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  本文關(guān)鍵詞：高產(chǎn)乳鏈菌肽基因工程菌株的構(gòu)建及其發(fā)酵條件優(yōu)化　出處：《山東大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 乳鏈菌肽 乳酸乳球菌 同源重組 過表達(dá) 工程菌株 發(fā)酵條件優(yōu)化

【摘要】：乳鏈菌肽(Nisin),是由某些乳酸乳球菌(Lactococcus lactis laactis)在其生長代謝過程中分泌的一種生物多肽類細(xì)菌素,具有良好的抑菌效應(yīng),對一些食源性腐敗菌或有害菌具有強(qiáng)烈的抑制或殺滅作用。長期以來,該抗菌肽主要作為一種安全高效的天然綠色生物防腐劑普遍用于食品加工企業(yè)。此外,在醫(yī)藥研究(抗生素替代品、生殖避孕、醫(yī)學(xué)移植、腫瘤研究)及農(nóng)業(yè)飼料等領(lǐng)域也極具廣闊的應(yīng)用前景。而在當(dāng)前經(jīng)濟(jì)與技術(shù)條件下,商品化Nisin的生產(chǎn)主要通過微生物發(fā)酵方法進(jìn)行,尚存在工業(yè)化產(chǎn)率低,生產(chǎn)成本較高等共性問題。隨著生物防腐劑市場需求的日益增大,合理選育Nisin高產(chǎn)菌株,優(yōu)化其生產(chǎn)菌發(fā)酵性能,提高Nisin工業(yè)生產(chǎn)的發(fā)酵水平,降低發(fā)酵生產(chǎn)成本,已然成為亟需解決的問題。盡管采用傳統(tǒng)誘變、基因組雜交等微生物篩選技術(shù)選育Nisin高產(chǎn)菌株已取得一定效果,但這些方法篩選過程復(fù)雜繁瑣,工作量較大,獲得高產(chǎn)菌株的效率低,而且所獲得高產(chǎn)菌株往往容易退化,不足以支持長期穩(wěn)定的發(fā)酵應(yīng)用。近些年來,隨著生物技術(shù)的快速發(fā)展與應(yīng)用,Nisin的生物合成基因簇及其代謝調(diào)控途徑已逐漸得以清晰闡明,越來越多的研究人員嘗試基于代謝途徑工程技術(shù)來構(gòu)建高效表達(dá)Nisin的基因工程菌株,以期望進(jìn)一步提高Nisin的工業(yè)化水平。目前Nisin產(chǎn)生菌的代謝工程改造主要通過對影響Nisin生物合成的相關(guān)負(fù)調(diào)控基因進(jìn)行敲除或改變其合成途徑中特定元件表達(dá)水平,或?qū)isin的生物合成途徑進(jìn)行異源表達(dá)來提高其發(fā)酵性能。本論文基于對Nisin生物合成途徑中關(guān)鍵基因的功能表達(dá)及其調(diào)控機(jī)制的認(rèn)識,以乳酸乳球菌ATCC11454基因組DNA為模板,利用PCR擴(kuò)增獲得Nisin生物合成途徑中的關(guān)鍵基因nisA(前體基因)ORF和nisRK(調(diào)控基因)片段,并通過同源重組技術(shù)將nisAORF和nisRK基因克隆至高拷貝的表達(dá)質(zhì)粒pMG36e中,構(gòu)建得到重組表達(dá)載體pMG36e-nisA和pMG36e-nisA-nisRK。以Nisin產(chǎn)生菌乳酸乳球菌LS01為受體菌,通過電轉(zhuǎn)化和抗性篩選,獲得過表達(dá)nisA的工程菌株LS01/pMG36e-nisA及聯(lián)合過表達(dá)nisA和nisRK的工程菌株LS01/pMG36e-nis4-nisRK。搖瓶發(fā)酵結(jié)果顯示,工程菌株LS01/pMG36e-nis4 的 Nisin 效價可達(dá) 1955 IU/mL,比原始菌株(1472 IU/mL)提高了 32.8%;工程菌株LS01/pMG36e-nisA-nisRK的Nisin效價峰值達(dá)到2470 IU/mL,比原始菌株提升了 67.8%。生物量測定分析表明,工程菌LS01/pMG36e-nisA和LS01/pMG36e-nisA-nisRK的生長狀況相對于原始菌株也發(fā)生了較明顯的變化,其最大生物量相對于原始菌株均略有降低,且后者下降更明顯。半定量RT-PCR檢測結(jié)果表明,目的基因nisA、nisR和nisK在工程菌株LS01/pMG36e-nisA-nisRK中的轉(zhuǎn)錄水平均得到了上調(diào),證實(shí)了基因nisA、nisR和nisK的過表達(dá)對Nisin的生物合成具有顯著促進(jìn)作用。利用響應(yīng)面實(shí)驗(yàn)設(shè)計(jì)對工程菌株LS01/pMG36e-nisA-nisRK進(jìn)行搖瓶發(fā)酵培養(yǎng)基的篩選與優(yōu)化,以進(jìn)一步提升工程菌株的發(fā)酵性能。結(jié)果獲得優(yōu)化后培養(yǎng)基組成為:蔗糖25.06 g/L,玉米漿粉末8.8 g/L,酵母粉10 g/L,KH2PO4 5 g/L,NaCl 2 g/L,MgS04-7H20 0.3 g/L,CaC033.5g/L,Tween-80 2.7 g/L。搖瓶驗(yàn)證結(jié)果表明,優(yōu)化后工程菌株LS01/pMG36e-nisA-nisRK的Nisin發(fā)酵水平可達(dá)到3037 IU/mL,比優(yōu)化處理前提升了 23%,比原始菌株產(chǎn)量更是提高了 106%。利用所得的最佳發(fā)酵培養(yǎng)基與工程菌株LS01/pMG36e-nisA-nisRK在10 L發(fā)酵罐中進(jìn)行分批補(bǔ)料發(fā)酵研究,結(jié)果表明優(yōu)產(chǎn)工程菌株在該發(fā)酵體系中Nisin發(fā)酵效價可達(dá)6380 IU/mL,比原始生產(chǎn)菌在初始發(fā)酵培養(yǎng)基條件中產(chǎn)量提高了 46.2%,較大地發(fā)揮了工程菌株的發(fā)酵潛力。綜上所述,利用基因工程手段結(jié)合發(fā)酵優(yōu)化不僅可以快速準(zhǔn)確篩選到目標(biāo)高產(chǎn)菌株,克服原始產(chǎn)生菌作為乳鏈菌肽生產(chǎn)菌的劣勢,而且對乳鏈菌肽的工業(yè)化擴(kuò)大生產(chǎn)及其應(yīng)用具有重要意義和潛在參考價值。
[Abstract]:Nisin (Nisin), is by some Lactococcus lactis (Lactococcus lactis laactis) is a kind of biological peptide bacteriocin secreted in the metabolism process, has good antibacterial effect, has a strong inhibitory or killing effects on some food borne spoilage bacteria or harmful bacteria. For a long time, the antimicrobial peptides are as a secure and efficient natural green biological preservatives commonly used in food processing enterprises. In addition, in the medical research (Research on antibiotic alternative, reproductive medicine, transplantation, tumor) and application prospect of agricultural feed and other fields is also very wide. But in the current economic and technological conditions, commercial production mainly through Nisin microbial fermentation method, there are industrial low yield, higher production cost and common problems. With Biopreservatives the increasing market demand, reasonable breeding of high yield Nisin strains and its optimization The production of fermentation performance, improve the fermentation level of the industrial production of Nisin, reduce the cost of fermentation production, has become the urgent problem. Although the traditional mutation, genome hybridization screening of microbial technology in breeding of high yield Nisin strains have achieved certain results, but the method of screening process is complex, large