Efficient Biosynthesis of (+)-α-Pinene and de Novo Synthesis of (+)-cis-Verbenol in Escherichia coli.

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 (P450camF89W,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.

Efficient myrcene production using linalool dehydratase isomerase and rational biochemical process in Escherichia coli.

Microbial synthesis of plant-based myrcene is of great interest because of its high demand, however, achieving high biosynthetic titers remains a great challenge. Previous strategies adopted for microbial myrcene production have relied on the recruitment of a multi-step biosynthetic pathway which requires complex metabolic regulation or high activity of myrcene synthase, hindering its application. Here, we present an effective one-step biotransformation system for myrcene biosynthesis from geraniol, using a linalool dehydratase isomerase (LDI) to overcome these limitations. The truncated LDI possesses nominal activity that catalyzes the isomerization of geraniol to linalool and the subsequent dehydration to myrcene in anaerobic environment. In order to improve the robustness of engineered strains for the efficient conversion of geraniol to myrcene, rational enzyme modification and a series of biochemical process engineering were employed to maintain and improve the anaerobic catalytic activity of LDI. Finally, by introducing the optimized myrcene biosynthetic capability in the existing geraniol-production strain, we achieve de novo biosynthesis of myrcene at 1.25 g/L from glycerol during 84 h aerobic-anaerobic two-stage fermentation, which is much higher than previously reported myrcene levels. This work highlights the value of dehydratase isomerase-based biocatalytic in establishing novel biosynthetic pathways and lays a reliable foundation for the microbial synthesis of myrcene.

Engineering Escherichia coli for production of geraniol by systematic synthetic biology approaches and laboratory-evolved fusion tags.

Geraniol is a valuable monoterpene extensively used in the fragrance, food, and cosmetic industries. Increasing environmental concerns and supply gaps have motivated efforts to advance the microbial production of geraniol from renewable feedstocks. In this study, we first constructed a platform geraniol Escherichia coli strain by bioprospecting the key enzymes geranyl diphosphate synthase (GPPS) and geraniol synthase (GES) and selection of a host cell background. This strategy led to a 46.4-fold increase in geraniol titer to 964.3 mg/L. We propose that the expression level of eukaryotic GES can be further optimized through fusion tag evolution engineering. To this end, we manipulated GES to maximize flux towards the targeted product geraniol from precursor geranyl diphosphate (GPP) via the utilization of fusion tags. Additionally, we developed a high-throughput screening system to monitor fusion tag variants. This common plug-and-play toolbox proved to be a robust approach for systematic modulation of protein expression and can be used to tune biosynthetic metabolic pathways. Finally, by combining a modified E1* fusion tag, we achieved 2124.1 mg/L of geraniol in shake flask cultures, which reached 27.2% of the maximum theoretical yield and was the highest titer ever reported. We propose that this strategy has set a good reference for enhancing a broader range of terpenoid production in microbial cell factories, which might open new possibilities for the bio-production of other valuable chemicals.