Terpenes constitute the largest class of natural products with over 55,000 compounds with versatile applications including drugs and biofuels. Introducing structural modifications to terpenes through metabolic engineering is an efficient and sustainable way to improve their properties. Here, we report the optimization of the lepidopteran mevalonate (LMVA) pathway towards the efficient production of isopentenyl pyrophosphate (IPP) analogs as terpene precursors. First, we linked the LMVA pathway to NudB, a promiscuous phosphatase, resulting in the production of the six-carbon analog of 3-methyl-3-buten-1-ol (isoprenol), 3-ethyl-3-buten-1-ol (C6-isoprenol). Using C6-isoprenol as the final product, we then engineered the LMVA pathway by redirecting its upstream portion from a thiolase-dependent pathway to a beta-oxidation pathway. The beta-oxidation LMVA pathway transforms valeric acid, a platform chemical that can be produced from biomass, into C6-isoprenol at a titer of 110.3 mg/L, improved from 5.5 mg/L by the thiolase LMVA pathway, which used propionic acid as a feedstock. Knockout of the E. coli endogenous thiolase genes further improved the C6-isoprenol titer to 390 mg/L, implying efficient production of homo isopentenyl pyrophosphate (HIPP). The beta-oxidation LMVA-NudB pathway also converts butanoic acid and hexanoic acid into isoprenol and isoprenol’s seven-carbon analog, 3-propyl-3-buten-1-ol (C7-isoprenol), respectively, suggesting the beta-oxidation LMVA pathway produces IPP and C7-IPP from the corresponding fatty acids. Fuel property tests revealed the longer chain isoprenol analogs have lower water solubilities, similar or higher energy densities, and comparable research octane number (RON) boosting effects to isopentenols. This work not only optimizes the LMVA pathway, setting the basis for homoterpene biosynthesis to expand terpene chemical space, but provides an efficient pathway to produce isoprenol analogs as next-generation biofuels from sustainable feedstocks.

萜烯是最大的一类天然产物，具有超过 55,000 种化合物，具有多种应用，包括药物和生物燃料。通过代谢工程对萜烯进行结构修饰是改善其特性的有效且可持续的方法。在这里，我们报告了鳞翅目甲羟戊酸 (LMVA) 途径的优化，以有效生产异戊烯焦磷酸 (IPP) 类似物作为萜烯前体。首先，我们将 LMVA 途径与 NudB（一种混杂的磷酸酶）联系起来，从而产生 3-methyl-3-buten-1-ol（异戊二烯醇）、3-ethyl-3-buten-1-的六碳类似物ol（C6-异戊二烯醇）。使用 C6-异戊二烯醇作为最终产品，然后我们通过将其上游部分从依赖硫解酶的途径重定向到 β-氧化途径来设计 LMVA 途径。β-氧化 LMVA 途径将戊酸（一种可从生物质生产的平台化学品）转化为 C6-异戊二烯醇，滴度为 110.3 mg/L，通过硫解酶 LMVA 途径从 5.5 mg/L 提高，该途径使用丙酸作为一种原料。淘汰赛大肠杆菌内源性硫解酶基因进一步将 C6-异戊二烯醇滴度提高到 390 mg/L，这意味着高异戊烯焦磷酸 (HIPP) 的高效生产。β-氧化 LMVA-NudB 途径还将丁酸和己酸分别转化为异戊二烯醇和异戊二烯醇的七碳类似物 3-丙基-3-丁烯-1-醇（C7-异戊二烯醇），表明β-氧化 LMVA 途径从相应的脂肪酸产生 IPP 和 C7-IPP。燃料特性测试表明，更长链的异戊二烯醇类似物具有更低的水溶性、相似或更高的能量密度以及与异戊烯醇相当的研究辛烷值 (RON) 提升效果。这项工作不仅优化了 LMVA 途径，为同萜生物合成奠定了基础，扩大了萜烯化学空间，