Disruptive & Sustainable Technologies for Agricultural Precision (DiSTAP), Singapore-MIT Alliance for Research and Technology, Singapore, Singapore.
Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore, Singapore.
Biotechnol Bioeng. 2023 Jan;120(1):230-238. doi: 10.1002/bit.28255. Epub 2022 Nov 1.
Isoprenoids are a large family of natural products with diverse structures, which allow them to play diverse and important roles in the physiology of plants and animals. They also have important commercial uses as pharmaceuticals, flavoring agents, fragrances, and nutritional supplements. Recently, metabolic engineering has been intensively investigated and emerged as the technology of choice for the production of isoprenoids through microbial fermentation. Isoprenoid biosynthesis typically originates in plants from acetyl-coA in central carbon metabolism, however, a recent study reported an alternative pathway, the isopentenol utilization pathway (IUP), that can provide the building blocks of isoprenoid biosynthesis from affordable C5 substrates. In this study, we expressed the IUP in Escherichia coli to efficiently convert isopentenols into geranate, a valuable isoprenoid compound. We first established a geraniol-producing strain in E. coli that uses the IUP. Then, we extended the geraniol synthesis pathway to produce geranate through two oxidation reactions catalyzed by two alcohol/aldehyde dehydrogenases from Castellaniella defragrans. The geranate titer was further increased by optimizing the expression of the two dehydrogenases and also parameters of the fermentation process. The best strain produced 764 mg/L geranate in 24 h from 2 g/L isopentenols (a mixture of isoprenol and prenol). We also investigated if the dehydrogenases could accept other isoprenoid alcohols as substrates.
异戊二烯类化合物是一大类具有多种结构的天然产物，它们在动植物的生理功能中扮演着多样化且重要的角色。它们还具有重要的商业用途，如药物、调味剂、香料和营养补充剂。最近，代谢工程已被广泛研究，并成为通过微生物发酵生产异戊二烯类化合物的首选技术。异戊二烯类化合物的生物合成通常起源于植物，由中心碳代谢中的乙酰辅酶 A 开始，但最近的一项研究报告了一种替代途径，即异戊烯醇利用途径（IUP），该途径可以从经济实惠的 C5 底物提供异戊二烯类化合物生物合成的构建块。在这项研究中，我们在大肠杆菌中表达了 IUP，以有效地将异戊烯醇转化为香叶醇，这是一种有价值的异戊二烯类化合物。我们首先在大肠杆菌中建立了使用 IUP 的香叶醇生产菌株。然后，我们通过两步氧化反应，将香叶醇合成途径扩展为生产香叶醇，这两步氧化反应由 Castellaniella defragrans 中的两种醇/醛脱氢酶催化。通过优化两种脱氢酶的表达以及发酵过程的参数，进一步提高了香叶醇的产量。最佳菌株能够从 2g/L 的异戊烯醇（异戊醇和烯丙醇的混合物）中在 24 小时内生产出 764mg/L 的香叶醇。我们还研究了脱氢酶是否可以接受其他异戊二烯醇作为底物。
