Department of Chemistry & Biotechnology, Faculty of Engineering, Tottori University, 4-101 Koyamacho-Minami, Tottori, 680-8552, Japan.
Department of Biotechnology, Graduate School of Engineering, Tottori University, 4-101 Koyamacho-Minami, Tottori, 680-8552, Japan.
Appl Microbiol Biotechnol. 2021 Aug;105(14-15):5821-5832. doi: 10.1007/s00253-021-11447-z. Epub 2021 Jul 29.
Pathway engineering is a useful technology for producing desired compounds on a large scale by modifying the biosynthetic pathways of host organisms using genetic engineering. We focused on acetoacetate esters as novel low-cost substrates and established an efficient terpenoid production system using pathway-engineered recombinant Escherichia coli. Functional analysis using recombinant E. coli proteins of 18 carboxylesterases identified from the microbial esterases and lipases database showed that the p-nitrobenzyl esterase (PnbA) from Bacillus subtilis specifically hydrolyzed two acetoacetate esters: methyl acetoacetate (MAA) and ethyl acetoacetate (EAA). We generated a plasmid (pAC-Mev/Scidi/Aacl/PnbA) co-expressing PnbA and six enzymes of the mevalonate pathway gene cluster from Streptomyces, isopentenyl diphosphate isomerase type I from Saccharomyces cerevisiae, and acetoacetyl-coenzyme A ligase from Rattus norvegicus. The plasmid pAC-Mev/Scidi/Aacl/PnbA was introduced into E. coli along with plasmid expressing carotenoid (lycopene) or sesquiterpene (β-bisabolene) biosynthesis genes, and the terpenoid production was evaluated following the addition of acetoacetate esters as substrates. These recombinant E. coli strains used MAA and EAA as substrates for the biosynthesis of terpenoids and produced almost equivalent concentrations of target compounds compared with the previous production system that used mevalonolactone and lithium acetoacetate. The findings of this study will enable the production of useful terpenoids from low-cost substrates, which may facilitate their commercial production on an industrial scale in the future. KEY POINTS: • PnbA from Bacillus subtilis exhibits acetoacetate hydrolysis activity. • A plasmid enabling terpenoid synthesis from acetoacetate esters was constructed. • Acetoacetate esters as substrates enable a low-cost production of terpenoids.
途径工程是一种有用的技术，通过使用遗传工程修饰宿主生物体的生物合成途径，可以大规模生产所需的化合物。我们专注于乙酰乙酸酯作为新型低成本底物，并使用途径工程改造的重组大肠杆菌建立了高效的萜类化合物生产系统。使用从微生物酯酶和脂肪酶数据库中鉴定的 18 种羧酸酯酶的重组大肠杆菌蛋白进行功能分析表明，来自枯草芽孢杆菌的对硝基苄基酯酶（PnbA）特异性水解两种乙酰乙酸酯：甲基乙酰乙酸酯（MAA）和乙基乙酰乙酸酯（EAA）。我们生成了一个质粒（pAC-Mev/Scidi/Aacl/PnbA），该质粒共表达了来自链霉菌的甲羟戊酸途径基因簇的六个酶、来自酿酒酵母的异戊二烯二磷酸异构酶 I 型和来自挪威鼠的乙酰乙酰辅酶 A 连接酶。将质粒 pAC-Mev/Scidi/Aacl/PnbA 与表达类胡萝卜素（番茄红素）或倍半萜烯（β- 法尼烯）生物合成基因的质粒一起引入大肠杆菌，并在添加乙酰乙酸酯作为底物后评估萜类化合物的生产情况。这些重组大肠杆菌菌株将 MAA 和 EAA 用作萜类化合物生物合成的底物，并产生与使用甲羟戊酸内酯和乙酰乙酸锂的先前生产系统相当的目标化合物浓度。这项研究的结果将能够从低成本底物生产有用的萜类化合物，这可能有助于它们在未来的工业规模上进行商业生产。 要点： • 枯草芽孢杆菌的 PnbA 表现出乙酰乙酸酯水解活性。 • 构建了能够从乙酰乙酸酯合成萜类化合物的质粒。 • 乙酰乙酸酯作为底物可实现萜类化合物的低成本生产。
