智能调控肺炎克雷伯氏菌甘油代谢的群体感应工程

Previous microbial breeding was confined to genetic modification at the single-cell level, ignoring the influences of cell-cell communications on desired metabolite and thereby leading to phenotypic variation when lab strains were subjected to real-world applications. To surmount this problem, in this study, we attempt to program the bacterial group behavior based on previous work. As it is known to all, 3-Hydroxypropionic acid (3-HP) is a top-valued platform chemical generated from glycerol reduction pathway in Klebsiella pneumoniae. To engineer high yield strain, the first step is to investigate the effects of glycerol consumption and signal molecule AI-2 on cell proliferation and 3-HP biosynthesis.The experimental data will be subjected to regression analysis to determine the time point at which 3-HP culminates. Next, we will rewire the type-2 quorum sensing (QS) in order to achieve the automuous control of 3-HP biosynthesis. The programmed QS network is able to simplify the fermentation process,attenuate the feedback inhibition occurred in late phase of fermentation, and thereby benefits the production of 3-HP. Moreover,the subsequent analysis of metabolic flux, optimization of fermentation parameters, as well as mathmatics modelling aim to unravel the underlying correlation among AI-2,cell proliferation,and 3-HP biosynthesis. Lastly, to dissect the modulation of QS on glycerol metabolism, we may recruit a panel of strategies, including the assay of enzymatic activities, isolating of differentially expressed genes, and locating the enzyme genes in KEGG pathways. In short, this protocol represents the frontier of microbial metabolic engineering because it for the first time bridges QS network with metabolite biosynthesis. Not only achieving high yield and novel expression system, this work may also provide solid basis for the forthcoming genetic modification of other strains together with automated control of the fermentation process.

以往的微生物育种局限于单细胞的基因改造，忽略了细胞间互作对目标产物的影响，导致实际生产产量低于实验室产量，本研究在种群水平上进行育种。3-羟基丙酸(3-HP)是重要平台化合物，产自肺炎克雷伯氏菌的甘油还原途径。首先考察甘油浓度、信号分子AI-2对生物量和3-HP产量的影响；重构其Ⅱ型群体感应（QS）网络，使之依照预定的逻辑程序智能合成3-HP，简化发酵控制并弱化发酵后期的反馈抑制，从而提高3-HP产量；分析工程菌的代谢流向变化及发酵动力学，结合数学模拟，解析AI-2、生物量和3-羟基丙酸这三者之间内在联系这一关键科学问题。通过关键酶活性分析、差异表达基因分离以及KEGG途径定位，解析QS调控甘油代谢的分子机理。该方案将QS用于工业育种，实现3-HP的智能合成和发酵过程简化，为微生物代谢工程的新思路，不仅获得高产菌和新型自动表达系统，而且为其它工业菌种的基因改造和发酵控制奠定理论和技术基础。

肺炎克雷伯氏菌(Klebsiella pneumoniae) 是近年工业微生物研究热点。为动态调控甘油代谢，本研究重构该菌的群体感应（QS）。克隆了QS信号分子AI-2的合成酶基因ygaG，用pET-pk载体过表达该基因，获得重组菌K. pneumoniae(pET-ygaG)；敲除该基因获得ygaG缺失突变株K. pneumoniaeΔygaG。SDS-PAGE分析显示该突变株在28 kDa和35 kDa左右出现差异条带，MALDI-TOF-TOF质谱和荧光定量PCR显示ygaG基因缺失导致膜蛋白和代谢相关基因的上调或下调。摇瓶发酵显示，过表达ygaG抑制K. pneumoniae生长，而敲除ygaG基因则促进生长。扫描电镜观察发现，与野生型相比突变株细胞被膜表面更光滑，褶皱更浅。药敏试验表明突变株抗药性增强。上述表明ygaG基因影响细胞被膜合成和致病性。为了动态调控乳酸代谢，构建4个QS工程菌，其一仅超表达信号分子酰基高丝氨酸内酯（AHL）的合成基因，另一菌同时表达AHL合成基因和其降解基因AiiA。相比于前者，后者对数生长期推迟。当表达乳酸脱氢酶ldhA时，与对照菌相比，前者的生长速度、生物量和甘油消耗速率分别提高了83.6%, 31.0% 和 36.0%, 而后者因动态协调细胞生长和乳酸积累而产生较多乳酸，D-乳酸产量和时空产率分别提高了231.9% 和117.3%。将QS元件和放大器耦合，构建了pBD-PhrpL-luxI-luxR-ParaC-hrpV和pcmR-15A-PluxI-hrpR- hrpS-PluxI-eGFP双质粒菌密度开关。首次证明CRISPR/Cas9系统在肺炎克雷伯氏菌中具有切割双链DNA功能。利用CRISPRi分别抑制该菌乳酸合成相关基因pmd、ldhA、aldA和mgsA, 或同时抑制这4个基因。发现ldhA为产乳酸关键基因。单独或同时抑制4基因都能抑制乳酸合成。通过同源重组敲除影响3-HP合成的8个副产物基因。将可特异性识别DNA序列的LacI与cAMP受体蛋白(CRP)融合表达，将操纵区lacO插入启动子上游，由融合蛋白引导CRP至目的基因。以此策略构建的双质粒工程菌在5 L发酵罐中3-HP产量较野生菌株提高523%。采用复合启动子和氮源补充策略3-HP发酵产量达到99 g/L，是目前报道的最高产量。