基于高产核黄素枯草芽孢杆菌全基因组突变分析的逆向代谢工程研究

Inverse metabolic engineering is an effective way to link genotype and phenotype of mutation strains with improved performance, revealing detailed knowledge of genetics or physiology of the producing strains. Based on comparative mutation analysis of riboflavin high-producing Bacillus subtilis strains with their reference through whole-genome sequencing, the present project aims to investigate the genotype-phenotype correlations of mutant riboflavin producing B.subtilis strains, to identify and evaluate the key relevant mutations or mutation combinations leading to riboflavin accumulation.The identified key mutations or mutation combinations will be further investigated on molecular basis for improved performance, preferably in order to understand the underlying genetics and biochemical mechanism of riboflavin prodction from B.subtilis. Finally, Only the relevant mutations are resconstructed to generate a defined mutant with the optimal mutation set that is necessary and sufficient for high-level riboflavin production in wild-type B.subtilis strain. The whole-genome mutation analysis, enabled by next-generation sequencing, not only can indentify relevant mutations for improved performance,but also link mutant genotype and phenotype from global metabolic network by a reverse metabolic engineering approach. Identification of these mutations could open the way to improved approaches for targeted and knowledge-based systems metabolic engineering of riboflavin in B.subtilis, and get more knowledge of ribofalvin production in B.subtilis. Additionally, secondary effects of mutations have a much broader impact, such as mutations in regulatory networks and proteins, can be engineered for other bioproducts.From the description above, we could conclude that this project shows great study signifinace and potential application in strain improvement.

通过逆向代谢工程重构目标表型菌株，是确定优良突变株基因型-表型关系，分析优良表型遗传机理的一种有效手段。实验室前期进行了高产核黄素枯草芽孢杆菌突变株全基因组测序和比较基因组分析。在此基础上,本项目拟利用无痕基因操作技术,分析突变枯草芽孢杆菌高产核黄素的优良表型与基因型之间对应关系，鉴定重要有利突变或突变组合，并评估其对高产表型的贡献度，探讨重要有利突变的调控机制，阐明枯草芽孢杆菌高产核黄素的遗传机理，最终重构只含有利突变的核黄素高产菌。基于全基因组尺度的突变分析，能够从整个代谢网络角度分析突变与表型的关系，发现有利突变或突变组合，进行枯草芽孢杆菌产核黄素的系统代谢工程，加深对枯草芽孢杆菌产核黄素遗传调控机理的认识；同时，新发现的优良表型突变还可应用到其它相关生物基产品的代谢工程研究，因此，本项目研究具有重要的科学意义和应用潜力。

随着第二代高通量测序技术的快速发展，可以方便快捷地测序或重测序突变菌株基因组和转录组信息，使基于比较基因组学的逆向代谢工程成为研究菌株遗传基础的重要策略。本项目以高产核黄素的枯草芽孢杆菌突变菌株为研究对象，基因组尺度分析了高产核黄素的优良表型与基因型之间对应关系，鉴定重要有利突变，探讨重要有利突变的调控机制，为阐明枯草芽孢杆菌高产核黄素的遗传机理提供依据，最终重构了只含有利突变的核黄素合成菌株。. 首次在枯草芽孢杆菌中建立以I-SceI介导DNA双链断裂诱导重组的无痕修饰技术，外源dsDNA片段的重组效率高达3000-5000 cfu/μg dsDNA，是传统Spizizen方法的10-100倍。该系统可以连续/迭代编辑基因组，包括等位基因置换、外源基因引入和基因敲除。该技术不仅为本项目研究提供有利工具，也为枯草芽孢杆菌的基因组编辑和分子改造提供技术平台。. 利用构建的基因组无痕修饰技术，将重要突变迭代引入野生型菌株，共筛选到7个有利突变基因，RibC(G199D)、ribD(G+39A)、PurAP(242L)、CcpN(A44S)、YvrH(R222Q)、YhcF(R90*)和YwaA(Q68*)。其中PurA(P242L)、CcpN(A44S)、YvrH(R222Q)、YhcF(R90*)和YwaA(Q68*)均为首次发现的高产核黄素有利突变；. 综合菌株生理与发酵特性，酶活性，转录组和代谢物组分析等组学手段，探讨主要有利突变导致核黄素高产的遗传机理。PurA(P242)调控嘌呤途径碳通量的重新分配，提高核黄素前体物GTP的供给；CcpN(A44S)引起中心代谢途径碳通量的重分配，提高戊糖磷酸途径通量和核黄素产量；YvrH(R222Q)影响细胞通透性，一定程度解调嘌呤途径调控，促进核黄素前体物合成；YwaA(Q68*)导致分支链氨基酸代谢途径部分基因表达水平下调，降低碳代谢流溢流代谢。新发现的有利突变调控机制，为阐明枯草芽孢杆菌生产核黄素的遗传基础提供依据。. 以野生型菌株B. subtilis 168为出发菌，将鉴定的有利突变引入出发菌株。进一步理性代谢工程解调嘌呤途径，使核黄素产量由0 mg/L 提高到980 mg/L。重构的菌株遗传背景清晰，只携带与目标表型相关的有利突变，保持了野生型菌株的生长良好、遗传稳定等优良表型。

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