Gallic acid fermentation by metabolically engineered Escherichia coli producing p-hydroxybenzoate hydroxylase from Hylemonella gracilis NS1.

IF 0.8 4区 生物学 Q4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Journal of General and Applied Microbiology Pub Date : 2024-05-02 Epub Date: 2023-08-30 DOI:10.2323/jgam.2023.08.004
Nozomi Katsuki, Shunsuke Masuo, Noriyuki Nukui, Hajime Minakawa, Naoki Takaya
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Abstract

Plant-derived phenolic gallic acid (GA) is an important raw material for antioxidants and food additives. Efforts to ferment GA using microbial processes have aimed at minimizing production costs and environmental load using enzymes that hydroxylate p-hydroxybenzoate and protocatechuate (PCA). Here, we found a p-hydroxybenzoate hydroxylase (PobA) in the bacterium Hylemonella gracilis NS1 (HgPobA) with 1.5-fold more hydroxylation activity than that from Pseudomonas aeruginosa PAO1 and thus converted PCA to GA more efficiently. The PCA hydroxylation activity of HgPobA was improved by introducing the amino acid substitutions L207V/Y393F or T302A/Y393F. These mutants had 2.9- and 3.7-fold lower Kmapp for PCA than wild-type HgPobA. An Escherichia coli strain that reinforces shikimate pathway metabolism and produces HgPobA when cultured for 60 h generated 0.27 g L-1 of GA. This is the first report of fermenting glucose to generate GA using a natural enzyme from the PobA family. The E. coli strain harboring the HgPobA L207V/Y393F mutant increased GA production to 0.56 g L-1. During the early stages of culture, GA was fermented at a 10-fold higher rate by a strain producing either HgPobA L207V/Y393F or T302A/Y393F compared with wild-type HgPobA, which agreed with the high kcatapp/Kmapp PCA values of this mutant. We enhanced a PobA isozyme and its PCA hydroxylating function to efficiently and cost-effectively ferment GA.

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代谢工程大肠杆菌产生来自 Hylemonella gracilis NS1 的对羟基苯甲酸羟化酶发酵没食子酸。
植物提取的酚类没食子酸(GA)是抗氧化剂和食品添加剂的重要原料。利用微生物工艺发酵没食子酸的努力旨在使用对羟基苯甲酸和原儿茶酸(PCA)羟化酶最大限度地降低生产成本和环境负荷。在这里,我们发现 Hylemonella gracilis NS1(HgPobA)细菌中的对羟基苯甲酸羟化酶(PobA)的羟化活性是铜绿假单胞菌 PAO1 的 1.5 倍,因此能更有效地将 PCA 转化为 GA。通过引入氨基酸替换 L207V/Y393F 或 T302A/Y393F,HgPobA 的 PCA 羟基化活性得到了提高。与野生型 HgPobA 相比,这些突变体的 PCA Kmapp 分别低 2.9 倍和 3.7 倍。一株加强莽草酸途径代谢并产生 HgPobA 的大肠杆菌在培养 60 小时后产生了 0.27 g L-1 的 GA。这是首次报道利用 PobA 家族的天然酶发酵葡萄糖产生 GA。携带 HgPobA L207V/Y393F 突变体的大肠杆菌菌株的 GA 产量增至 0.56 g L-1。在培养初期,产生 HgPobA L207V/Y393F 或 T302A/Y393F 的菌株发酵 GA 的速率是野生型 HgPobA 的 10 倍,这与该突变体的高 kcatapp/Kmapp PCA 值一致。我们增强了 PobA 同工酶及其 PCA 羟基化功能,以高效、低成本地发酵 GA。
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来源期刊
Journal of General and Applied Microbiology
Journal of General and Applied Microbiology 生物-生物工程与应用微生物
CiteScore
2.40
自引率
0.00%
发文量
42
审稿时长
6-12 weeks
期刊介绍: JGAM is going to publish scientific reports containing novel and significant microbiological findings, which are mainly devoted to the following categories: Antibiotics and Secondary Metabolites; Biotechnology and Metabolic Engineering; Developmental Microbiology; Environmental Microbiology and Bioremediation; Enzymology; Eukaryotic Microbiology; Evolution and Phylogenetics; Genome Integrity and Plasticity; Microalgae and Photosynthesis; Microbiology for Food; Molecular Genetics; Physiology and Cell Surface; Synthetic and Systems Microbiology.
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