Metabolite Profiling Identified Methylerythritol Cyclodiphosphate Efflux as a Limiting Step in Microbial Isoprenoid Production

Isoprenoids are natural products that are all derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). These precursors are synthesized either by the mevalonate (MVA) pathway or the 1-Deoxy-D-Xylulose 5-Phosphate (DXP) pathway. Metabolic engineering of microbes has enabled overproduction of various isoprenoid products from the DXP pathway including lycopene, artemisinic acid, taxadiene and levopimaradiene. To date, there is no method to accurately measure all the DXP metabolic intermediates simultaneously so as to enable the identification of potential flux limiting steps. In this study, a solid phase extraction coupled with ultra performance liquid chromatography mass spectrometry (SPE UPLC-MS) method was developed. This method was used to measure the DXP intermediates in genetically engineered E. coli. Unexpectedly, methylerythritol cyclodiphosphate (MEC) was found to efflux when certain enzymes of the pathway were over-expressed, demonstrating the existence of a novel competing pathway branch in the DXP metabolism. Guided by these findings, ispG was overexpressed and was found to effectively reduce the efflux of MEC inside the cells, resulting in a significant increase in downstream isoprenoid production. This study demonstrated the necessity to quantify metabolites enabling the identification of a hitherto unrecognized pathway and provided useful insights into rational design in metabolic engineering.     

Reconstruction and Evaluation of the Synthetic Bacterial MEP Pathway in Saccharomyces cerevisiae

Isoprenoids, which are a large group of natural and chemical compounds with a variety of applications as e.g. fragrances, pharmaceuticals and potential biofuels, are produced via two different metabolic pathways, the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we attempted to replace the endogenous MVA pathway in Saccharomyces cerevisiae by a synthetic bacterial MEP pathway integrated into the genome to benefit from its superior properties in terms of energy consumption and productivity at defined growth conditions. It was shown that the growth of a MVA pathway deficient S. cerevisiae strain could not be restored by the heterologous MEP pathway even when accompanied by the co-expression of genes erpA, hISCA1 and CpIscA involved in the Fe-S trafficking routes leading to maturation of IspG and IspH and E. coli genes fldA and fpr encoding flavodoxin and flavodoxin reductase believed to be responsible for electron transfer to IspG and IspH.     

Combining Genotype Improvement and Statistical Media Optimization for Isoprenoid Production in E. coli

Isoprenoids are a large and diverse class of compounds that includes many high value natural products and are thus in great demand. To meet the increasing demand for isoprenoid compounds, metabolic engineering of microbes has been used to produce isoprenoids in an economical and sustainable manner. To achieve high isoprenoid yields using this technology, the availability of metabolic precursors feeding the deoxyxylulose phosphate (DXP) pathway, responsible for isoprenoid biosynthesis, has to be optimized. In this study, phosphoenolpyruvate, a vital DXP pathway precursor, was enriched by deleting the genes encoding the carbohydrate phosphotransferase system (PTS) in E. coli. Production of lycopene (a C40 isoprenoid) was maximized by optimizing growth medium and culture conditions. In optimized conditions, the lycopene yield from PTS mutant was seven fold higher than that obtained from the wild type strain. This resulted in the highest reported specific yield of lycopene produced from the DXP pathway in E. coli to date (20,000 µg/g dry cell weight). Both the copy number of the plasmid encoding the lycopene biosynthetic genes and the expression were found to be increased in the optimized media. Deletion of PTS together with a similar optimization strategy was also successful in enhancing the production of amorpha-1,4-diene, a distinct C15 isoprenoid, suggesting that the approaches developed herein can be generally applied to optimize production of other isoprenoids.     

Reconstruction of a bacterial isoprenoid biosynthetic pathway in Saccharomyces cerevisiae

A eukaryotic mevalonate pathway transferred and expressed in Escherichia coli, and a mammalian hydrocortisone biosynthetic pathway rebuilt in Saccharomyces cerevisiae are examples showing that transferring metabolic pathways from one organism to another can have a powerful impact on cell properties. In this study, we reconstructed the E. coli isoprenoid biosynthetic pathway in S. cerevisiae. Genes encoding the seven enzymatic steps of the pathway were cloned and expressed in S. cerevisiae. mRNA from the seven genes was detected, and the pathway was shown able to sustain growth of yeast in conditions of inhibition of its constitutive isoprenoid biosynthetic pathway.     

