嗜盐嗜碱多能硫碱弧菌D306基因组编辑和脱硫途径工程研究

硫化氢通常存在于石油和天然气及其下游工艺中，由于其可形成酸雨，对环境的危害很大。多能硫碱弧菌 D306是一种化能自养型、GC含量高的革兰氏阴性嗜盐嗜碱细菌，对硫化氢有较强的脱硫作用，产物是单质硫和硫酸根。为了加强多能硫碱弧菌 D306对天然气的脱硫作用和单质硫的生成，本论文努力解决三个问题，即缺乏基因组编辑方法、硫代谢不明确和硫酸根的产生、 对工业设备和细胞的毒害作用。首先，利用已知的菌株信息，采用细胞死亡诱导 (cell death induced, CDI) 策略，开发出了编辑效率为8.2%的多能硫碱弧菌基因组编辑系统。建立了嗜盐嗜碱多能硫碱弧菌 D306降落PCR (touchdown PCR) 技术，利用不同表达水平的Cas9蛋白和sgRNA对基因组编辑系统进行优化，并通过CDI策略进行筛选，优化后的系统效率提高到41.2%。因此，CDI策略成功简化了多能硫碱弧菌CRISPR系统开发的工作流程，并可以应用于其他极端微生物。第二，我们在多能硫碱弧菌 D306中鉴定了约50个硫代谢相关蛋白，并推测了它们的定位，深化了对D306菌株硫代谢途径的认识。利用CRISPR系统敲除了产生硫酸根的四个关键基因，强化了单质硫的生成。与野生型菌株相比，HdrB基因的敲除使多能硫碱弧菌在以硫代硫酸钠和硫化钠为硫源时，硫酸盐的产量分别下降了20.8%和55.1%。这意味着HdrB的敲除可以防止菌株消耗硫作为能量来源，可以产生更多的单质硫。这些结果也被电子显微镜观察和菌株生长曲线所证实。第三，构建了严紧调控的诱导表达体系，设计、构建、检测和验证了Cas9蛋白、Ⅱ类CRISPR主效应蛋白等天然毒性蛋白的表达。采用补料分批策略提高了多能硫碱弧菌D306的生长速度和生物量，为提高生物脱硫效率奠定了基础。所构建的系统使用铁吸收调节剂调控基因转录水平，使蛋白表达量增加到对照组的27倍。利用该系统成功表达了一种剧毒蛋白，验证了系统的功能。第四，初步构建了具有通用性的多基因编辑载体系统。选择具有通用性的tRNA作为基因编辑靶标，在大肠杆菌中针对11种不同的tRNAs进行了多基因编辑研究，其中，1种tRNAs来源于多能硫碱弧菌D306，1种来源于水稻，9种来源于大肠杆菌。研究结果显示，所构建的多基因编辑载体对LeuW，ValU和LysV的编辑效率分别达到100%，66.7% 和33.3%。综上所述，我们首次在嗜盐嗜碱多能硫碱弧菌 D306中构建了一个基于CRISPR- cas9系统和细胞死亡诱导策略的敲除系统。该体系通过敲除硫酸盐产生途径的基因来提高脱硫效率。结果表明，该方法具有较好的应用价值。经过设计、构建、测试和验证的过程，开发了另一种利用铁抑制蛋白诱导蛋白表达的系统。最后，在大肠杆菌中进行了多重CRISPR的研究，为将来在嗜盐嗜碱多能硫碱弧菌 D306中的应用提供参考。;Hydrogen sulfide, normally found in oil and gas and their down streaming processes, represents a highly toxic environmental threat by forming acid rain. Thioalkalivibrio versutus D306, an autotrophic; high GC content and gram-negative polyextremophile, can strongly desulfurize natural gas and produce sulfur and the less favorable, sulfate. To strengthen the power of T. versutus D306 as a natural gas desulfurizing bacterium, this thesis aimed to solve three different obstacles for this objective, which are lack of genome editing method, unclear sulfur metabolism and unfavorable sulfate production which is both; harmful to industrial equipment and toxic to the cells. To develop efficient genome editing strategy for T. versutus D306 using CRISPR-cas9 system based upon known strain information, cell death induced (CDI) strategy was followed. The strategy used cell death induction, calculated as colony forming units, as an indicator for screening the best knockout plasmid without the need to check colonies using PCR.Firstly, a CRISPR-cas9 system with editing efficiency of 8.2% of the screened colonies was first developed. Further optimization of the system, using different expression levels of Cas9 protein and sgRNA, was screened again by the CDI strategy. The editing efficiency was increased to 41.2% using touchdown PCR technique developed especially for T. versutus D306. Therefore, CDI strategy showed a success in simplifying workflow for CRISPR system development in T. versutus D306 and can be applied to other autotrophs or polyextremophiles. Secondly, 50 sulfur related proteins were identified in T. versutus D306 and their localization were assumed, which gave us a clear picture for different sulfur metabolism steps in the strain. The CRISPR system was used to knockout four sulfate producing key genes to enhance the desulfurization process. HdrB protein knockout enabled us to decrease sulfate production by 20.8 % and 55.1 % for thiosulfate and sulfide grown T. versutus, respectively, as compared to the native strain, which means more sulfur, was produced. It also prevented the strain to consume sulfur as the strain energy substrate. These results were also confirmed by electron microscope and strain growth. The desulfurization process profitability was further improved by proving the ability of T. versutus D306 to produce nano sulfur (less than 50 nm) from sulfide as a substrate using XRD, electron microscope and grain size measurements.Thirdly, a tightly inducible expression system was built using fed-batch and Design, build, test and validate approach for expressing toxic proteins by nature like Cas9 protein, Class 2 CRISPR main effector protein. The fed batch strategy improved the growth of T. versutus D306 to facilitate expression system measurements. The final constructed system, using ferric uptake regulator, enabled the expression to be repressed near the control strain values and increased again to 27 times the control. The system successfully expressed a highly toxic protein as a validation step.Fourthly, a multi gene editing CRISPR-cas9 system was constructed. The universal tRNA was selected as the target of gene editing, and 13 different tRNAs, which were 1 native T. versutus D306, 1 rice native one and 11 E. coli ones, were studied in E. coli. The results showed that the editing efficiency of the constructed system for LeuW, ValU and LysV was 100%, 66.7% and 33.3% respectively.In conclusion, a first knockout system, based on CRISPR- cas9 system and cell- death induced strategy, was built in T. versutus D306. The system was used to improve productivity of desulfurization process by knocking out unfavorable sulfate production pathway genes. The nanometric characteristics of T. versutus sulfur was proved for more profitable bio desulfurization process. Another system for inducible protein expression was developed using ferric repression protein depending upon design, build, test and validate approach. Finally, multiplexing CRISPR was studied in E. coli for future application in T. versutus D306.