红球菌具有强有机溶剂耐受性、能实现功能基因的尿素诱导高表达等多种优势，逐渐成为一种应用价值巨大的重要微生物。本论文以红球菌启动子为研究对象，从红球菌启动子特征解析、高强度启动子改造以及独特的尿素诱导型启动子诱导机理发现这几方面入手，开展了一系列基础和应用研究。首先对红球菌启动子特征进行了解析并进行了高强度启动子改造和工业应用。利用转录组测序和报告基因分析挖掘了红球菌的内源型启动子，筛选获得了7个具有代表性的梯级强度启动子。对这些启动子的核心区序列分析得到红球菌-35区和-10区保守序列。将其中的两个强启动子Pnh和Pami的核心区序列突变为保守序列后，启动子强度分别提升至原来的2.2倍和7.7倍。建立了包含15个启动子-RBS组合的表达元件库，使得LacZ的最高表达量和最低表达量之间相差137倍。利用强启动子PamiM表达强稳定性腈水合酶的基因工程红球菌R. ruber TH8，在丙烯酰胺的工业化生产中表现出优异的催化性能。进一步建立了红球菌高效基因敲除方法。研究发现，利用同源重组单交换和双交换的基因敲除方法不能够实现R. ruber TH中有效的基因敲除。通过在R. ruber TH中使用尿素诱导启动子诱导表达重组酶Che9c60&Che9c61，并利用dsDNA提供同源臂，当诱导剂尿素浓度为3 g/L、同源臂长度为750 bp时，实现了高效的基因敲除。通过对R. ruber TH中两个主要的酰胺酶基因的同时敲除，在丙烯酰胺生产过程，避免了副产物丙烯酸的生成。该方法的建立为尿素诱导调控机理解析奠定了方法基础。针对红球菌独特的尿素诱导高表达腈水合酶和酰胺酶基因的现象进行了重点研究。基于转录组分析和56 kb腈水合酶-酰胺酶基因簇上下游27个基因的大片段分区逐一敲除和收缩定位，利用腈水合酶和绿色荧光蛋白GFP双报告基因体系分析尿素诱导表达结果，挖掘并发现了发挥正/负调控作用的三个调控基因。其中，nhhC基因起正调控作用，nhhD和amiE基因起负调控作用。进一步通过对这些调控基因的组合敲除得到了各调控蛋白之间的相互作用关系，发现红球菌尿素诱导调控需要三个调控因子精细合作实现。最后，基于本论文研究结果，提出了红球菌三调控因子精细协作的尿素诱导调控机理。红球菌尿素诱导具有调控严谨、诱导剂廉价的优势，具备在不同宿主菌中广泛应用的潜力。
The genus Rhodococcus is a kind of important microorganism platform with great industrial application value because of the advantages such as high tolerance to organic solvents and the ability of high protein expression level with the urea-inducible promoters. This work focused on Rhodococcus promoters and a series of basic and application researches were carried out on promoter characterization, strong promoter design and explanation of the unique urea induction mechanism.Firstly, we analyzed the characteristics of Rhodococcus promoters, artificially designed the strong promoters and applied them in industry. Transcriptome analysis and reporter gene measurement of the native Rhodococcus promoters revealed 7 representative promoters with different activity levels. The ＆＃8722;35 and ＆＃8722;10 core elements of these promoters were analyzed, and the conserved sequences were obtained. By mutating the core elements of the two strong promoters, Pnh and Pami, into the consensus sequence, the activities of the promoters were improved to 2.2 and 7.7 times, respectively. A fine-tuning promoter-RBS combination min-pool with different activity levels was successfully established and the highest LacZ expression level was 137 folds of the lowest expression level. By expressing the highly stable nitrile hydratase （NHase） with the stronger promoter PamiM, the recombinant R. ruber TH8 exhibited superior catalytic performance in the industrial production of acrylamide.Next, a high efficient gene konockout method of Rhodococcus was established. Single-crossover and double-crossover recombinations were investigated and these two methods could hardly be achieved to delete genes in Rhodococcus. By expression of the recombinases Che9c60＆Che9c61 with the urea-inducible promoter and introduction of homogenous arm with dsDNA in R. ruber TH, high gene knockout efficiency was accomplished in the optimum condition: 3 g/L urea inducer and 750 bp homogenous arm length. The two major amidases were deleted in R. ruber TH and the byproduct acrylic acid synthesis was blocked in the acrylamide production. This method forms a solid foundation for the urea induction mechanism research.Finally, we focused on the phenomenon of high NHase and amidase expression with the unique urea-inducible promoters in Rhodococcus. Based on transcriptome analysis, large fragment deletion of the 27 genes in the 56 kb NHase-amidase gene cluster and contractile localization of the regulators, three regulatory genes were found to play negative or positive roles using the NHase and green fluorescent protein GFP dual reporters as the indicator for urea-inducible expression. nhhC gene is the positive regulator and nhhD and amiE genes are the negative regulators. Furthermore, the interaction between the regulators was obtained by combinatorial knockout of the regulatory genes and it was found that the urea-inducible regulation requires fine cooperation of the three regulators. In summary, the “Lock-Key-Controller” mechanism of urea-inducible regulation was proposed which involves the fine coordination of three regulators in Rhodococcus. Because of the advantages of tight regulation and cheap urea inducer, the urea-inducible regulation of Rhodococcus has the potential to be widely used in different hosts.Firstly, we analyzed the characteristics of Rhodococcus promoters, artificially designed the strong promoters and applied them in industry. Transcriptome analysis and reporter gene measurement of the native Rhodococcus promoters revealed 7 representative promoters with different activity levels. The ＆＃8722;35 and ＆＃8722;10 core elements of these promoters were analyzed, and the conserved sequences were obtained. By mutating the core elements of the two strong promoters, Pnh and Pami, into the consensus sequence, the activities of the promoters were improved to 2.2 and 7.7 times, respectively. By expressing the highly stable nitrile hydratase （NHase） with the stronger promoter PamiM, the recombinant R. ruber TH8 exhibited superior catalytic performance in the industrial production of acrylamide.Next, a high efficient gene konockout method of Rhodococcus was established. Single-crossover and double-crossover recombinations were investigated and these two methods could hardly be achieved to delete genes in Rhodococcus. By expression of the recombinases Che9c60＆Che9c61 with the urea-inducible promoter and introduction of homogenous arm with dsDNA in R. ruber TH, high gene knockout efficiency was accomplished in the optimum condition: 3 g/L urea inducer and 750 bp homogenous arm length. The two major amidases were deleted in R. ruber TH and the byproduct acrylic acid synthesis was blocked in the acrylamide production.Finally, we focused on the phenomenon of high NHase and amidase expression with the unique urea-inducible promoters in Rhodococcus. Based on transcriptome analysis, large fragment deletion of the 27 genes in the 56 kb NHase-amidase gene cluster and contractile localization of the regulators, three regulatory genes were found to play negative or positive roles using the NHase and green fluorescent protein GFP dual reporters as the indicator for urea-inducible expression. nhhC gene is the positive regulator and nhhD and amiE genes are the negative regulators. Furthermore, the interaction between the regulators was obtained by combinatorial knockout of the regulatory genes and it was found that the urea-inducible regulation requires fine cooperation of the three regulators. In summary, the “Lock-Key-Controller” mechanism of urea-inducible regulation was proposed which involves the fine coordination of three regulators in Rhodococcus. Because of the advantages of tight regulation and cheap urea inducer, the urea-inducible regulation of Rhodococcus has the potential to be widely used in different hosts.
