不依赖Ca2+的L-赖氨酸合成酶系胞内自组装及分子机制研究

The microbial synthesis of L-lysine is a sequential reaction involving multiple enzymes and the synergistic catalytic efficiency of multiple enzymes is the key factor of determining the rate of L-lysine synthesis. The cellulosome can use the dockerin and a variety of enzyme molecules for high efficient self-assembly. The proximity effect is shown in multienzyme polymer after assembled, so it has high efficiency in catalyzing products with the characteristics of a multistep sequence synthesis. The synthesis of L-lysine requires the support of the microbial intracellular environment, while the lack of intracellular Ca2+ dont't cause a role which is played by the key component of the cellulosome (the dockerin protein), which limited the application of cellulosome components in the cell. In this study, firstly, a mutant of a dockerin that does not depend on Ca2+ was obtained by semi-rational design and high-throughput screening, the crystal structure of the mutant was analyzed and further the mechanism of intercellular assemble of dockerin without Ca2+ was elaborated . Secondly, the key enzymes involved in L- lysine are linked to the dockerin by molecular biological methods. Multi enzyme catalysis of simulated extracellular cellulosome. Construction of high efficient L- lysine synthesis system for intracellular self-assembly, and anchored to the cell membrane. The synthesis of L-lysine and the simultaneous enhancement of the rate of extracellular transport were realized. The successful implementation of this study will provide new ideas for the improvement of functional food fermented microorganisms.

L-赖氨酸的微生物合成是一种多酶参与的顺序反应，而多酶间的协同催化效率是决定L-赖氨酸合成速率的关键因素。纤维小体在胞外能够利用对接蛋白与多种酶分子进行高效自组装，所得多酶聚合体由于“临近效应”，会在催化多步顺序合成反应时具备较高的效率。但目前L-赖氨酸的合成需要胞内环境的支持，因胞内Ca2+的匮乏导致纤维小体的关键元件--对接蛋白无法在胞内发挥作用，限制了纤维小体元件在胞内的应用。本研究拟通过半理性设计及高通量筛选，获得不依赖Ca2+的对接蛋白突变体，同时解析其晶体结构，阐述不依赖Ca2+的对接蛋白胞内组装机制；其次通过分子生物学手段将参与L-赖氨酸催化的关键酶与对接蛋白相连，模拟胞外纤维小体的多酶催化方式，构建胞内自组装的高效L-赖氨酸合成体系，并锚定于细胞膜上，实现L-赖氨酸合成及胞外转运速率的同步提升。本研究的成功实施将为功能食品发酵微生物的改良提供新的思路。

L-赖氨酸的微生物合成是一种多酶参与的顺序反应，而多酶间的协同催化效率是决定L-赖氨酸合成速率的关键因素。纤维小体在胞外能够利用对接蛋白与多种酶分子进行高效自组装，所得多酶聚合体由于“临近效应”，会在催化多步顺序合成反应时具备较高的效率。但目前L-赖氨酸的合成需要胞内环境的支持，因胞内钙离子的匮乏导致纤维小体的关键元件--对接蛋白无法在胞内发挥作用，限制了纤维小体元件在胞内的应用。本项目通过理性设计、半理性设计等方法，结合自主构建的基于流式细胞仪的BIFC-FC高通量筛选系统，构建、筛选了共计57种正向突变对接蛋白基因。利用生物大分子相互作用仪分析，发现其中突变体D3与黏连蛋白的结合能力较未突变提高了5.9倍，同时在细胞内微钙环境具有了良好的组装活性；结构分析发现，对接突变体D3在没有Ca2+的情况下形成了较为稳定的loop区域；分子动力学模拟分析发现，对接蛋白L1区域Ca2+结合位点可能是对接蛋白-粘连蛋白相互结合的关键点；正向突变库测序分析发现利用芳香族氨基酸及大侧链非带电氨基酸替换带电荷氨基酸是解除对接蛋白钙离子依赖的关键之一。以突变体D3为研究基础，以7种L-赖氨酸合成途径基因为研究对象，综合分析游离酶组装及内膜锚定等多种组装方式，构建7株L-赖氨酸工程菌，其中摇瓶发酵条件下组装工程菌株QDE-AspC-Coh/DocA-D3-LysC的L-赖氨酸36h浓度累计达到60.3g/L，比出发菌株QDE高24.1%，产业化应用发现该工程菌糖酸转化率达70%以上，L-赖氨酸产量提高到252g/L，发酵时间降低至44h，综合成本降低3.1%。首次实现了纤维小体关键元件在细胞内微钙环境的组装活性，并在L-赖氨酸合成途径上进行了充分的测试。该结果是对细胞内组装方式的有效补充，其灵活多样的组装设计模式也为未来细胞内多步合成途径效率的提升提供了新的有效的途径，具有良好的应用前景。