Efficient Biosynthesis of (+)-α-Pinene and de Novo Synthesis of (+)-cis-Verbenol in Escherichia coli,Journal of Agricultural and Food Chemistry

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Efficient Biosynthesis of (+)-α-Pinene and de Novo Synthesis of (+)-cis-Verbenol in Escherichia coli
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2024-08-14 , DOI: 10.1021/acs.jafc.4c05387
Yujunjie Zhou ₁ , Tao Li ₁ , Xilong He ₁ , Xun Wang ₁ , Fei Wang ₁ , Xun Li ₁

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Bark beetles, major pests that bore into forest stems, cause significant economic damage to forests globally. (+)-α-Pinene is the precursor to (+)-cis-verbenol, a crucial component of the aggregation pheromones produced by bark beetles. This paper describes the de novo synthesis of (+)-cis-verbenol in Escherichia coli. Initially, the truncation position of (+)-α-pinene synthase (PtPS30 from Pinus taeda) and monoterpene precursor (geranyl diphosphate/neryl diphosphate) synthases were evaluated. Neryl diphosphate synthase from Solanum lycopersicum (SlNPPS1) and truncated (+)-α-pinene synthase (PtPS30–39) were selected as promising candidates. Subsequently, the titer of (+)-α-pinene was significantly increased 8.9-fold by using the fusion tag CM29, which enhanced the solubility of PtPS30–39. In addition, by optimizing expression elements (ribosomal binding sites, linkers, and up elements) and overexpressing CM29*PtPS30–39, a yield of 134.12 mg/L (+)-α-pinene was achieved. Finally, the first de novo synthesis of enantiopure (+)-cis-verbenol was achieved by introducing a cytochrome P450 mutant from Pseudomonas putida (P450_cam^{F89W,Y98F,L246A}), resulting in a yield of 11.13 mg/L. This study lays the groundwork for developing verbenol-based trapping technology for controlling bark beetles.

中文翻译：

大肠杆菌中 (+)-α-蒎烯的高效生物合成和 (+)-顺式马鞭草醇的从头合成

树皮甲虫是钻入森林茎的主要害虫，对全球森林造成重大经济损失。 (+)-α-蒎烯是 (+)-顺式-马鞭草酚的前体，而 (+)-顺式-马鞭草酚是树皮甲虫产生的聚集信息素的重要组成部分。本文描述了在大肠杆菌中从头合成(+)-顺式-马鞭草醇。最初，评估了(+)-α-蒎烯合酶（来自火炬松的Pt PS30）和单萜前体（香叶基二磷酸/橙花基二磷酸）合酶的截短位置。来自番茄的橙花基二磷酸合酶 ( Sl NPPS1) 和截短的 (+)-α-蒎烯合酶 ( Pt PS30–39) 被选为有前途的候选酶。随后，通过使用融合标签 CM29，(+)-α-蒎烯的效价显着增加了 8.9 倍，从而增强了Pt PS30-39 的溶解度。此外，通过优化表达元件（核糖体结合位点、连接子和up元件）和过表达CM29* Pt PS30–39，获得了134.12 mg/L (+)-α-蒎烯的产量。最后，通过引入恶臭假单胞菌细胞色素P450突变体（P450 _cam ^{F89W，Y98F，L246A} ），首次从头合成了对映体纯（+）-顺式-马鞭草醇，产量为11.13 mg/L。这项研究为开发基于马鞭草酚的捕获技术来控制树皮甲虫奠定了基础。

更新日期：2024-08-14

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