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.     

Utilizing a Dynamical Description of IspH to Aid in the Development of Novel Antimicrobial Drugs

The nonmevalonate pathway is responsible for isoprenoid production in microbes, including H. pylori, M. tuberculosis and P. falciparum, but is nonexistent in humans, thus providing a desirable route for antibacterial and antimalarial drug discovery. We coordinate a structural study of IspH, a [4Fe-4S] protein responsible for converting HMBPP to IPP and DMAPP in the ultimate step in the nonmevalonate pathway. By performing accelerated molecular dynamics simulations on both substrate-free and HMBPP-bound [Fe₄S₄]²⁺ IspH, we elucidate how substrate binding alters the dynamics of the protein. Using principal component analysis, we note that while substrate-free IspH samples various open and closed conformations, the closed conformation observed experimentally for HMBPP-bound IspH is inaccessible in the absence of HMBPP. In contrast, simulations with HMBPP bound are restricted from accessing the open states sampled by the substrate-free simulations. Further investigation of the substrate-free simulations reveals large fluctuations in the HMBPP binding pocket, as well as allosteric pocket openings – both of which are achieved through the hinge motions of the individual domains in IspH. Coupling these findings with solvent mapping and various structural analyses reveals alternative druggable sites that may be exploited in future drug design efforts.     

Production of β-carotene and acetate in recombinant Escherichia coli with or without mevalonate pathway at different culture temperature or pH

Natural β-carotene has received much attention as consumers have become more health conscious. Its production by various microorganisms including metabolically engineered Escherichia coli or Saccharomyces cerevisiae has been attempted. We successfully created a recombinant E. coli with an engineered whole mevalonate pathway in addition to β-carotene biosynthetic genes and evaluated the engineered cells from the aspects of metabolic balance between central metabolism and β-carotene production by comparison with conventional β-carotene producing recombinant E. coli (control) utilizing a native methylerythritol phosphate (MEP) pathway using bioreactor cultures generated at different temperatures or pHs. Better production of β-carotene was obtained in E. coli cultured at 37°C than at 25°C. A two-fold higher titer and 2.9-fold higher volumetric productivity were obtained in engineered cells compared with control cells. Notably, a marginal amount of acetate was produced in actively growing engineered cells, whereas more than 8 g/L of acetate was produced in control cells with reduced cell growth at 37°C. The data indicated that the artificial operon of the whole mevalonate pathway operated efficiently in redirecting acetyl-CoA into isopentenyl pyrophosphate (IPP), thereby improving production of β-carotene, whereas the native MEP pathway did not convert a sufficient amount of pyruvate into IPP due to endogenous feedback regulation. Engineered cells also produced lycopene with a reduced amount of β-carotene in weak alkaline cultures, consistent with the inhibition of lycopene cyclase.     

A closer look at the spectroscopic properties of possible reaction intermediates in wild-type and mutant (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase

(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (IspH or LytB) catalyzes the terminal step of the MEP/DOXP pathway where it converts (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into the two products, isopentenyl diphosphate and dimethylallyl diphosphate. The reaction involves the reductive elimination of the C4 hydroxyl group, using a total of two electrons. Here we show that the active form of IspH contains a [4Fe-4S] cluster and not the [3Fe-4S] form. Our studies show that the cluster is the direct electron source for the reaction and that a reaction intermediate is bound directly to the cluster. This active form has been trapped in a state, dubbed FeS(A), that was detected by electron paramagnetic resonance (EPR) spectroscopy when one-electron-reduced IspH was incubated with HMBPP. In addition, three mutants of IspH have been prepared and studied, His42, His124, and Glu126 (Aquifex aeolicus numbering), with particular attention paid to the effects on the cluster properties and possible reaction intermediates. None of the mutants significantly affected the properties of the [4Fe-4S](+) cluster, but different effects were observed when one-electron-reduced forms were incubated with HMBPP. Replacing His42 led to an increased K(M) value and a much lower catalytic efficiency, confirming the role of this residue in substrate binding. Replacing the His124 also resulted in a lower catalytic efficiency. In this case, however, the enzyme showed the loss of the [4Fe-4S](+) EPR signal upon addition of HMBPP without the subsequent formation of the FeS(A) signal. Instead, a radical-type signal was observed in some of the samples, indicating that this residue plays a role in the correct positioning of the substrate. The incorrect orientation in the mutant leads to the formation of substrate-based radicals instead of the cluster-bound intermediate complex FeS(A). Replacing the Glu126 also resulted in a lower catalytic efficiency, with yet a third type of EPR signal being detected upon incubation with HMBPP. (31)P and (2)H ENDOR measurements of the FeS(A) species incubated with regular and (2)H-C4-labeled HMBPP reveal that the substrate binds to the enzyme in the proximity of the active-site cluster with C4 adjacent to the site of linkage between the FeS cluster and HMBPP. Comparison of the spectroscopic properties of this intermediate to those of intermediates detected in (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase and ferredoxin:thioredoxin reductase suggests that HMBPP binds to the FeS cluster via its hydroxyl group instead of a side-on binding as previously proposed for the species detected in the inactive Glu126 variant. Consequences for the IspH reaction mechanism are discussed.     

