【作者】 王超；

【导师】 崔艳华；

【作者基本信息】 哈尔滨工业大学 ， 化学工程与技术， 2020， 博士

【摘要】 德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckii subsp.bulgaricus,L.bulgaricus)广泛应用于世界食品发酵工业中,具有重要经济价值。菌株自身生长和应用过程中经常遇到酸性环境,低p H值对细胞生理产生不利影响,包括膜损伤、抑制酶表达和生存能力降低等。因此,明晰菌株内的耐酸调控机制,建立菌株的耐酸调控网络对于细菌的生存和菌株在工业应用都至关重要。本研究以德氏乳杆菌保加利亚亚种野生型CH3菌株、组氨酸蛋白激酶HPK1突变体CH3-H、应答调节蛋白RR1突变体CH3-R为研究对象,采用双向电泳和实时定量PCR技术,研究了在自然发酵和酸诱导条件下,由双组分信号转导系统HPK1/RR1调控的与菌株耐酸能力相关的基因;以乳酸乳球菌NZ9000为宿主菌,对关键差异基因进行异源表达和功能鉴定;采用酵母双杂交技术初步研究了双组分信号转导系统TCS1中HPK1和RR1与关键差异蛋白之间的互作关系,并初步确定了蛋白间的互作部位。研究内容及结果如下:酸胁迫处理可以有效模拟菌株在生长过程中受到的酸刺激环境。本研究采用双向电泳方法和质谱技术,分离鉴定了正常发酵和酸胁迫条件下L.bulgaricus CH3中差异蛋白,并进行了转录水平分析。在自然发酵条件下,突变体CH3-H和CH3-R与野生型CH3相比,差异表达蛋白分别为22个和24个;在酸诱导条件下,突变体CH3-H和CH3-R相比于野生型CH3,差异表达的蛋白分别为6个和20个。进一步采用实时定量PCR技术研究了差异蛋白在转录水平的表达量变化。研究确定了由双组分信号转导系统中HPK1或RR1调控的4个关键差异蛋白,即Aprt、Ddl、Opp DII和Tsf。综合以上结果表明,双组分信号转导系统HPK1/RR1通过调节多个代谢途径来实现对菌株耐酸能力的调控。本研究以乳酸乳球菌NZ9000为宿主,异源表达关键差异蛋白Aprt、Ddl、Opp DII和Tsf,旨在阐明上述关键差异蛋白在耐酸调控中的功能。四个关键差异蛋白在乳酸乳球菌NZ9000中成功表达。同时评价了异源表达菌株在酸胁迫和其他环境胁迫条件下的耐受性。研究发现,表达蛋白Aprt和Ddl显著提高了菌株的耐酸能力。异源表达蛋白Aprt和Ddl的菌株的耐酸能力,与对照菌株NZCK相比,分别提高了75倍和114倍。Opp DII和Tsf的表达对重组菌株的耐酸能力没有明显影响。此外,携带Aprt的重组菌株的胆盐耐受力增强了5倍,携带Ddl的重组菌株胆盐耐受力和对冷胁迫的耐受力分别增强了12倍和了8倍。由此可见,Aprt和Ddl是提高菌株在酸性环境中耐受性的原因之一。同时,研究表明,菌株中的耐酸调控涉及到多种代谢途径的协同作用。本研究采用酵母双杂交系统,研究了双组分信号转导系统TCS1中HPK1和RR1与菌株耐酸能力调控的关键蛋白Aprt、Ddl之间的相互作用,并确定了互作蛋白之间的互作部位。本研究初步明晰了HPK1和Ddl之间的互作关系,并初步确定HPK1-C是与Ddl发生互作的关键部位,为深入解析L.bulgaricus CH3中双组分信号转导系统HPK1/RR1在菌株耐酸调控网络与调控机制中的作用奠定了理论基础,为进一步研究HPK1、Ddl蛋白的功能提供了一些新的思路和方向。更多还原

