Enhanced fermentative γ-aminobutyric acid production by a metabolic engineered Corynebacterium glutamicum

IF 2.5 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Biotechnology and Bioprocess Engineering Pub Date : 2024-02-12 DOI:10.1007/s12257-024-00008-6

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引用次数: 0

Abstract

γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid with important physiological functions, which has been widely used in food, pharmaceuticals, and polyamides production. The fermentative GABA production by Corynebacterium glutamicum was recognized as one of the most promising methods. However, the problems of low catalytic activity of the heterologously expressed glutamate decarboxylase (GAD) and the imbalanced carbon flux between cell growth and GABA synthesis severely limited the GABA production by C. glutamicum. This study applied combinational metabolic engineering and catalytic condition optimization strategies to solve these two major obstacles. The secretory expression of GAD was enhanced using a bicistronic-designed expression cassette. This bicistronic expression cassette was further triply inserted into the genome by substituting the ldhA, pqo, and ack genes, thus stabilizing the expression of GAD and reducing the accumulation of by-products of lactate and acetate. A growth-regulated promoter P_{CP_2836} was applied to dynamically control the expression of odhA, thus controlling the α-oxoglutarate dehydrogenase complex activity for balanced cell growth and GABA production. The glutamate precursor synthesis and pyridoxal 5′-phosphate supply were also strengthened by promoter substitution. Finally, through a two-stage pH-controlled fed-batch fermentation under optimized conditions, the engineered strain reached GABA titer of 81.31 ± 1.31 g/L with a yield and productivity of 0.50 ± 0.01 g/g and 1.36 ± 0.23 g L⁻¹ h⁻¹, which was 4.8%, 13.6%, and 11.2% higher than that of the original strain. This study laid a solid foundation for industrial fermentative GABA production by engineered C. glutamicum.

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经代谢工程改造的谷氨酸棒状杆菌提高了γ-氨基丁酸的发酵产量

摘要 γ-氨基丁酸（GABA）是一种非蛋白源氨基酸，具有重要的生理功能，已被广泛应用于食品、药品和聚酰胺的生产。谷氨酸棒杆菌发酵生产 GABA 被认为是最有前途的方法之一。然而，异源表达的谷氨酸脱羧酶（GAD）催化活性低以及细胞生长与 GABA 合成之间的碳通量不平衡等问题严重限制了谷氨酸棒杆菌生产 GABA。本研究采用组合代谢工程和催化条件优化策略来解决这两大障碍。使用双螺旋设计的表达盒增强了 GAD 的分泌表达。通过替换ldhA、pqo和ack基因，进一步将该双螺旋表达盒三重插入基因组，从而稳定了GAD的表达，并减少了副产物乳酸和乙酸的积累。应用生长调控启动子 PCP_2836 动态控制 odhA 的表达，从而控制 α-氧谷氨酸脱氢酶复合物的活性，以平衡细胞生长和 GABA 的产生。通过启动子替换，谷氨酸前体合成和 5′-磷酸吡哆醛供应也得到了加强。最后，通过优化条件下的两阶段 pH 控制饲料批量发酵，工程菌株的 GABA 滴度达到 81.31 ± 1.31 g/L，产量和生产率分别为 0.50 ± 0.01 g/g 和 1.36 ± 0.23 g L-1 h-1，比原始菌株分别高出 4.8%、13.6% 和 11.2%。这项研究为利用工程谷氨酸棒状杆菌发酵生产 GABA 奠定了坚实的基础。

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来源期刊

Biotechnology and Bioprocess Engineering 工程技术-生物工程与应用微生物

CiteScore

5.00

自引率

12.50%

发文量

审稿时长

3 months

期刊介绍： Biotechnology and Bioprocess Engineering is an international bimonthly journal published by the Korean Society for Biotechnology and Bioengineering. BBE is devoted to the advancement in science and technology in the wide area of biotechnology, bioengineering, and (bio)medical engineering. This includes but is not limited to applied molecular and cell biology, engineered biocatalysis and biotransformation, metabolic engineering and systems biology, bioseparation and bioprocess engineering, cell culture technology, environmental and food biotechnology, pharmaceutics and biopharmaceutics, biomaterials engineering, nanobiotechnology, and biosensor and bioelectronics.