Engineering central metabolic modules of Escherichia coli for improving β-carotene production

ATP and NADPH are two important cofactors for production of terpenoids compounds. Here we have constructed and optimized β-carotene synthetic pathway in Escherichia coli, followed by engineering central metabolic modules to increase ATP and NADPH supplies for improving β-carotene production. The whole β-carotene synthetic pathway was divided into five modules. Engineering MEP module resulted in 3.5-fold increase of β-carotene yield, while engineering β-carotene synthesis module resulted in another 3.4-fold increase. The best β-carotene yield increased 21%, 17% and 39% after modulating single gene of ATP synthesis, pentose phosphate and TCA modules, respectively. Combined engineering of TCA and PPP modules had a synergistic effect on improving β-carotene yield, leading to 64% increase of β-carotene yield over a high producing parental strain. Fed-batch fermentation of the best strain CAR005 was performed, which produced 2.1 g/L β-carotene with a yield of 60 mg/g DCW.     

Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae

Transfer of a biosynthetic pathway between evolutionary distant organisms can create a metabolic shunt capable of bypassing the native regulation of the host organism, hereby improving the production of secondary metabolite precursor molecules for important natural products. Here, we report the engineering of Escherichia coli genes encoding the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway into the genome of Saccharomyces cerevisiae and the characterization of intermediate metabolites synthesized by the MEP pathway in yeast. Our UPLC-MS analysis of the MEP pathway metabolites from engineered yeast showed that the pathway is active until the synthesis of 2-C-methyl-d-erythritol-2,4-cyclodiphosphate, but appears to lack functionality of the last two steps of the MEP pathway, catalyzed by the [4Fe–4S] iron sulfur cluster proteins encoded by ispG and ispH. In order to functionalize the last two steps of the MEP pathway, we co-expressed the genes for the E. coli iron sulfur cluster (ISC) assembly machinery. By deleting ERG13, thereby incapacitating the mevalonate pathway, in conjunction with labeling experiments with U–¹³C₆ glucose and growth experiments, we found that the ISC assembly machinery was unable to functionalize ispG and ispH. However, we have found that leuC and leuD, encoding the heterodimeric iron–sulfur cluster protein, isopropylmalate isomerase, can complement the S. cerevisiae leu1 auxotrophy. To our knowledge, this is the first time a bacterial iron–sulfur cluster protein has been functionally expressed in the cytosol of S. cerevisiae under aerobic conditions and shows that S. cerevisiae has the capability to functionally express at least some bacterial iron–sulfur cluster proteins in its cytosol.     

Construction of an alternative glycerol-utilization pathway for improved β-carotene production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Glycerol, which is an inevitable by-product of biodiesel production, is an ideal carbon source for the production of carotenoids due to its low price, good availability and chemically reduced status, which results in a low requirement for additional reducing equivalents. In this study, an alternative carbon-utilization pathway was constructed in Escherichia coli to enable more efficient β-carotene production from glycerol. An aldehyde reductase gene (alrd) and an aldehyde dehydrogenase gene (aldH) from Ralstonia eutropha H16 were integrated into the E. coli chromosome to form a novel glycerol-utilization pathway. The β-carotene specific production value was increased by 50% after the introduction of alrd and aldH. It was found that the glycerol kinase gene (garK), alrd and aldH were the bottleneck of the alternative glycerol metabolic pathway, and modulation of garK gene with an mRS library further increased the β-carotene specific production value by 13%. Finally, co-modulation of genes in the introduced aldH–alrd operon led to 86% more of β-carotene specific production value than that of the strain without the alternative glycerol-utilization pathway and the glycerol-utilization rate was also increased. In this work, β-carotene production of E. coli was significantly improved by constructing and optimizing an alternative glycerol-utilization pathway. This strategy can potentially be used to improve the production of other isoprenoids using glycerol as a cheap and abundant substrate, and therefore has industrial relevance.     

Structure of the GcpE (IspG)-MEcPP complex from Thermus thermophilus

Isoprenoid precursor biosynthesis occurs through the mevalonate or the methylerythritol phosphate (MEP) pathway, used i.e., by humans and by many human pathogens, respectively. In the MEP pathway, 2-C-methyl-d-erythritol-2,4-cyclo-diphosphate (MEcPP) is converted to (E)-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) by the iron-sulfur cluster enzyme HMBPP synthase (GcpE). The presented X-ray structure of the GcpE-MEcPP complex from Thermus thermophilus at 1.55 Å resolution provides valuable information about the catalytic mechanism and for rational inhibitor design. MEcPP binding inside the TIM-barrel funnel induces a 60° rotation of the [4Fe-4S] cluster containing domain onto the TIM-barrel entrance. The apical iron of the [4Fe-4S] cluster ligates with the C3 oxygen atom of MEcPP.     