【Abstract】 Lactobacillus delbrueckii subsp.bulgaricus is a kind of lactic acid bacteria which is widely used in the world food fermentation industry.Lactic acid bacteria often encounter acid environment during their growth and application,which has adverse effects on cell physiology,including membrane damage,inhibition of enzyme expression and transport system.Therefore,it is very important to clarify the mechanisms of acid resistance regulation and establish acid resistance regulation network for the survival and industrial application of bacteria.In this study,two-dimensional electrophoresis and real-time quantitative PCR were used to screen the genes related to acid tolerance regulated by two-component system HPK1/RR1 in histidine protein kinase HPK1 mutant CH3-H and response regulator protein RR1 mutant CH3-R compared with wild-type CH3 strain under the conditions of natural fermentation and acid induction.Lactococcus lactis NZ9000 was used for heterogenous expression and functional identification of key differentially expressed genes.The interaction between HPK1 or RR1 in two-component signal transduction system TCS1 and key differential proteins was preliminarily validated by yeast two-hybrid technique,and the key interaction sites were initially identified.The research contents and results are as follows:Acid-induced treatment can better simulate a series of genes that may be involved in the improvement of viability of strains when encounter ed with acid.Therefore,differentially expressed proteins in L.bulgaricus CH3 under normal fermentation and acid stress conditions were isolated,identified and the expression of transcriptional level was analyzed.Two-dimensional electrophoresis and mass spectrometry were used to identify the different proteins under normal fermentation and acid stress.Under natural fermentation,there were 22 differentially expressed proteins in mutant CH3-H and 24 differentially expressed proteins in mutant CH3-R compared with wild type CH3.Under acid induction,there were 6 differentially expressed proteins in mutant CH3-H and 20 differentially expressed proteins in mutant CH3-R compared with wild type CH3.Real-time quantitative PCR was used to study the expression of differentially expressed proteins related to acid stress at the transcriptional level.The above results indicate that the acid tolerance regulation of CH3 strain by two-component signal transduction system HPK1/RR1 is achieved through comprehensive regulation of multiple metabolic pathways.Heterogenous expression and functional validation of differentially expressed genes related to acid tolerance were carried out.The differentially expressed proteins Aprt,Ddl,Opp DII and Tsf regulated by HPK1 or RR1 i n the two-component signal transduction system of L.bulgaricus CH3 were successfully expressed in L.lactis NZ9000.The tolerance of the recombinant strains to acid stress and other environmental stresses were also studied compared with the control strain NZCK.Expression of Aprt and Ddl proteins had significant effects on acid tolerance of the strain,which increased 75 times and 114 times,respectively.The expression of Opp DII and Tsf had no significant effect on the acid resistance of the recombinant strains.Moreover,the bile salt tolerance of the recombinant strain carrying Aprt increased by 5 times,while that of the recombinant strain carrying Ddl increased by 12 times,and the tolerance to cold stress increased by 8 times.Thus,Aprt and Ddl are one of the reasons for the increased tolerance of strains in acidic environment.At the same time,acid-tolerance regulation in strains is a complex regulation process involving multiple metabolic pathways,and various metabolic processes play a synergistic role to achieve self-protection of strains.The interactions between two-component signal transduction proteins and acid-tolerance regulatory proteins were studied.In this project,yeast two-hybrid system was used to screen the interactions between two-component signal transduction system HPK1 or RR1 and Aprt or Ddl related to acid tolerance of CH3 strain,which were screened by two-dimensional electrophoresis,and the key interaction sites between the interacting proteins were verified.The results of this study preliminarily clarify the interaction between HPK1 and Ddl,and initially identify that HPK1-C was the key interaction site with Ddl.This study laid a theoretical foundation for understanding the acid tolerance regulatory network and mechanism regulated by the two-component signal transduction system HPK1/RR1 in L.bulgaricus CH3,and provided some new ideas and directions for further study of the functions of HPK1 and Ddl.更多还原