黑曲霉菌株柠檬酸合成酶基因的敲除及功能分析

【背景】柠檬酸合成酶是碳代谢途径的中心酶,其在三羧酸循环(tricarboxylic acid cycle,TCA)、氨基酸合成和乙醛酸循环中发挥着重要作用,是柠檬酸合成的关键酶。本论文所选用的是一株高产柠檬酸的黑曲霉菌株CGMCC10142。【目的】克隆柠檬酸合成酶关键基因,构建柠檬酸合成酶的敲除菌株并鉴定其在黑曲霉菌株高产柠檬酸过程中的功能及影响。【方法】采用根癌农杆菌转化方法并利用同源重组原理,采用抗性筛选和致死型反向筛选的双重筛选方法获得正确敲除株。对转化子在不同碳源下的生长情况进行观察并对柠檬酸发酵过程中菌丝球变化和产酸量进行分析,最后通过荧光定量PCR分析柠檬酸合成酶基因对黑曲霉积累柠檬酸的影响,及其对主要代谢途径中重要酶相关基因和其他的表达量的影响。【结果】以柠檬酸高产菌株黑曲霉CGMCC10142为出发菌,构建一株遗传稳定的柠檬酸合成酶敲除的菌株T1-2。结果发现该菌株在以葡萄糖为碳源的培养基上生长缓慢并且产生孢子量减少。通过摇瓶发酵产酸实验,结果表明敲除菌在84 h产酸量为64.3 g/L,相对于出发菌的98.7g/L降低了34.85%。通过荧光定量PCR发现柠檬酸合成酶的表达量是下降的,同时重要酶的表达量都下降。【结论】该菌株的柠檬酸合成酶基因对柠檬酸积累具有重要作用,但存在其他同工酶基因,该基因敲除仅使产酸合成降低34.85%,同时发现该柠檬酸合成酶的顺畅表达有助于主代谢途径中各关键酶的高效表达,本研究可为研究黑曲霉高产柠檬酸机理奠定基础。

Knock-out and functional analysis of citrate synthase gene from Aspergillus niger

[Background] Citric acid synthase is the central enzyme of carbon metabolism. It is important in the tricarboxylic acid (TCA) cycle, amino acid synthesis and glyoxylate cycle, and is a key enzyme in the synthesis of citric acid. In this paper, a citric acid-producing Aspergillus niger strain CGMCC10142 was selected. [Objective] Cloning of key genecitrate synthase, constructing the knockout strain of citrate synthase and identifying its function and influence in the process of high yield of citric acid by Aspergillus niger strain. [Methods] In this experiment, the Agrobacterium tumefaciens transformation method and the principle of homologous recombination were used, and the correct knockout strain was obtained by the dual screening method of resistance screening and lethal type and reverse screening. Observation of growth of different carbon sources by transformants and the changes of mycelial spheres and acid production during citric acid fermentation were analyzed. Finally, the effect of citrate synthase gene on the accumulation of citric acid in Aspergillus niger and its effect on the expression of important enzyme-related genes and other expressions in the main metabolic pathways were analyzed by real-time PCR. [Results] A strain T1-2 with a genetically stable knockout citrate synthase was constructed based on the high-yield citric acid strain Aspergillus niger CGMCC10142. The results showed that the strain grew slowly on the medium with glucose as carbon source and produced fewer spores. The results of shaking flask fermentation showed that the acid yield of knockout bacteria was 64.3 g/L at 84 h, which was 34.85% lower than that of the original bacteria at 98.7 g/L. Real-time quantitative PCR results showed that the expression level of citrate synthase was decreased, and the expression levels of other important enzymes were decreased. [Conclusion] The citrate synthase gene of this strain plays an important role in the accumulation of citric acid, but there are other isozyme genes. When this gene knockout, only the acid production synthesis is reduced by 34.85%. At the same time, it was found that the smooth expression of the citrate synthase facilitated the high-efficiency expression of each key enzyme in the main metabolic pathway, which laid a foundation for studying the mechanism of high-yield citric acid in Aspergillus niger.