Carbon partitioning to the terpenoid biosynthetic pathway enables heterologous β-phellandrene production in Escherichia coli cultures

Abstract

Escherichia coli was used as a microbial system for the heterologous synthesis of β-phellandrene, a monoterpene of plant origin with several potential commercial applications. Expression of Lavandula angustifolia β-phellandrene synthase (PHLS), alone or in combination with Picea abies geranyl-diphosphate synthase in E. coli, resulted in no β-phellandrene accumulation, in sharp contrast to observations with PHLS-transformed cyanobacteria. Lack of β-phellandrene biosynthesis in E. coli was attributed to the limited endogenous carbon partitioning through the native 2-C-methylerythritol-4-phosphate (MEP) pathway. Heterologous co-expression of the mevalonic acid pathway, enhancing cellular carbon partitioning and flux toward the universal isoprenoid precursors, isopentenyl-diphosphate and dimethylallyl-diphosphate, was required to confer β-phellandrene production. Differences in endogenous carbon flux toward the synthesis of isoprenoids between photosynthetic (Synechocystis) and non-photosynthetic bacteria (E. coli) are discussed in terms of differences in the regulation of carbon partitioning through the MEP biosynthetic pathway in the two systems.

Photosynthetic generation of heterologous terpenoids in cyanobacteria

“…15 ml of heptane were then added to the surface of the culture to help collect the floating terpene hydrocarbon products. The concentration of monoterpenes in the heptane extracts was quantified by UV‐absorbance spectrophotometry and sensitive flame ionization detector gas chromatography (GC‐FID), as previously described (Betterle & Melis, ; Formighieri & Melis, ; Formighieri and Melis, ). β‐Phellandrene has a known absorbance spectrum with a primary peak at 232.4 nm in heptane.…”

“…β‐Phellandrene has a known absorbance spectrum with a primary peak at 232.4 nm in heptane. The concentration of this monoterpene was calculated applying Lambert‐Beer's law ( ε [232.4 nm] = 15.7 mM −1 cm −1 ; Formighieri & Melis, ). Total absorbance in the UV region for the various samples was then normalized to the amount of biomass (OD/w) generated during the 48‐hr incubation in the bioreactor, as previously described (Betterle & Melis, ; Formighieri & Melis, ).…”

Photosynthetic terpene hydrocarbon production for fuels and chemicals

“…Compared to chemoheterotrophic organisms, photosynthetic microbes already have elevated terpene carbon partition due to their extra needs in MEP‐derived terpenoids and derivatives such as carotenoids, prenylated plastoquinones and phytol moieties of chlorophylls (Formighieri and Melis, ; Melis, ). More importantly, photosynthetic terpenoid production through MEP could intercept glyceraldehyde 3‐phosphate (G3P) directly from the photosynthetic carbon reduction cycle within the eukaryotic chloroplast or cyanobacteria (Figure ).…”

Advances in the Metabolic Engineering of Escherichia coli for the Manufacture of Monoterpenes

“…Three strategies are available: (1) insertion of stronger promoters into the operons directing the enzyme gene expression; (2) codon optimization of enzyme-coding regions; and (3) screening of enzymes from different species for a higher compatible and efficient homolog [51,52]. For the integration of MEV pathway and monoterpene synthases, a series of strong promoters, including T7, lacUV5, and trc, was utilized [32,33]. In another way, the codon optimization scheme is also essential for the heterologous metabolic system due to relatively diminished expression efficiency by biased codon usage [53].…”

“…However, after introducing the MEV pathway in collaboration with the GPPS and the PHLS, the output of β-phellandrene reached 11 mg/g dcw after over 20 h of incubation. This titer could be further improved to 25 mg/g dcw by optimizing LB broth with 1% glucose supplement and then extending the incubation time to over 72 h [33]. Notably, when the endogenous MEP pathway and the exogenous MEV pathway were used individually, the production of sabinene generated from integrated GPPS and sabinene synthase in the MEV pathway was 20-fold higher than that in the MEP pathway.…”