新型天冬氨酸激酶别构调控机制研究及高产蛋氨酸工程菌构建

L-methionine is the only sulphur-containing essential amino acid in human and animals, only could be produced by chemical synthesis with the disadvantages of low safety and high cost, which lead to the methionine production by biological fermentation is imminent. Aspartate kinase(AK) is the first key rate-limiting enzyme in the biosynthesis pathway of methionine, and strictly regulated by cooperative feedback inhibition, which severely restricts methionine fermentation. Based on the high expression of AK gene from Corynebacterium pekinense(CP), a novel monomer AK with high enzyme activity and relieved feedback inhibition was obtained by research group. In this research, using molecular biology and bioinformatics technology, such as molecular dynamics simulation, coevolution analysis and virtual site saturation mutation to determine the important residues that binding inhibitor; using fluorescent high-throughput screening technique to screen the saturation mutagenesis library, the mutant enzyme with good performance can be obtained with enzymatic properties characterized; through crystal structure and quantum chemical analysis, the allosteric regulation model can be built to reveal molecular mechanism of monomer AK allosteric regulation; finally the highly expressed AK gene without allosteric regulation can be introduced into CP, with the purpose of obtaining engineering strain for producing high-yield methionine. The research lays a solid theoretical basis for improvement of strain producing amino acid and push the fermentation industrialization process of methionine production.

L-蛋氨酸是人和动物体内唯一含硫，却不能以发酵法生产的必需氨基酸，目前只能采用安全性低、成本高的化学合成法生产，致使生物发酵法生产蛋氨酸迫在眉睫。蛋氨酸生物合成途径中的第一限速别构酶--天冬氨酸激酶（AK）受严格协同反馈抑制，致使发酵法生产蛋氨酸严重受限。项目组在北京棒杆菌（CP）AK基因产物高效表达的基础上，获得了高酶活、有效解除反馈抑制的新型单体AK。本研究拟利用分子生物学及生物信息学技术，通过分子动力学模拟、协同进化分析及虚拟定点饱和突变，进一步确定AK与抑制剂结合的重要残基位点；采用荧光高通量筛选技术对饱和突变库进行筛选，获得优良突变体并表征酶学性质；通过晶体结构和量子化学分析，建立AK别构调控模型，进而揭示单体AK分子别构调控机制；通过将别构调控解除的高活力AK基因导入CP，获取高产量蛋氨酸工程菌。本研究成果将为氨基酸生产菌种改造奠定基础，进而推动发酵法生产蛋氨酸工业化进程。

L-蛋氨酸合成途径中的第一限速别构酶--天冬氨酸激酶（AK）受严格协同反馈抑制，致使发酵法生产蛋氨酸受限。项目以新型单体AK为研究对象，构建了构象合理且稳定的新型单体AK 三维晶体结构模型；确定了13个AK 别构抑制剂Lys和Thr结合区域关键残基位点，16个催化活性中心ATP和底物Asp结合区域关键氨基酸残基位点。构建了不同程度解除AK别构抑制、酶活力提高的134株突变体饱和突变库，酶活力最大提高了99.43倍，完成了115株突变体AK酶学性质表征。从原子水平揭示了抑制剂Lys对AK构象别构调控机制：一方面是通过ATP途径，αA、αB区域发生偏移，导致ATP结合区域（tC）的整体移动，使得ATP远离催化底物Asp；一方面是通过Asp路径，tA区域发生了偏移，引起了dB区域较大相对运动，导致tB区域发生了整个AK内部最大的运动和局部结构改变，关键氨基酸残基Gly170 和 Gly171与底物Asp间的氢键受到破坏，降低了催化反应的可能；明确了AK催化过程的催化反应物、过渡态产物和最终产物的结构。构建了高产蛋氨酸工程菌17株，优化后工程菌PEC-lysCm-homm-metX最高产量为6.85g/L，较原菌提高了274.32%。本研究丰富了AK酶理论研究，为天冬氨酸族氨基酸合成路径上所有酶的催化反应及相关产物的代谢过程提供参考，为发酵法生产蛋氨酸菌种的选育提供了借鉴，推进了蛋氨酸生物发酵生产进程。.依托本项目发表学术论文12篇，其中SCI收录文章4篇，EI收录文章6篇，核心期刊文章2篇，获授权发明专利1件，获吉林省科技进步二等奖1项。参加国际国内学术会议9次，项目负责人入选国家高层次人才和吉林省“长白山人才工程”科技创新领军人才；核心成员1人晋升副教授职称，并主持国家自然科学基金青年基金1项（31901695）；1人晋升高级实验师职称；培养博士研究生1名，硕士研究生10名。