Search

Geranyl diphosphate (GPP), the unique precursor for all monoterpenoids, is biosynthesized from isopentenyl diphosphate and dimethylallyl diphosphate via the head-to-tail condensation reaction catalyzed by GPP synthase (GPPS). Herein a homomeric GPPS from Camptotheca acuminata, a camptothecin-producing plant, was obtained from 5'- and 3'-rapid amplification of cDNA ends and subsequent overlap extension and convenient PCR amplifications. The truncate CaGPPS was introduced to replace ispA of pBbA5c-MevT(CO)-MBIS(CO, ispA), a de novo biosynthetic construct for farnesyl diphosphate generation, and overexpressed in Escherichia coli, together with the truncate geraniol synthase-encoding gene from C. acuminata (tCaGES), to confirm CaGPPS-catalyzed reaction in vivo. A 24.0 ± 1.3 mg L -1 of geraniol was produced in the recombinant E. coli. The production of GPP was also validated by the direct UPLC-HRMS E analyses. The tCaGPPS and tCaGES genes with different copy numbers were introduced into E. coli to balance their catalytic potential for high-yield geraniol production. A 1.6-fold increase of geraniol production was obtained when four copies of tCaGPPS and one copy of tCaGES were introduced into E. coli. The following fermentation conditions optimization, including removal of organic layers and addition of new n-decane, led to a 74.6 ± 6.5 mg L -1 of geraniol production. The present study suggested that the gene copy number optimization, i.e., the ratio of tCaGPPS and tCaGES, plays an important role in geraniol production in the recombinant E. coli. The removal and addition of organic solvent are very useful for sustainable high-yield production of geraniol in the recombinant E. coli in view of that the solubility of geraniol is limited in the fermentation broth and/or n-decane.

Terpenes have numerous applications, ranging from pharmaceuticals to fragrances and biofuels. With increasing interest in producing terpenes sustainably and economically, there has been significant progress in recent years in developing methods for their production in microorganisms. In Saccharomyces cerevisiae, production of the 20-carbon diterpenes has so far proven to be significantly less efficient than production of their 15-carbon sesquiterpene counterparts. In this report, we identify the modular structure of geranylgeranyl diphosphate synthesis in yeast to be a major limitation in diterpene yields, and we engineer the yeast farnesyl diphosphate synthase Erg20p to produce geranylgeranyl diphosphate. Using a combination of protein and genetic engineering, we achieve significant improvements in the production of sclareol and several other isoprenoids, including cis-abienol, abietadiene and β-carotene. We also report the development of yeast strains carrying the engineered Erg20p, which support efficient isoprenoid production and can be used as a dedicated chassis for diterpene production or biosynthetic pathway elucidation. The design developed here can be applied to the production of any GGPP-derived isoprenoid and is compatible with other yeast terpene production platforms., (Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

We report the production of taxadiene by transformation of N. benthamiana with a taxadiene synthase gene. The production was significantly increased by an elicitor treatment or metabolic pathway shunting. Paclitaxel (Taxol(®)) was first isolated from the bark of the pacific yew tree as an anticancer agent and has been used extensively to treat various types of cancer. Taxadiene, the first committed product of paclitaxel synthesis is cyclized from geranylgeranyl diphosphate (GGPP), and further complex hydroxylation and acylation processes of the unique taxane core skeleton produce paclitaxel. To accomplish de novo production of taxadiene, we transformed Nicotiana benthamiana with a taxadiene synthase (TS) gene. The introduced TS gene under the transcriptional control of the CaMV 35S promoter was constitutively expressed in N. benthamiana, and the de novo production of taxadiene was confirmed by mass spectroscopy profiling. Transformed N. benthamiana homozygous lines produced 11-27 μg taxadiene/g of dry weight. The highest taxadiene production line TSS-8 was further treated with an elicitor, methyl jasmonate, and metabolic pathway shunting by suppression of the phytoene synthase gene expression which resulted in accumulation of increased taxadiene accumulation by 1.4- or 1.9-fold, respectively. In summary, we report that the production of taxadiene in N. benthamiana was possible by the ectopic expression of the TS gene, and higher accumulation of taxadiene could be achieved by elicitor treatment or metabolic pathway shunting of the terpenoid pathway.

Isoprenyl diphosphate synthases (IDSs) produce the ubiquitous branched-chain diphosphates of different lengths that are precursors of all major classes of terpenes. Typically, individual short-chain IDSs (scIDSs) make the C10, C15, and C20 isoprenyl diphosphates separately. Here, we report that the product length synthesized by a single scIDS shifts depending on the divalent metal cofactor present. This previously undescribed mechanism of carbon chain-length determination was discovered for a scIDS from juvenile horseradish leaf beetles, Phaedon cochleariae. The recombinant enzyme P. cochleariae isoprenyl diphosphate synthase 1 (PcIDS1) yields 96% C10-geranyl diphosphate (GDP) and only 4% C15-farnesyl diphosphate (FDP) in the presence of Co(2+) or Mn(2+) as a cofactor, whereas it yields only 18% C10 GDP but 82% C15 FDP in the presence of Mg(2+). In reaction with Co(2+), PcIDS1 has a Km of 11.6 μM for dimethylallyl diphosphate as a cosubstrate and 24.3 μM for GDP. However, with Mg(2+), PcIDS1 has a Km of 1.18 μM for GDP, suggesting that this substrate is favored by the enzyme under such conditions. RNAi targeting PcIDS1 revealed the participation of this enzyme in the de novo synthesis of defensive monoterpenoids in the beetle larvae. As an FDP synthase, PcIDS1 could be associated with the formation of sesquiterpenes, such as juvenile hormones. Detection of Co(2+), Mn(2+), or Mg(2+) in the beetle larvae suggests flux control into C10 vs. C15 isoprenoids could be accomplished by these ions in vivo. The dependence of product chain length of scIDSs on metal cofactor identity introduces an additional regulation for these branch point enzymes of terpene metabolism.

