads to environmental acidification that significantly inhibits glycolysis,resulting in limited biomass accumulation and nisin biosynthetic capability.Additionally,the optimum neutral pH(6.0-7.0)for L.lactis growth seriously conflicts with the acidic conditions that optimize nisin activity which further increases complexity of the production process.Here,we report simultaneously improved energy supply and acid resistance of L.lactis through reprogramming glycolytic flux towards alternative metabolites.The target metabolites are selected through transcriptome analysis and experimental verification,and then redirect the carbon flux to selected metabolites in L.lactis.To diminish redox imbalance of the engineered strains,we decrease NADH/NAD~+ratio through overexpressing NADH oxidase and increasing oxygen supply.The results indicate that decreasing NADH/NAD~+ratio can serve as a driving force for carbon redistribution.We also demonstrate the utility of pH-responsive promoters for stepwise carbon redistribution,leading to a shortened fermentation period and increased nisin yield.The L.lactis platforms developed here have potential to be extended to produce other valuable food-grade compounds such as vaccines,vitamins and exopolysaccharides.The long-term adaptation of L.lactis in nutrient-rich environments including milk,meat and fruits has resulted in the reduction of biosynthetic pathways of many metabolites.Therefore L.lactis has fastidious growth requirements for exogenous nutritions.In nisin fermentation process,refined sugars(sucrose or glucose)and arge amounts of expensive high-quality nitrogen sources(peptone,corn steep liquor and yeast extract)are applied to support L.lactis growth and nisin production.Therefore,applying agricultural wastes as cheaper substrates will be an effective strategy to reduce the production costs.In this research,defatted rice bran(DRB)and defatted soybean meal(DSM)are used as carbon source and nitrogen source,respectively.We develop three processes including SHRSF process(separated hydrolysis of DRB and DSM for fermentation),CHRSF process(Combined hydrolysis of DRB and DSM for nisin fermentation)and SHFRS process(Simultaneous hydrolysis and fermentation of DRB and DSM).These three processes yield 3629,3579 and 1870 IU/mL nisin.The nisin yield of SHFRS process is further increased 70%through overexpressing NADH oxidase and adding hemin in the fermentation system which activates the respiration chain of L.lactis.To reduce the cost of nisin extraction and purification,semi-purified nisin or even the fermentation broth is directly used in food preservation in many cases.Thus,the reduced cost is at the expense of decreased preservation performance.To further improve the effectiveness of the fermentation product in food preservation,we develop a L.lactis fermentation system that can simultaneously produce nisin and GABA.As one of the GRAS non-protein amino acids,?-aminobutyric acid(GABA)is recently proven to be very effective in maintaining quality and improving shelf-life of foods through alleviate fruit chilling injury,oxidative damage and induce resistance against pathogens.In the nisin and GABA co-producing system,we firstly metabolically engineer a nisin producing and non-GABA producing strain L.lactis F44 for GABA production by overexpressing GadCB complex.The GABA titer reaches 2.26 g/L at 24h in shake flask fermentation and 84.2%of sodium glutamate covertion rate is attained.GadCB overexpression also facilitate nisin production through enhancing acid resistance of the strain.By applying a two-stage pH control fermentation strategy,the resulting strain yields up to 9.12 g/L GABA,which is 2.2 times more than that in the fermentation system maintaining pH 6.And the covertion rate of sodium glutamate is74.4%.Meanwhile,this strategy decreases the degradation rate of nisin.The freeze-dried fermentation product of F44/GadB1C1 exhibits excellent antioxidant capacity.Its free radical scavenging capacity of DPPH and ABTS~+are both better than BHA with the same concentration.Subsequently,we demonstrate the potential application of the fermentation product as a preservative to improve the storage performance of meat and fruit.The fermentation product can effectively alleviate lipid oxidation and microbiological contamination of pork,as well as decrease the rotting rate and surface color change of strawberry.