典型产纤维小体梭菌遗传操作平台的建立
文献类型：学位论文
| 作者 | 张杰 |
| 学位类别 | 博士 |
| 答辩日期 | 2015-05 |
| 授予单位 | 中国科学院大学 |
| 授予地点 | 北京 |
| 导师 | 崔球 |
| 关键词 | 热纤梭菌 解纤维梭菌 遗传改造 木质纤维素 纤维小体 ClosTron |
| 学位专业 | 生物化学与分子生物化学 |
| 中文摘要 | 木质纤维素生物质原料充足，来源广泛，环境友好，因此，生物质能有可能成为全面替代化石能源的新一代绿色能源，从而解决目前能源紧缺、环境污染等国际性问题。整合生物加工技术（Consolidated bioprocessing, CBP）可以实现从木质纤维素原料到生物能源产品的直接转化，是目前最有希望实现纤维素生物质能源工业化的技术之一。以解纤维梭菌（Clostridium cellulolyticum）和热纤梭菌（Clostridium thermocellum）为代表的产纤维小体梭菌具有高效的木质纤维素降解能力，是优良的生物质CBP转化候选菌株。然而，产纤维小体梭菌的遗传改造手段相对缺乏，已有技术仍存在缺陷，这严重阻碍了产纤维小体梭菌的基因工程改造，以及木质纤维素生物转化的进程。为了建立和完善产纤维小体梭菌的遗传改造平台，本论文以解纤维梭菌和热纤梭菌为研究对象，主要在可控基因表达工具的构建以及无疤基因组编辑工具的建立两个部分开展工作，具体如下： I. 可控基因操作系统的建立 我们在解纤维梭菌中建立了一个新型的阿拉伯糖诱导的可控操作系统（Arabinose-inducible genetic operation system, ARAi），其核心是一种阿拉伯糖诱导启动子，由来源于丙酮丁醇梭菌ptk（磷酸转酮酶）基因的启动子和抑制蛋白AraR表达基因组成。ARAi包括基于不依赖于氧气的荧光蛋白报告系统、灵敏高效的反向筛选标记，以及可诱导的基因打靶系统。 首先，我们以PpFbFPm（厌氧绿色荧光蛋白）和GusA（β-葡糖醛酸酶）作为报告基因对ARAi系统的效率和严谨性进行了研究。结果显示，ARAi系统在解纤维梭菌中具有很好的严谨性，同时确定了L-阿拉伯糖的最佳诱导浓度为0.1 g/L，最佳诱导时间为2 h。在最佳诱导条件下，GusA活性可以达到9.0×104 U/mg，诱导强度高达800倍，是目前梭菌中所报道的诱导强度最高的诱导表达系统。在确立了最佳诱导条件后，我们又对ARAi系统的诱导物特异性、诱导的持续性及其它单糖对诱导效果的影响进行了研究。实验证明，除了L-阿拉伯糖外，其它测试单糖对ARAi系统都没有诱导效果，但是多数单糖会对L-阿拉伯糖的诱导起到抑制作用。这说明ARAi系统具有诱导物的专一性，并且在其诱导过程中可以通过添加其它单糖来抑制目标基因的表达，增加了诱导的灵活性。由于L-阿拉伯糖可以被解纤维梭菌作为碳源所代谢，诱导的持续性也是我们所必须考虑的。结果表明，解纤维梭菌可以在12 h内将1 g/L的L-阿拉伯糖消耗完毕，因此在诱导过程中应依据诱导时间的长短确定L-阿拉伯糖的诱导浓度。 其次，我们利用该阿拉伯糖诱导系统建立了基于mazF基因的反向筛选标记，并对已有的ClosTron系统进行改良，显著提高其打靶特异性。我们将控制Intron表达的组成型表达启动子换成ARAi启动子后获得了新型的可诱导ClosTron系统（inducible ClosTron, iClosTron）。以解纤维梭菌mspI和cipC为目标靶基因，我们对iClosTron系统的打靶效率和打靶特异性进行了研究。实验发现，与传统的ClosTron系统相比，虽然iClosTron系统的打靶效率出现了较大程度的降低，由原来的接近100%降低到了小于10%，但是其打靶特异性却从之前的0升高到了接近100%。这说明，iClosTron系统通过对Intron表达量的控制成功解决了传统ClosTron系统在解纤维梭菌中的脱靶问题，实现了基因的精确敲除。 II. 无疤遗传操作系统的建立 无疤遗传操作可实现基因的敲除、敲入、替换、点突变等精准的基因组编辑工作，是代谢工程及合成生物学研究不可缺少的遗传改造工具。我们利用前期构建的mazF以及tdk等反向筛选标记，并借鉴ACE（Allele-Coupled Exchange）技术可以通过长短同源臂控制同源重组顺序的方法，在嗜中温的解纤维梭菌和嗜高温的热纤梭菌中都成功建立了无疤遗传操作系统。 在构建热纤梭菌的无疤遗传操作系统过程中，我们发现了热纤梭菌胞内可能存在着基因和蛋白质水平上的“自我保护”系统。已知氟-脱氧尿嘧啶（Fluoro-deoxyuracil, FUDR）经Tdk的催化会生成有毒的氟尿嘧啶脱氧核苷酸（F-dUMP）进而造成细胞的死亡。我们在进行基于tdk的反向筛选时发现，外源基因tdk在FUDR的筛选压力下会被热纤梭菌胞内的转座酶或整合酶所失活，从而失去反筛的效果。已经敲入到基因组中的外源基因cglT在热纤梭菌胞内表达后却遭到了降解，其降解机制尚不清楚。这说明，热纤梭菌的生理生化及遗传体系中还有很多奥妙等待挖掘。 综上所述，我们建立了解纤维梭菌的ARAi系统，升级了传统的ClosTron技术，并在解纤维梭菌和热纤梭菌中构建了无疤遗传操作系统。这些遗传改造平台的建立为最终获得高效的CBP菌株及实现木质纤维素生物能源的工业化奠定了基础。 |
| 英文摘要 | Biomass-based energy has the potential to fully replace the fossil fuels and solve the international energy crisis and environmental pollution, because lignocellulosic biomass is the most abundant and sustainable organic feedstocks on the Earth. Consolidated bioprocessing (CBP) is an outstanding strategy for industrialized production of cellulosic fuels because it converts lignocellulosic raw materials directly to bio-based energy products. Clostridium cellulolyticum and Clostridium thermocellum are typical cellulolytic cellulosome-producing strains, and are considered promising candidates for CBP production. However, the lack of genetic tools and the drawbacks of existing technologies hinder the process of genetic engineering of cellulosome-producing strains for lignocellulose bioconversion. Therefore, the goals of this study are to develop and improve the genetic modification platform for C. cellulolyticum and C. thermocellum, and the dissertation includes two main contents: the development of controllable gene expression tool, and the construction of markerless genetic manipulation system. I. Development of controllable genetic operation system We constructed a novel arabinose-inducible genetic operation system (ARAi) in C. cellulolyticum, which was derived from an arabinose-inducible promoter containing the promoter of ptk gene and the araR regulator expression cassette of C. acetobutylicum. The ARAi system includes an oxygen-independent fluorescent protein reporting system, a sensitive counterselection genetic marker, and an inducible gene targeting system. Firstly, we used PpFbFPm and GusA as reporter genes to investigate the efficiency and stringency of the ARAi system. The results indicated that the ARAi system had high stringency in C. cellulolyticum, and the optimal induction condition was using 0.1 g/L L-arabinose as inducer and incubing for 2 h. Under the optimal induction condition, the GusA activity was approximately 9.0×104 U/mg and