workload, low efficiency and high yield strains. The high yield strains are often susceptible to degradation, not enough to support the long-term stability of fermentation applications. In recent years, with the rapid development and application of biological technology, biosynthetic gene cluster and its metabolic regulation way Nisin has gradually clear, more and more researchers try based on metabolic pathway engineering technology to construct high expression gene engineering strain Nisin in order to further improve the Nisin, the level of industrialization. The metabolic engineering for the production of Nisin transformation The relevant negative regulation of Nisin biosynthesis genes affect the knockout levels of specific expression elements of the pathway or change, or on the biosynthesis pathway of Nisin for heterologous expression to improve the fermentation performance. This paper understanding function on the expression of Nisin gene in the key biosynthetic pathway and its regulation mechanism based on Lactococcus lactis ATCC11454 genome DNA as template, amplified the key gene nisA in the Nisin biosynthetic pathway by PCR (precursor gene) and nisRK (ORF gene) fragment, and through homologous recombination pMG36e expression plasmid nisAORF and gene cloning of nisRK high copy, to construct the recombinant expression vector of pMG36e-nisA and pMG36e-nisA-nisRK. in Nisin producing strains of Lactococcus lactis LS01 is the receptor bacteria by electroporation and resistance screening, get the overexpression of nisA and associated engineering strain LS01/pMG36e-nisA Over expression of nisA and nisRK engineering strain LS01/pMG36e-nis4-nisRK. fermentation results showed that the engineering strain LS01/pMG36e-nis4 Nisin titer of 1955 IU/mL, than the original strain (1472 IU/mL) increased by 32.8%; the Nisin titer peak engineering strain LS01/pMG36e-nisA-nisRK up to 2470 IU/mL, compared with the original strain analysis showed that 67.8%. enhance the determination of biomass, growth status of engineering strains LS01/pMG36e-nisA and LS01/pMG36e-nisA-nisRK compared to the original strain also changed obviously, the maximum biomass decreased slightly compared to the original strain, and the latter decreased more significantly. Semi quantitative RT-PCR results showed that nisA gene, the transcriptional level of nisR and nisK in engineering strain LS01/pMG36e-nisA-nisRK were up-regulated genes was confirmed by nisA for over expression, significantly promote the biosynthesis of Nisin with nisR and nisK. The response surface experimental design for screening and optimization of fermentation medium of strain in LS01/pMG36e-nisA-nisRK use, to further enhance the performance of fermentation engineering strains. The results obtained the optimized medium was: sucrose 25.06 g/L, corn steep powder 8.8 g/L, yeast powder 10 g/L, KH2PO4 5 g/L, NaCl 2 g/L, MgS04-7H20 0.3 g/L CaC033.5g/L, Tween-80, 2.7 g/L. shake flask test showed that the Nisin level of fermentation engineering strain LS01/pMG36e-nisA-nisRK after optimization can reach 3037 IU/mL, increased by 23% than the original strain before the optimization process, the yield is improved by using 106%. optimal fermentation medium and engineering strain LS01/pMG36e-nisA-nisRK by fed batch fermentation in 10 L fermentation the results show that the tank, excellent production engineering strain Nisin in the fermentation system in the fermentation titer of 6380 IU/mL, than the original producing bacteria in the early fermentation culture of origin A medium in yield increased by 46.2%, played a fermentation potential engineering strain greatly. In summary, the use of genetic engineering techniques combined with optimization of fermentation can not only quickly and accurately to the target screening of high yield strains, overcome the original producing bacteria as nisin producing strain and disadvantages of nisin industrialization to expand production and its application has the important significance and potential value.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：TQ920.6

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