脯氨酸在大豆异黄酮转化菌株耐氧突变株菌体增殖中的作用及调控机制研究

In our previous study, several oxygen-tolerant bacterial mutant strains were derived from the obligate anaerobic wild-type bacteria after a long-term oxygen-tolerant domestication process. Due to the fact that the oxygen-tolerant mutant strains are capable of producing proline, both the function and the regulatory mechanisms of proline in process of proliferation of the oxygen-tolerant mutant strains when grown in the presence of atmospheric oxygen will be investigated. The regulatory mechanisms of the key enzymes responsible for both biosynthesis and biodegradation of proline will be studied on the bases of the change of the genes in mRNA and protein levels. Moreover, the initial sequence for different metabolic pathways of carbohydrates, which include glycolysis (i.e. EMP pathway), gluconeogenesis, and pentose phosphate pathway (PPP), will be determined based on the change of the cloned key enzymes in mRNA and protein levels as well as the amount of the intermediates produced in each metabolic pathway. The mechanisms responding for the growth of the oxygen-tolerant mutant strain will be elucidated on the basis of the metabolism of proline and glucose. The results will provide an important guide for better understanding of fast and efficient growth of other prokaryotic cells (like the opportunistic pathogens) or eukaryotic cells (like cancer cells).

课题组前期对分离得到的严格厌氧大豆异黄酮转化菌株进行了耐氧驯化，并成功得到其耐氧突变株。本项目基于耐氧突变株产脯氨酸的前期发现，拟研究脯氨酸在耐氧突变株菌体增殖中的作用与调控机制：根据不同条件下脯氨酸合成与降解酶基因以及脯氨酸产生量的变化，阐明脯氨酸合成与降解的调控机制；通过克隆糖酵解、糖异生以及磷酸戊糖途径中不可逆反应的关键酶基因，检测关键酶基因在转录和翻译水平变化规律，结合测定糖代谢关键中间代谢产物的含量，分析不同糖代谢途径发生顺序，揭示脯氨酸和葡萄糖代谢对耐氧突变株生长的调控机制。研究结果可为其他原核细胞（如条件致病菌）或真核细胞（如癌变细胞）的高效快速增殖提供重要理论参考。

课题组前期发现，原出发厌氧梭菌AUH-JLC108在厌氧条件下不产脯氨酸，但该菌株的耐氧突变株Aeroto-AUH-JLC108在有氧条件下则产生大量脯氨酸。项目对脯氨酸在耐氧突变株菌体增殖中的作用和机制进行探索。研究发现：脯氨酸和羟基脯氨酸均具有一定的DPPH自由基和羟基自由基清除能力，其中相同浓度的脯氨酸和羟脯氨酸对DPPH自由基的清除能力要高于羟基自由基。耐氧突变株Aeroto-AUH-JLC108产脯氨酸最大量接近0.3mg/mL，该浓度的脯氨酸明显具有降低培养基氧化还原电位的功效。因此，脯氨酸可通过降低培养基氧化还原电位为耐氧突变株提供适宜的“低氧微环境”来促进菌体生长和增殖。另一方面，耐氧突变株Aeroto-AUH-JLC108脯氨酸脱氢酶和P5C脱氢酶变异失活以及P5C还原酶上调表达，导致脯氨酸大量生成。脯氨酸大量积累促进了谷氨酸、谷氨酸酰胺、精氨酸等的积累，其中有研究报道谷氨酰胺和精氨酸有促进细胞增殖和动物生长的作用。另外，本研究还检测到耐氧突变株在培养基中葡萄糖耗尽后，天冬氨酸、组氨酸、苯丙氨酸、甘氨酸等氨基酸有明显积累。以质粒pNICKclos2.0和pMK4为模板和载体，基于CRISPR技术构建基因敲除载体pNICK-Pcat-E，结果酶带大小与酶切验证结果总是与理论值不符。经总体测序后发现，缺失了酶切位点Hpa I至Cas9之间共5683bp的碱基序列（包括sgRNA、酶切位点SpeI和XhoI、以及Cas9的部分序列）。课题组改用不含脯氨酸的化学限定培养基培养耐氧突变株Aeroto-AUH-JLC108，发现部分糖酵解和糖异生的关键酶基因均有差异表达，但目前结果还难以准确判定不同糖代谢发生顺序。此外，本项目还发现参与TCA循环的部分关键酶基因以及与氧气刺激相关的代谢产物，这为今后进一步研究氧气对耐氧突变株的全面影响提供了重要依据。