| Nisin is a kind of antibacterial peptides produced by Lactococcus lactis.It is generally used in meat processing,dairy fermentation,canned food production,etc.Compared with synthetic chemical preservatives,nisin can be decomposed by proteases in the gastrointestinal tract so it is widely used as a natural and safe biological preservative.Besides,it is the only one approved for food addition.In addition,nisin has attracted mounting attention by researchers because of its potential application in the fields of healthcare and veterinary medicine.However,the low yield of Nisin fermentation in industrial production leads to high cost of use,which limits the large-scale application of Nisin.Therefore,breeding high-yield strains as well as optimizing fermentation conditions is of great significance in industrial production.There are two types of strain breeding methods-mutation breeding and genetic engineering breeding.Mutation breeding is time-consuming while the latter one is targeted and efficient,so it is more prevalent in practical application.Lactococcus lactis is a kind of gram-positive bacteria with dense cell walls which are an obstacle for genes entering.It increased the difficulty of genetic engineering and reduced the transformation efficiency.Therefore,it is necessary to optimize the transfer conditions to increase transformation efficiency.In this paper,we studied the effect of different types of cell wall weakening agents on transformation efficiency and selected ampicillin sodium as the weakening agent.After that,electric field strength,frequency and pulse duration were studied one by one.Finally,we found that the highest transformation efficiency appeared when the electric field strength was 10 kV/cm,frequency was 1500 Hz,and pulse durations was 5 milliseconds.Furthermore,the effect of different recovery time on transformation efficiency was tested,and the optimal time was determined to be 4 hours.We constructed an electrical transformation system of nisin high-yield strain LS03.Basing on previous studies,the expression levels of nisin biosynthesis genes are closely related to fermentation yield.So,we took the genome DNA of Lactococcus lactis ATCC11454 as a template to clone gene nisA.We construct a recombinant plasmid pMG36e-nisA and transformed it into LS03.Genetically engineered bacteria LS03/pMG36e-hisA was constructed.After related tests we found that the expression level of nisA has been increased by two times.Comparing the growth curve and fermentation yield before and after genetic engineering,we found that the overexpression of nisA could reduce the biomass of bacteria because of the overexpression of nisA has little effect on growth.The experimental results of fermentation yield showed that the up-regulation of the prenisin gene nisA increased nisin fermentation yield by 25%,from 1901 IU/mL to 2386 IU/mL.The up-regulation of nisA gene puts forward higher requirements on bacterial metabolism so we optimized the composition of fermentation medium.The optimal medium composition was determined by Plackett-Burman design and response surface methodology.The optimal medium composition is sucrose 26.50 g/L,yeast extract 10 g/L,corn steep powder 10.1 g/L,KH2PO4 5 g/L,NaCl 2 g/L,MgSO4ยท7H2O 0.2 g/L,tween-80 1.79 mL/L.Further fermentation verification showed that nisin production was increased to 2875 IU/mL after optimizing the medium,which was 20%higher compared to original yield.We improved the fermentation capacity of genetically engineered strain. |
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