1-Deoxy-D-xylulose 5-phosphate synthase catalyzes a novel random sequential mechanism

Emerging resistance of human pathogens to anti-infective agents make it necessary to develop new agents to treat infection. The methylerythritol phosphate pathway has been identified as an anti-infective target, as this essential isoprenoid biosynthetic pathway is widespread in human pathogens but absent in humans. The first enzyme of the pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase, catalyzes the formation of DXP via condensation of D-glyceraldehyde 3-phosphate (D-GAP) and pyruvate in a thiamine diphosphate-dependent manner. Structural analysis has revealed a unique domain arrangement suggesting opportunities for the selective targeting of DXP synthase; however, reports on the kinetic mechanism are conflicting. Here, we present the results of tryptophan fluorescence binding and kinetic analyses of DXP synthase and propose a new model for substrate binding and mechanism. Our results are consistent with a random sequential kinetic mechanism, which is unprecedented in this enzyme class.     

Engineering Saccharomyces cerevisiae for isoprenol production

Isoprenol (3-methyl-3-butene-1-ol) is a valuable drop-in biofuel and an important precursor of several commodity chemicals. Synthetic microbial systems using the heterologous mevalonate pathway have recently been developed for the production of isoprenol in Escherichia coli, and a significant yield and titer improvement has been achieved through a decade of research. Saccharomyces cerevisiae has been widely used in the biotechnology industry for isoprenoid production, but there has been no good example of isoprenol production reported in this host. In this study, we engineered the budding yeast S. cerevisiae for improved biosynthesis of isoprenol. The strain engineered with the mevalonate pathway achieved isoprenol production at the titer of 36.02 ± 0.92 mg/L in the flask. The IPP (isopentenyl diphosphate)-bypass pathway, which has shown more efficient isoprenol production by avoiding the accumulation of the toxic intermediate in E. coli, was also constructed in S. cerevisiae and improved the isoprenol titer by 2-fold. We further engineered the strains by deleting a promiscuous endogenous kinase that could divert the pathway flux away from the isoprenol production and improved the titer to 130.52 ± 8.01 mg/L. Finally, we identified a pathway bottleneck using metabolomics analysis and overexpressed a promiscuous alkaline phosphatase to relieve this bottleneck. The combined efforts resulted in the titer improvement to 383.1 ± 31.62 mg/L in the flask. This is the highest isoprenol titer up to date in S. cerevisiae and this work provides the key strategies to engineer yeast as an industrial platform for isoprenol production.     

Mutations in Escherichia coli aceE and ribB Genes Allow Survival of Strains Defective in the First Step of the Isoprenoid Biosynthesis Pathway

A functional 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is required for isoprenoid biosynthesis and hence survival in Escherichia coli and most other bacteria. In the first two steps of the pathway, MEP is produced from the central metabolic intermediates pyruvate and glyceraldehyde 3-phosphate via 1-deoxy-D-xylulose 5-phosphate (DXP) by the activity of the enzymes DXP synthase (DXS) and DXP reductoisomerase (DXR). Because the MEP pathway is absent from humans, it was proposed as a promising new target to develop new antibiotics. However, the lethal phenotype caused by the deletion of DXS or DXR was found to be suppressed with a relatively high efficiency by unidentified mutations. Here we report that several mutations in the unrelated genes aceE and ribB rescue growth of DXS-defective mutants because the encoded enzymes allowed the production of sufficient DXP in vivo. Together, this work unveils the diversity of mechanisms that can evolve in bacteria to circumvent a blockage of the first step of the MEP pathway.     

Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

Terpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO₂. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C₅ sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of a dxs deletion in Escherichia coli grown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-type E. coli yajO gene, annotated as a putative xylose reductase, or via various mutations in the native ribB gene. In vitro analysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway in E. coli for production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.     

Advances in the Plant Isoprenoid Biosynthesis Pathway and Its Metabolic Engineering

Although the cytosolic isoprenoid biosynthetic pathway, mavolonate pathway, in plants has been known for many years, a new plastidial 1-deoxyxylulose-5-phosphate (DXP) pathway was identified in the past few years and its related intermediates, enzymes, and genes have been characterized quite recently.With a deep insight into the biosynthetic pathway of isoprenoids, investigations into the metabolic engineering of isoprenoid biosynthesis have started to prosper. In the present article, recent advances in the discoveries and regulatory roles of new genes and enzymes in the plastidial isoprenoid biosynthesis path way are reviewed and examples of the metabolic engineering of cytosolic and plastidial isoprenoids biosnthesis are discussed.     