the gene expression was up-regulated over 800-fold, which was the highest inducing activity in Clostridium so far according to our knowledge. Afterwords, the inducer specificity, induction stability, and inhibition effect of the ARAi system were tested in C. cellulolyticum. The results demonstrated that all selected sugars had no inductive effect on the ARAi system except L-arabinose. Surprisingly, the addition of some sugars significantly inhibited the induction activity of L-arabinose. These indicated that L-arabinose was the specific inducer of the ARAi system, and the addition of some other monoses can ‘turn down’ the inducing activity of L-arabinose in C. cellulolyticum, which may benefit the large range of flexible regulation. To test the induction stability of the ARAi system, we also analyzed the utilization of L-arabinose by C. cellulolyticum. The results showed that 1 g/L L-arabinose could be completely utilized in 12 h. So the concentration of L-arabinose used in C. cellulolyticum should be adjusted based on the induction period. Secondly, we established a MazF-based counterselection marker on the basis of ARAi, and improved the traditional ClosTron system by significantly increasing the target specificity. We developed a novel inducible ClosTron (iClosTron) by replacing the constitutive promoter of the intron with the newly developed arabinose-inducible promoter. Using mspI and cipC as the target genes, the target efficiency and specificity of iClosTron were investigated in C. cellulolyticum. Compared with the traditional ClosTron, the target efficiency of iClosTron was decreased, but the target specificity was dramatically increased from 0 to approximately 100%. These results proved that iClosTron could avoid off-target integrations by controlling the intron expression, and can be used for the precise gene targeting in C. cellulolyticum. II. Development of markerless genetic manipulation system Markerless genetic manipulation system is an indispensable genetic modification tool for metabolic engineering and synthetic biology research, which can fulfill precise genome editing, such as gene knock-out, gene knock-in, gene replacement and site-directed mutation. We developed two counterselection markers mazF and tdk in our early work. Based on the two markers and the ACE (Allele-Coupled Exchange) technology which can control the order of homologous recombination by determining the length of the homologous arms, markerless genetic manipulation systems were constructed in both mesophilic C. cellulolyticum and thermophilic C. thermocellum. In the process of the construction of the markerless genetic manipulation system, we found that the “self-protection” system might exist in C. thermocellum. Tdk can catalyze the conversion of F-dUMP from FUDR (Fluoro-deoxyuracil), while F-dUMP is a cytotoxin that can cause cell death. We found the exogenous gene tdk was inactivated by intracellular transposase or integrase under the selection pressure of FUDR. In addition, the exogenous protein CglT was degraded after its expression in C. thermocellum. Although the degradation mechanism is unclear by now, these results indicated the “self-protection” system in C. thermocellum. The results also suggested that the improvement of genetic modification platform in C. thermocellum still has a long way to go. In conclusion, we have constructed an novel arabinose-inducible gene operation system ARAi in C. cellulolyticum, with which we have improved the traditional ClosTron technology by decreasing the off-target frequency, and developed the markerless genetic manipulation system both in C. cellulolyticum and C. thermocellum. All of these genetic tools constitute a genetic modification platform for cellulosome-producing Clostridium strains, and will contribute to the construction of recombinant strains for the industrialization of lignocellulose-based biofuels via CBP route. |
| 学科主题 | 分子生物学 |
| 语种 | 中文 |
| 公开日期 | 2020-06-30 |
| 源URL | [http://ir.qibebt.ac.cn/handle/337004/8084]  |
| 专题 | 青岛生物能源与过程研究所_代谢物组学团队 |
| 作者单位 | 中国科学院青岛生物能源与过程研究所 |
| 推荐引用方式 GB/T 7714 | 张杰. 典型产纤维小体梭菌遗传操作平台的建立[D]. 北京. 中国科学院大学. 2015. |
入库方式： OAI收割
来源：青岛生物能源与过程研究所
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