Glucoamylase is one of the most industrially applied enzymes, produced by Aspergillus species, like Aspergillus niger. Compared to the traditional ways of process optimization, the metabolic engineering strategies to improve glucoamylase production are relatively scarce. In the previous study combined multi-omics integrative analysis and amino acid supplementation experiment, we predicted four amino acids (alanine, glutamate, glycine and aspartate) as the limited precursors for glucoamylase production in A. niger. To further verify this, five mutants namely OE-ala, OE-glu, OE-gly, OE-asp1 and OE-asp2, derived from the parental strain A. niger CBS 513.88, were constructed respectively for the overexpression of five genes responsible for the biosynthesis of the four kinds of amino acids (An11g02620, An04g00990, An05g00410, An04g06380 and An16g05570). Real-time quantitative PCR revealed that all these genes were successfully overexpressed at the mRNA level while the five mutants exhibited different performance in glucoamylase production in shake flask cultivation. Notably, the results demonstrated that mutant OE-asp2 which was constructed for reinforcing cytosolic aspartate synthetic pathway, exhibited significantly increased glucoamylase activity by 23.5% and 60.3% compared to CBS 513.88 in the cultivation of shake flask and the 5 L fermentor, respectively. Compared to A. niger CBS 513.88, mutant OE-asp2 has a higher intracellular amino acid pool, in particular, alanine, leucine, glycine and glutamine, while the pool of glutamate was decreased. Our study combines the target prediction from multi-omics analysis with the experimental validation and proves the possibility of increasing glucoamylase production by enhancing limited amino acid biosynthesis. In short, this systematically conducted study will surely deepen the understanding of resources allocation in cell factory and provide new strategies for the rational design of enzyme production strains.

中文翻译：

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改善氧气限制下黑曲霉胞质中天冬氨酸的生物合成会增加葡糖淀粉酶的产量

葡糖淀粉酶是工业上应用最广泛的酶之一，由曲霉菌种（如黑曲霉）产生。与传统的过程优化方法相比，用于改善葡糖淀粉酶生产的代谢工程策略相对稀缺。在先前的研究中，结合多组学综合分析和氨基酸补充实验，我们预测了四种氨基酸（丙氨酸，谷氨酸，甘氨酸和天冬氨酸）是黑曲霉生产葡糖淀粉酶的有限前体。为了进一步证实这一点，分别构建了五个突变体，分别来自亲本菌株黑曲霉CBS 513.88的OE-ala，OE-glu，OE-gly，OE-asp1和OE-asp2，用于五个基因的过表达。四种氨基酸（An11g02620，An04g00990，An05g00410，An04g06380和An16g05570）的生物合成。实时定量PCR显示，所有这些基因均在mRNA水平成功过表达，而这五个突变体在摇瓶培养中在葡糖淀粉酶生产中表现出不同的性能。值得注意的是，结果表明，在摇瓶和5 L发酵罐的培养中，与BSBS 513.88相比，构建用于增强胞质天冬氨酸合成途径的突变OE-asp2的葡糖淀粉酶活性分别显着提高了23.5％和60.3％。与黑曲霉CBS 513.88相比，突变OE-asp2具有更高的细胞内氨基酸库，尤其是丙氨酸，亮氨酸，甘氨酸和谷氨酰胺，而谷氨酸库却减少了。我们的研究将多组学分析的目标预测与实验验证相结合，并证明了通过增强有限的氨基酸生物合成来增加葡糖淀粉酶产量的可能性。简而言之，这项系统地进行的研究必将加深对细胞工厂资源分配的理解，并为合理设计酶生产菌株提供新的策略。