红曲霉ATP柠檬酸裂解酶促进红曲色素生成基因表达调控机制研究

Monascus pigment was the metabolites of Monascus spp. which fermented on rice. The cytoplasm of acetyl coenzyme A was used as a precursor in Monascus pigment synthesis, which was converted from citric acid by a key enzyme of ATP-citrate lyase (ACL). In our previous studies, both the genes acl1 and acl2 encoding ACL protein were expressed in M. ruber, respectively. And they could improve the amount of Monascus pigment, especially in red pigment. At the same time, it was firstly found significantly promoting the starch degradation of rice. In this project, the genetic engineering strains for the single and double acl genes of over-expression, knock-out mutant and revertant strain were constructed. Based on transcriptome sequencing technology combined with metabolome analyses of sugar, organic acid and amino acid; In addition, determination of the Monascus pigment value and tone, ACL enzyme activity, the content of acetyl coenzyme A, fatty acid content, starch content, and qRT-PCR. The relationship between acl gene expression level and ACL enzyme activity, acetyl coenzyme A, starch, amino acid metabolites change, Monascus pigment and tone was established. From the three levels of starch, sugar, organic acids as well as amino acid metabolism - acetyl coenzyme A- Monascus pigment, it will reveal the gene expression regulation mechanism of ACL promote monascus pigment. It will provide new ideas for study the mechanism of Monascus pigment formation, and has important theoretical guiding significance to improve the production of Monascus pigment.

红曲色素是红曲霉以大米为原料发酵而成的代谢产物，其合成原料来自胞质乙酰辅酶A。ATP柠檬酸裂解酶（ACL）是催化柠檬酸生成乙酰辅酶A的关键酶。本课题组过表达红色红曲霉ACL编码基因acl1与acl2，均能够大幅提高红曲色素的产量，特别是红色素；并且首次发现显著促进大米中淀粉的降解。但是其分子机理不明确。本项目通过构建acl单基因及双基因过表达工程菌株、基因缺失突变株、回补菌株；采用转录组测序联合代谢组重点分析糖、有机酸、氨基酸；测定红曲色素色价与色调、ACL酶活性、乙酰辅酶A和脂肪酸以及淀粉含量，qRT-PCR验证。建立acl基因表达量与ACL酶活性、乙酰辅酶A、淀粉、有机酸、氨基酸代谢变化规律、红曲色素之间内在联系。从淀粉、糖、有机酸、氨基酸代谢-乙酰辅酶A-红曲色素三个层次，揭示ACL促进红曲色素形成基因表达调控机理，为研究红曲色素代谢机制提供新思路，对提高其产量具有重要理论指导意义。

红曲色素是红曲霉以大米为原料发酵而成的代谢产物，其合成原料来自胞质乙酰辅酶A，而ATP柠檬酸裂解酶（ACL）是催化柠檬酸生成乙酰辅酶A的关键酶。本研究从红曲霉CICC41233中克隆ATP柠檬酸裂解酶编码基因acl1和acl2，并分别构建基因表达和敲除载体，通过根癌农杆菌介导分别转化红曲霉CICC41233获得基因过表达菌株红曲霉ACL438469-4和红曲霉ACL501956-11；以及基因敲除菌株红曲霉△ACL438469-1和红曲霉△ACL501956-16。将以上红曲霉菌种进行红曲色素发酵，结果表明相比红曲霉CICC41233，过表达菌株红曲霉ACL438469-4和红曲霉ACL501956-11，第6 d红曲色素色价分别提高92.69%和122.64%，ACL酶活性分别提高11.77%和37.25%，乙酰辅酶A的含量分别提高87.30%和45.21%。过表达菌株显著促进淀粉降解，发酵4d大米淀粉完全降解，而野生株在第6天仍剩有淀粉。过表达菌株对葡萄糖和柠檬酸的含量没有改变，但均降低了硬脂酸和亚麻酸含量。相比红曲霉CICC41233，基因敲除菌株红曲霉△ACL438469-1和红曲霉△ACL501956-16，第6d红曲色素色价分别降低22.03%和25.92%；淀粉降解速率低于野生株，剩余淀粉含量比野生株分别还高10.85%和13.29%。突变株ACL酶活性没有测出，但乙酰辅酶A的含量分别降低20.61%和35.57%。转录组学和代谢组学分析表明红曲霉红曲色素合成中ACL反馈促进淀粉和蔗糖代谢、糖酵解和糖异生；而氨基酸代谢如甘氨酸-丝氨酸-苏氨酸和精氨酸代谢，缬氨酸、亮氨酸和异亮氨酸降解代谢，赖氨酸和苯丙氨酸合成代谢对红曲色素红色素和黄色素合成有促进作用。本项目从淀粉-乙酰辅酶A-红曲色素揭示ACL促进红曲色素合成机理，对提高其产量具有重要理论指导意义。