Isolated hepatocytes from rat liver were incubated with [3H]mevalonate, and the labeling of polyprenols in the microsomal fraction was followed. After a 1-min incubation the alpha-unsaturated forms of polyprenyl-P2, -P, and polyprenol were mainly labeled and at this time point only 2, 8, and 17%, respectively, of the label was associated with the saturated forms. In the case of the free alcohol 2 h of incubation was required before all the labeling was recovered in the saturated form. After 1 min polyprenols and polyprenyl-P with 20 and 21 isoprene residues demonstrated much higher specific labeling than the shorter compounds, but after 5 min these differences were greatly reduced. In experiments utilizing short incubation times and chasing no evidence has been obtained that the phosphorylated form is a precursor of the free alcohol or vice versa, that the free alcohol is a precursor of the phosphorylated form. In human liver about 1% of the dolichol is present in the alpha-unsaturated form. These experiments suggest that: 1) the alpha-unsaturated form is the precursor of the alpha-saturated free alcohol, 2) dolichol does not necessarily arise directly from dephosphorylation of the phosphorylated form, and 3) the free alcohol is for the most part not phosphorylated under in vivo conditions in rat liver.

The regenerating liver presents a changed ability to use mevalonate 16 hr after partial hepatectomy. The dolichol content and its synthesis from mevalonate is increased, while no variation of dolichyl-P and ubiquinone parameters are detectable. The greater amount of mevalonate utilized to form dolichol, but not dolichyl-P, in this proliferating system, raises some questions about the physiological significance of these isoprenoid compounds and about their biosynthetic sequence.

The biosynthesis of dolichol and dolichylmonophosphate in rat liver was studied using [3H]mevalonate as precursor. The radioactive precursor was either injected into the portal vein of the rat or added to the incubation medium containing isolated hepatocytes, followed by the isolation of microsomes and mitochondria from the liver or the hepatocytes. In both systems dolichol in microsomes was highly labeled after a short labeling period followed by a rapid decrease. During this period the labeling of mitochondrial dolichol was low. The specific radioactivity of dolichyl-P in microsomes of both systems was higher in the initial phase than in dolichol and increased further with time. The mitochondrial labeling was also increased but was at a much lower level.

Two independent experiments indicated that the major subcellular site of de novo dolichyl phosphate biosynthesis in rat liver is in the microsomes. First, when purified subcellular fractions were assayed for long chain prenyltransferase activity, the profile obtained indicated exclusively a microsomal localization. Second, when liver slices were incubated 60 min with [14C]acetate and subcellular fractions were subsequently isolated, the microsomal fraction was found to contain greater than two-thirds of the total radioactivity incorporated into dolichyl phosphate. Chemical analysis of dolichyl phosphate in subcellular fractions showed substantial quantities in both the microsomal and mitochondrial-lysosomal fractions. In contrast, dolichol was found primarily in the mitochondrial-lysosomal fraction. The absolute in vitro rate of hepatic dolichyl phosphate synthesis was determined in liver slices and isolated hepatocytes by [14C]acetate labeling using [3H] water incorporation into cholesterol to correct for the dilution of the labeled acetate by the endogenous acetyl-CoA pool. From this value, an in vivo rate of de novo synthesis was estimated as 1.7-2.6 nmol of dolichyl phosphate/liver/day. This rate is more than 30 times the maximum possible rate of accumulation in liver of dietary dolichol (Keller, R. K., Jehle, E., and Adair, W. L., Jr. (1982) J. Biol. Chem. 257, 8985-8989). From the previously determined concentration of dolichyl phosphate in rate liver (Keller, R. K. Tamkun, J. M., and Adair, W. L. (1981) Biochemistry 20, 5831-5836) and the de novo rate of synthesis, it appears that the half-life of hepatic dolichyl phosphate is on the order of days.

The effects of addition of 1 microM-dexamethasone on the rate of transfer of mannose from GDP-mannose into mannolipid have been studied in HeLa cell cultures. Concurrent with an increase in incorporation of mannose into glycoproteins, the incorporation of mannose from GDP-mannose in vitro into mannolipid and dolichol-linked oligosaccharides was increased after dexamethasone treatment. Stimulation of mannolipid synthesis showed a correlation with the 11 beta, 17 alpha, 21-trihydroxy structure of C21 steroids. Dexamethasone treatment also resulted in an increased incorporation of acetate into dolichol and dolichyl phosphate. The results suggest that the effect of dexamethasone on the cell-surface glycoprotein accumulation is related to increased sugar-linked dolichol synthesis.