<Emphasis Type="Italic">In silico</Emphasis> analysis and experimental improvement of taxadiene heterologous biosynthesis in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The biosynthesis of terpenoids in heterologous hosts has become increasingly popular. Isopentenyl diphosphate (IPP) is the central precursor of all isoprenoids, and the synthesis can proceed via two separate pathways in different organisms: The 1-deoxylulose 5-phosphate (DXP) pathway and the mevalonate (MVA) pathway. In this study, an in silico comparison was made between the maximum theoretical IPP yields and the thermodynamic properties of the DXP and MVA pathways using different hosts and carbon sources. We found that Escherichia coli and its DXP pathway have the most potential for IPP production. Consequently, codon usage redesign, and combinations of chromosomal engineering and various strains were considered for optimizing taxadiene biosynthesis through the endogenic DXP pathway. A high production strain yielding 876 ± 60 mg/L taxadiene, with an overall volumetric productivity of 8.9 mg/(L × h), was successfully obtained by combining the chromosomal engineered upstream DXP pathway and the downstream taxadiene biosynthesis pathway. This is the highest yield thus far reported for taxadiene production in a heterologous host. These results indicate that genetic manipulation of the DXP pathway has great potential to be used for production of terpenoids, and that chromosomal engineering is a powerful tool for heterologous biosynthesis of natural products.     

Advances in the Plant Isoprenoid Biosynthesis Pathway and Its Metabolic Engineering

Although the cytosolic isoprenoid biosynthetic pathway, mavolonate pathway, in plants has been known for many years, a new plastidial 1-deoxyxylulose-5-phosphate (DXP) pathway was identified in the past few years and its related intermediates, enzymes, and genes have been characterized quite recently. With a deep insight into the biosynthetic pathway of isoprenoids, investigations into the metabolic engineering of isoprenoid biosynthesis have started to prosper. In the present article, recent advances in the discoveries and regulatory roles of new genes and enzymes in the plastidial isoprenoid biosynthesis pathway are reviewed and examples of the metabolic engineering of cytosolic and plastidial isoprenoids biosynthesis are discussed.     

1-Deoxy-D-xylulose 5-phosphate reductoisomerase and plastid isoprenoid biosynthesis during tomato fruit ripening

The recently discovered 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the biosynthesis of plastid isoprenoids (including carotenoids) is not fully elucidated yet despite its central importance for plant life. It is known, however, that the first reaction completely specific to the pathway is the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) into MEP by the enzyme DXP reductoisomerase (DXR). We have identified a tomato cDNA encoding a protein with homology to DXR and in vivo activity, and show that the levels of the corresponding DXR mRNA and encoded protein in fruit tissues are similar before and during the massive accumulation of carotenoids characteristic of fruit ripening. The results are consistent with a non-limiting role of DXR, and support previous work proposing DXP synthase (DXS) as the first regulatory enzyme for plastid isoprenoid biosynthesis in tomato fruit. Inhibition of DXR activity by fosmidomycin showed that plastid isoprenoid biosynthesis is required for tomato fruit carotenogenesis but not for other ripening processes. In addition, dormancy was reduced in seeds from fosmidomycin-treated fruit but not in seeds from the tomato yellow ripe mutant (defective in phytoene synthase-1, PSY1), suggesting that the isoform PSY2 might channel the production of carotenoids for abscisic acid biosynthesis. Furthermore, the complete arrest of tomato seedling development using fosmidomycin confirms a key role of the MEP pathway in plant development.     

(R)-Mevalonate 3-Phosphate Is an Intermediate of the Mevalonate Pathway in Thermoplasma acidophilum

The lack of a few conserved enzymes in the classical mevalonate pathway and the widespread existence of isopentenyl phosphate kinase suggest the presence of a partly modified mevalonate pathway in most archaea and in some bacteria. In the pathway, (R)-mevalonate 5-phosphate is thought to be metabolized to isopentenyl diphosphate via isopentenyl phosphate. The long anticipated enzyme that catalyzes the reaction from (R)-mevalonate 5-phosphate to isopentenyl phosphate was recently identified in a Cloroflexi bacterium, Roseiflexus castenholzii, and in a halophilic archaeon, Haloferax volcanii. However, our trial to convert the intermediates of the classical and modified mevalonate pathways into isopentenyl diphosphate using cell-free extract from a thermophilic archaeon Thermoplasma acidophilum implied that the branch point intermediate of these known pathways, i.e. (R)-mevalonate 5-phosphate, is unlikely to be the precursor of isoprenoid. Through the process of characterizing the recombinant homologs of mevalonate pathway-related enzymes from the archaeon, a distant homolog of diphosphomevalonate decarboxylase was found to catalyze the phosphorylation of (R)-mevalonate to yield (R)-mevalonate 3-phosphate. The product could be converted into isopentenyl phosphate, probably through (R)-mevalonate 3,5-bisphosphate, by the action of unidentified T. acidophilum enzymes fractionated by anion-exchange chromatography. These findings demonstrate the presence of a third alternative “Thermoplasma-type” mevalonate pathway, which involves (R)-mevalonate 3-phosphotransferase and probably both (R)-mevalonate 3-phosphate 5-phosphotransferase and (R)-mevalonate 3,5-bisphosphate decarboxylase, in addition to isopentenyl phosphate kinase.     

