Nisin A前體基因nisA的過量表達對Nisin A產量的影響

樊苗苗，邱一敏，劉晨，冀志霞，馬昕，虞沂，陳守文

1 華中農業大學 農業微生物學國家重點實驗室，湖北 武漢 430070

2 武漢大學藥學院，湖北 武漢 430071

工業生物技術

Nisin A前體基因nisA的過量表達對Nisin A產量的影響

樊苗苗1，邱一敏1，劉晨1，冀志霞1，馬昕1，虞沂2，陳守文1

1 華中農業大學 農業微生物學國家重點實驗室，湖北 武漢 430070

2 武漢大學藥學院，湖北 武漢 430071

Nisin是一種廣泛應用于食品工業的抗菌素。通過基因工程手段分別構建了Nisin A前體結構基因nisA的穿梭表達載體 pMG36ek-nisA和整合型載體 pDG780-nisA，并轉入 Nisin A產生菌乳酸乳球菌Lactococcus lactisATCC 11454中，得到兩株基因工程菌FMM1和FMM2。對比工程菌和原始產生菌的生長狀態及Nisin A產量，結果表明FMM1的生長速度、生物量以及發酵液的酸堿度沒有顯著變化，而Nisin A產量提高了31%；相反，FMM2的生物量較原始菌株顯著降低，但Nisin A產量也有一定程度的提高。通過RT-PCR檢測工程菌與原始產生菌Nisin A生物合成基因簇中11個基因的轉錄水平，結果顯示FMM1和FMM2的11個基因的轉錄水平均有提高，其中FMM1提高更為顯著。因此推測，nisA是Nisin A高產的關鍵因素之一，其游離型過量表達能顯著提高Nisin A的產量。該研究為采用基因工程手段提高Nisin A的產量提供了新的思路，并對Nisin A的大規模工業生產有指導意義，同時，也為其他抗菌肽產生菌的基因工程改造提供了參考。

Nisin A，nisA，乳酸乳球菌ATCC 11454，過量表達

Abstract:Nisin is an antimicrobial peptide widely used in food industry. In this study, Nisin A production inLactococcus lactisATCC 11454 was improved by overexpression of Nisin A structural genenisAthrough introducing a shuttle expression vector pMG36ek-nisAand anintegrated vector pDG780-nisAinto 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 replicatingnisAgene. These data suggest that expression ofnisAmay 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 ofnisA, paving the way to further large-scale industrial production of Nisin A.

Keywords:Nisin A,nisA,Lactococcus lactisATCC 11454, overexpression

乳鏈菌肽 (Nisin)，亦稱乳酸鏈球菌素或尼生素，是乳酸乳球菌Lactococcus lactis某些亞種在生長過程中所產生的一類屬于羊毛硫抗生素家族的天然活性多肽類細菌素。它對許多革蘭氏陽性菌有強烈的抑制作用。……