Improving Nitrogen Source Utilization From Defatted Soybean Meal For Nisin Production By Enhancing Proteolytic Function Of Lactococcus Lactis F44

Nisin,one kind of natural antimicrobial peptide,is produced by certain strains of Lactococcus lactis.It has been widely used as a safe and effective food preservative and a potential agent in veterinary and pharmaceutical products.L.lactis is a nutritionally fastidious microorganism due to the lack of many metabolic pathways,especially the biosynthetic pathways of amino acids.Therefore,the nitrogen source of fermentation medium is important in the production of nisin.L.lactis has intricate machinery for proteolysis which converts exogenous proteins to peptides and then to free amino acids.The proteolytic system has great significance for improving L.lactis growth and promoting the formation metabolite.Here we use defatted soybean meal(DSM)as sole nitrogen source to support L.lactis growth and nisin production.To further increase nitrogen utilization of DSM hydrolysates,promote strain growth and increase nisin production,we enhanced the proteolytic function in L.lactis through expressing the related enzymes.Taking the pH change of L.lactis growth and the molecular weight distribution of peptides in DSM hydrolysates into consideration,to further degrade protein and macromolecule peptides in DSM medium,improve the DSM degree of hydrolysis,promote strain growth and increase nisin production,we introduced several extracellular protease genes from Bacillus subtilis 168 to F44.Engineered strains were fermented in DSM medium for detecting their growth and nisin production.The results indicated that F44/pnprB exhibited the highest biomass accumulation and nisin titer.Since the content of some amino acids such as glutamate and methionine in DSM hydrolysates was very low,it is necessary to increase the supply of these amino acids.To further degrade the peptides which were rich in these amino acids,we overexpressed several peptidases for enhancing the activity of some specific peptidases.The resulting strains with higher nisin productions were selected,peptidases that resulted to a better nisin production is PepF and PepM,the nisin yield was increased from 3879.58 IU / mL to 4445.34IU/mL and 4389.91 IU/mL,respectively.In addition,we investigated the cell density and nisin production of engineered strains in DSM medium with 25% addition of DSM hydrolysates.The results showed that NprB,OppA,PepF and PepM expressed strains were capable of achieving efficient nisin production,indicating that enhancing the activity of these proteolysis related genes could significantly increase the utilization efficiency of DSM proteins.Furthermore,we constructed an artificial proteolytic system through combined expression of four genes nprB,oppA,pepF and pepM to enhance the function of the proteolytic system to further promote cell growth and increase nisin production.The highest nisin titer of the engineered strain BAFM was up to 5288.89 IU/mL,increased by 36.3% compared with F44 strain in DSM medium.BAFM was significantly reduced and 30% of DSM hydrolysates could be saved.Thus the cost of nisin production could be further reduced.The strategy described here offers opportunities for low-cost L.lactis fermentation and large-scale nisin production in industry.