A Synechococcus leopoliensis SAUG 1402-1 operon harboring the 1-deoxyxylulose 5-phosphate synthase gene and two additional open reading frames is functionally involved in the dimethylallyl diphosphate synthesis.

Experiments have been performed to prove the existence and the functionality of the novel mevalonate independent 1-deoxyxylulose 5-phosphate isoprenoid biosynthesis pathway in cyanobacteria. For this purpose, a segment of the 1-deoxyxylulose 5-phosphate synthase gene (dxs) was amplified from Synechococcus leopoliensis SAUG 1402-1 DNA via PCR using oligonucleotides for conserved regions of dxs. Subsequent hybridization screening of a genomic cosmid library of S. leopoliensis with this segment has led to the identification of an 18.7 kbp segment of the S. leopoliensis genome on which a dxs homologous gene and two adjacent open reading frames organized in one operon could be localized by DNA sequencing. The three genes of the operon were separately expressed in Escherichia coli, proving that the identified cyanobacterial dxs is functionally involved in the formation of dimethylallyl diphosphate, one basic intermediate of isoprenoid biosynthesis.     

Precise precursor rebalancing for isoprenoids production by fine control of gapA expression in Escherichia coli

Biosynthesis of isoprenoids via the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway requires equimolar glyceraldehyde 3-phosphate and pyruvate to divert carbon flux toward the products of interest. Here, we demonstrate that precursor balancing is one of the critical steps for the production of isoprenoids in Escherichia coli. First, the implementation of the synthetic lycopene production pathway as a model system and the amplification of the native DXP pathway were accomplished using synthetic constitutive promoters and redesigned 5′-untranslated regions (5′-UTRs). Next, fine-controlled precursor balancing was investigated by tuning phosphoenolpyruvate synthase (PpsA) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The results showed that tuning-down of gapA improved the specific lycopene content by 45% compared to the overexpression of ppsA. The specific lycopene content in the strains with down-regulated gapA increased by 97% compared to that in the parental strain. Our results indicate that gapA is the best target for precursor balancing to increase biosynthesis of isoprenoids.     

Structure of 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase involved in mevalonate-independent biosynthesis of isoprenoids

Isoprenoids are biosynthesized from isopentenyl diphosphate and the isomeric dimethylallyl diphosphate via the mevalonate pathway or a mevalonate-independent pathway that was identified during the last decade. The non-mevalonate pathway is present in many bacteria, some algae and in certain protozoa such as the malaria parasite Plasmodium falciparum and in the plastids of higher plants, but not in mammals and archaea. Therefore, these enzymes have been recognised as promising drug targets. We report the crystal structure of Escherichia coli 2C- methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF), which converts 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate into 2C-methyl-d-erythritol 2,4-cyclodiphosphate and CMP in a Mg-dependent reaction. The protein forms homotrimers that tightly bind one zinc ion per subunit at the active site, which helps to position the substrate for direct attack of the 2-phosphate group on the beta-phosphate.     

The interplay between classical and alternative isoprenoid biosynthesis controls gammadelta T cell bioactivity of Listeria monocytogenes

Isoprenoids are synthesised either through the classical, mevalonate pathway, or the alternative, non-mevalonate, 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The latter is found in many microbial pathogens and proceeds via (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), a potent activator of human Vgamma9/Vdelta2 T cells. Listeria monocytogenes is the only pathogenic bacterium known to contain both pathways concurrently. Strategic gene knockouts demonstrate that either pathway is functional but dispensable for viability. Yet, disrupting the mevalonate pathway results in a complementary upregulation of the MEP pathway. Vgamma9/Vdelta2 T cell bioactivity is increased in DeltalytB mutants where HMB-PP accumulation is expected, and lost in DeltagcpE mutants which fail to produce HMB-PP.     

Interrelation between the pathways of isoprenoid biosynthesis and carbon source catabolism in anaerobic and facultatively anaerobic bacteria

Data on the interrelation between the pathways of the carbon source catabolism and isoprenoid biosynthesis in anaerobic and facultatively anaerobic bacteria were obtained. Two pathways of isoprenoid biosynthesis (nonmevalonate and mevalonate) were revealed in the representatives of the genus Clostridium. The nonmevalonate pathway of isoprenoid biosynthesis and the glycolytic pathway of substrate oxidation are typical of glucose-grown bacteria, whereas the pentose phosphate cycle operates in xylose-grown bacteria. The mevalonate pathway of isoprenoid biosynthesis was revealed in strain Clostridium thermosaccharolyticum DSM 571 grown in the presence of mevinolin, as well as in a number of lactic acid bacteria. Mevinolin is known to react with the lactate dehydrogenase complex, preventing reduction of pyruvate. The nonmevalonate pathway of isoprenoid biosynthesis was revealed in Bifidobacterium bifidum. The role of different metabolic pathways in isoprenoid biosynthesis is discussed.