高产乳链菌肽基因工程菌株的构建及其发酵条件优化

发布时间：2018-01-18 06:08

本文关键词：高产乳链菌肽基因工程菌株的构建及其发酵条件优化　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：乳链菌肽(Nisin),是由某些乳酸乳球菌(Lactococcus lactis laactis)在其生长代谢过程中分泌的一种生物多肽类细菌素,具有良好的抑菌效应,对一些食源性腐败菌或有害菌具有强烈的抑制或杀灭作用。长期以来,该抗菌肽主要作为一种安全高效的天然绿色生物防腐剂普遍用于食品加工企业。此外,在医药研究(抗生素替代品、生殖避孕、医学移植、肿瘤研究)及农业饲料等领域也极具广阔的应用前景。而在当前经济与技术条件下,商品化Nisin的生产主要通过微生物发酵方法进行,尚存在工业化产率低,生产成本较高等共性问题。随着生物防腐剂市场需求的日益增大,合理选育Nisin高产菌株,优化其生产菌发酵性能,提高Nisin工业生产的发酵水平,降低发酵生产成本,已然成为亟需解决的问题。尽管采用传统诱变、基因组杂交等微生物筛选技术选育Nisin高产菌株已取得一定效果,但这些方法筛选过程复杂繁琐,工作量较大,获得高产菌株的效率低,而且所获得高产菌株往往容易退化,不足以支持长期稳定的发酵应用。近些年来,随着生物技术的快速发展与应用,Nisin的生物合成基因簇及其代谢调控途径已逐渐得以清晰阐明,越来越多的研究人员尝试基于代谢途径工程技术来构建高效表达Nisin的基因工程菌株,以期望进一步提高Nisin的工业化水平。目前Nisin产生菌的代谢工程改造主要通过对影响Nisin生物合成的相关负调控基因进行敲除或改变其合成途径中特定元件表达水平,或对Nisin的生物合成途径进行异源表达来提高其发酵性能。本论文基于对Nisin生物合成途径中关键基因的功能表达及其调控机制的认识,以乳酸乳球菌ATCC11454基因组DNA为模板,利用PCR扩增获得Nisin生物合成途径中的关键基因nisA(前体基因)ORF和nisRK(调控基因)片段,并通过同源重组技术将nisAORF和nisRK基因克隆至高拷贝的表达质粒pMG36e中,构建得到重组表达载体pMG36e-nisA和pMG36e-nisA-nisRK。以Nisin产生菌乳酸乳球菌LS01为受体菌,通过电转化和抗性筛选,获得过表达nisA的工程菌株LS01/pMG36e-nisA及联合过表达nisA和nisRK的工程菌株LS01/pMG36e-nis4-nisRK。摇瓶发酵结果显示,工程菌株LS01/pMG36e-nis4 的 Nisin 效价可达 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效价峰值达到2470 IU/mL,比原始菌株提升了 67.8%。生物量测定分析表明,工程菌LS01/pMG36e-nisA和LS01/pMG36e-nisA-nisRK的生长状况相对于原始菌株也发生了较明显的变化,其最大生物量相对于原始菌株均略有降低,且后者下降更明显。半定量RT-PCR检测结果表明,目的基因nisA、nisR和nisK在工程菌株LS01/pMG36e-nisA-nisRK中的转录水平均得到了上调,证实了基因nisA、nisR和nisK的过表达对Nisin的生物合成具有显著促进作用。利用响应面实验设计对工程菌株LS01/pMG36e-nisA-nisRK进行摇瓶发酵培养基的筛选与优化,以进一步提升工程菌株的发酵性能。结果获得优化后培养基组成为:蔗糖25.06 g/L,玉米浆粉末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。摇瓶验证结果表明,优化后工程菌株LS01/pMG36e-nisA-nisRK的Nisin发酵水平可达到3037 IU/mL,比优化处理前提升了 23%,比原始菌株产量更是提高了 106%。利用所得的最佳发酵培养基与工程菌株LS01/pMG36e-nisA-nisRK在10 L发酵罐中进行分批补料发酵研究,结果表明优产工程菌株在该发酵体系中Nisin发酵效价可达6380 IU/mL,比原始生产菌在初始发酵培养基条件中产量提高了 46.2%,较大地发挥了工程菌株的发酵潜力。综上所述,利用基因工程手段结合发酵优化不仅可以快速准确筛选到目标高产菌株,克服原始产生菌作为乳链菌肽生产菌的劣势,而且对乳链菌肽的工业化扩大生产及其应用具有重要意义和潜在参考价值。
[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.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ920.6

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