Nisin A前体基因nisA的过量表达对Nisin A产量的影响

Effect of overexpressing Nisin A structural gene nisA on Nisin A production

摘要:

Nisin是一种广泛应用于食品工业的抗菌素。通过基因工程手段分别构建了Nisin A前体结构基因nisA的穿梭表达载体pMG36ek-nisA和整合型载体pDG780-nisA，并转入Nisin A产生菌乳酸乳球菌Lactococcus lactis ATCC 11454中，得到两株基因工程菌FMM1和FMM2。对比工程菌和原始产生菌的生长状态及Nisin A产量，结果表明FMM1的生长速度、生物量以及发酵液的酸碱度没有显著变化，而Nisin A产量提高了31％；相反，FMM2的生物量较原始菌株显著降低，但Nisin A产量也有一定程度的提高。通过RT-PCR检测工程菌与原始产生菌Nisin A生物合成基因簇中11个基因的转录水平，结果显示FMM1和FMM2的11个基因的转录水平均有提高，其中FMM1提高更为显著。因此推测，nisA是Nisin A高产的关键因素之一，其游离型过量表达能显著提高Nisin A的产量。该研究为采用基因工程手段提高Nisin A的产量提供了新的思路，并对Nisin A的大规模工业生产有指导意义，同时，也为其他抗菌肽产生菌的基因工程改造提供了参考。

Abstract:

Nisin is an antimicrobial peptide widely used in food industry. In this study, Nisin A production in Lactococcus lactis ATCC 11454 was improved by overexpression of Nisin A structural gene nisA through introducing a shuttle expression vector pMG36ek-nisA and an integrated vector pDG780-nisA into the host strain. The differences of growth profiles and Nisin A production level between the two obtained genetic engineering strains FMM1/FMM2 and the parent strain were investigated. Our results show that while the growth profile (the growth rate, biomass and ph) of FMM1 was similar to the parent strain, its Nisin A production increased 31%. In contrast, the biomass of FMM2 was notably lower than the parent strain, while its yield of Nisin A enhanced slightly. The transcription level of genes involved in Nisin A biosynthesis in both engineering strains was further detected by RT-PCR. We found that all the 11 Nisin A biosynthetic genes in FMM1 and FMM2 had a higher transcription level than those in the parent strain, and these genes exhibited more significant increasing degree of transcription level in FMM1 which hosted the autonomous replicating nisA gene. These data suggest that expression of nisA may act as a rate-limit factor in Nisin A biosynthesis. In conclusion, this work provides a new method to improve Nisin A production by increasing the transcription level of nisA, paving the way to further large-scale industrial production of Nisin A.