Two-step pathway for isoprenoid synthesis

Abstract

Isoprenoids comprise a large class of chemicals of significant interest due to their diverse properties. Biological production of isoprenoids is considered to be the most efficient way for their large-scale production. Isoprenoid biosynthesis has thus far been dependent on pathways inextricably linked to glucose metabolism. These pathways suffer from inherent limitations due to their length, complex regulation, and extensive cofactor requirements. Here, we present a synthetic isoprenoid pathway that aims to overcome these limitations. This isopentenol utilization pathway (IUP) can produce isopentenyl diphosphate or dimethylallyl diphosphate, the main precursors to isoprenoid synthesis, through sequential phosphorylation of isopentenol isomers isoprenol or prenol. After identifying suitable enzymes and constructing the pathway, we attempted to probe the limits of the IUP for producing various isoprenoid downstream products. The IUP flux exceeded the capacity of almost all downstream pathways tested and was competitive with the highest isoprenoid fluxes reported.

Keywords: biosynthesis; isopentenol; isoprenoids; pathway; utilization.

Fig. 1.

Development of the isopentenol utilization pathway and in vitro characterization of choline kinase. (A) The IUP can produce the basic isoprenoid metabolic intermediates IPP and DMAPP in two steps using isoprenol or prenol, respectively, as feedstock. The steps are catalyzed by a promiscuous kinase and isopentenyl phosphate kinase (IPK). IPP and DMAPP can then be interconverted via IDI. IPP and DMAPP act as the precursor molecules for larger prenyl diphosphates and eventually isoprenoids. (B) Enzymes screened in this work. (C) Results of overnight screen to identify a suitable promiscuous kinase using isoprenol (IP and IPP) or prenol (DMAP and DMAPP) as a substrate, respectively. (D) Kinetic analysis of choline kinase from S. cerevisiae (ScCK) at a fixed ATP concentration for the determination of k_cat and K_m with regard to isopentenol substrate. Reaction rates are reported as means ± SD (n = 3).

Fig. 2.

Characterization of the IUP using the lycopene pathway encoded by pAC-LYCipi in M9 media. (A) Lycopene content in strains containing the IUP under the control of a low strength constitutive promoter (pro4) or a strong inducible promoter (pTET) induced with 20 ng/mL aTc. This was compared with the control strain containing only the lycopene production plasmid (pAC-LYCipi), without the IUP plasmid or addition of either isopentenol. (B) The effect of ipk gene on the synthesis of lycopene. (C) The effect of the pro4IUP pathway on intracellular levels of IPP/DMAPP compared with the control strain containing only pAC-LYCipi. All experimental values are normalized to dry cell weight and represent the mean ± SD of three biological replicates.

Fig. 3.

Isoprenol pulse–chase experiment for metabolite monitoring. (A) Levels of labeled MEC. (B) Levels of labeled IPP/DMAPP. (C) Levels of unlabeled IP/DMAP. (D) Levels of unlabeled IPP/DMAPP. (E and F) Labeling patterns for 3-phosphoglycerate (3PG) and phosphoenolpyruvate (PEP), respectively, in the pro4IUPi strain over the first 60 min. pTET cultures were induced with 10 ng/mL aTC. All experimental values are normalized to dry cell weight and represent the mean ± SD of three biological replicates.

Fig. 4.

Use of the isopentenol utilization pathway for the production of isoprenoids. (A) Isoprenoid product titers after culturing for 48 h expressing the IUP under the control of the pro4 or pTET promoters (10 ng/mL aTC), along with a control expressing only the downstream cassette. Concentrations are expressed as equivalents of the internal standard caryophyllene. (B) Lycopene content in strains using an endogenous constitutive promoter with various copy number plasmids (pAC-LYCipi ∼ 15, p20-LYCipi ∼ 20, pUC-LYCipi > 100) and under the control of a strong inducible promoter in a plasmid with copy number ∼5 (p5T7-LYCipi). (C and D) Concentrations of metabolic intermediates for strains expressing the IU pathway along with a plasmid for the production of (C) lycopene (p5T7-LYCipi) or (D) taxadiene (p5T7tds-ggpps), respectively. All metabolite and product concentrations are reported as means ± SD of three biological replicates. Conc., concentration.

Fig. 5.

Metabolite levels and products from cultures with taxadiene-producing strains growing in U-¹³C–labeled glucose. The cultures differ on whether they express the IU pathway (±IUP) and on whether the culture media were supplemented with unlabeled isoprenol at t = 0 (±ISO). Taxadiene and metabolic intermediate pools were analyzed after 48 h of culture. Shown are concentrations and labeling patterns for metabolic intermediates (A) IP, (B) IPP/DMAPP, (C) GPP, (D) FPP, and (E) GGPP, respectively. (F) Titers of taxadiene produced after 48 h. (G) Labeled taxadiene mass spectrum. (H) Unlabeled taxadiene mass spectrum. All metabolite and product concentrations are reported as means ± SD of three biological replicates.

Fig. 6.

Batch bioreactor cultivation of lycopene production utilizing the IUP. The IUP was expressed under the control of the pro4 promoter along with a p5T7-LYC vector containing either crtE or ggpps. (A) Glucose concentration and optical density over time. (B) Lycopene content over time. (C) Cumulative IPP flux calculated from lycopene productivity and comparison with some of the highest reported isoprenoid fluxes (29, 30) in the literature. (D) Cell pellets taken from one CrtE bioreactor at different time points. All values represent the mean ± SD based on samples taken from three bioreactor runs. A.U., absorbance units.