Predictive modeling of β-carotene accumulation by Dunaliella salina as a function of pH,NaCI, and irradiance

Predictive modeling of β-carotene accumulation by Dunaliella salina as a function of NaCI, pH, and irradiance was studied. Modified Logistic, Gompertz, Schnute, Richards, and Stannard models were fitted to describe β-carotene accumulation by the alga under various environmental conditions. Lag time (λ, days), maximum accumulation (A, pg/cell), and the maximum production rate (μ, 1/day) for β-carotene accumulation were calculated by modified Logistic and Gompertz models. Values of λ, A, and μ for β-carotene accumulation varied between 0.26 and 20.14 days, 57.48 to 198.76 pg β-carotene/cell, and 1.80 to 3.68 1/day, respectively. Results revealed that Logistic and Gompertz models could be used to describe the accumulation of β-carotene by D. salina as a function of salt concentrations, pH, and irradiance. The highest asymptotic value was predicted from Logistic and Gompertz models at pH 9.0, 48 kerg/(cm² s) light intensity, and 20% NaCl concentration.     

Influence of oxidative and nitrosative stress on accumulation of diphosphate intermediates of the non-mevalonate pathway of isoprenoid biosynthesis in corynebacteria and mycobacteria

Artificial generation of oxygen superoxide radicals in actively growing cultures of Mycobacterium tuberculosis, Myc. smegmatis, and Corynebacterium ammoniagenes is followed by accumulation in the bacterial cells of substantial amounts of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcDP) — an intermediate of the non-mevalonate pathway of isoprenoid biosynthesis (MEP) — most possibly due to the interaction of the oxygen radicals with the 4Fe-4S group in the active center and inhibition of the enzyme (E)-4-oxy-3-methylbut-2-enyl diphosphate synthase (IspG). Cadmium ions known to inhibit IspG enzyme in chloroplasts (Rivasseau, C., Seemann, M., Boisson, A. M., Streb, P., Gout, E., Douce, R., Rohmer, M., and Bligny, R. (2009) Plant Cell Environ., 32, 82–92), when added to culture of Myc. smegmatis, substantially increase accumulation of MEcDP induced by oxidative stress with no accumulation of other organic phosphate intermediates in the cell. Corynebacterium ammoniagenes, well-known for its ability to synthesize large amounts of MEcDP, was also shown to accumulate this unique cyclodiphosphate in actively growing culture when NO at low concentration is artificially generated in the medium. A possible role of the MEP-pathway of isoprenoid biosynthesis and a role of its central intermediate MEcDP in bacterial response to nitrosative and oxidative stress is discussed.     

Reconstruction of the astaxanthin biosynthesis pathway in rice endosperm reveals a metabolic bottleneck at the level of endogenous β-carotene hydroxylase activity

Astaxanthin is a high-value ketocarotenoid rarely found in plants. It is derived from β-carotene by the 3-hydroxylation and 4-ketolation of both ionone end groups, in reactions catalyzed by β-carotene hydroxylase and β-carotene ketolase, respectively. We investigated the feasibility of introducing an extended carotenoid biosynthesis pathway into rice endosperm to achieve the production of astaxanthin. This allowed us to identify potential metabolic bottlenecks that have thus far prevented the accumulation of this valuable compound in storage tissues such as cereal grains. Rice endosperm does not usually accumulate carotenoids because phytoene synthase, the enzyme responsible for the first committed step in the pathway, is not present in this tissue. We therefore expressed maize phytoene synthase 1 (ZmPSY1), Pantoea ananatis phytoene desaturase (PaCRTI) and a synthetic Chlamydomonas reinhardtii β-carotene ketolase (sCrBKT) in transgenic rice plants under the control of endosperm-specific promoters. The resulting grains predominantly accumulated the diketocarotenoids canthaxanthin, adonirubin and astaxanthin as well as low levels of monoketocarotenoids. The predominance of canthaxanthin and adonirubin indicated the presence of a hydroxylation bottleneck in the ketocarotenoid pathway. This final rate-limiting step must therefore be overcome to maximize the accumulation of astaxanthin, the end product of the pathway.     

Efficient production of retinol in Yarrowia lipolytica by increasing stability using antioxidant and detergent extraction

The demand for bio-based retinol (vitamin A) is currently increasing, however its instability represents a major bottleneck in microbial production. Here, we developed an efficient method to selectively produce retinol in Yarrowia lipolytica. The β-carotene 15,15′-dioxygenase (BCO) cleaves β-carotene into retinal, which is reduced to retinol by retinol dehydrogenase (RDH). Therefore, to produce retinol, we first generated β-carotene-producing strain based on a high-lipid-producer via overexpressing genes including heterologous β-carotene biosynthetic genes, GGS1^F43I mutant of endogenous geranylgeranyl pyrophosphate synthase isolated by directed evolution, and FAD1 encoding flavin adenine dinucleotide synthetase, while deleting several genes previously known to be beneficial for carotenoid production. To produce retinol, 11 copies of BCO gene from marine bacterium 66A03 (Mb.Blh) were integrated into the rDNA sites of the β-carotene overproducer. The resulting strain produced more retinol than retinal, suggesting strong endogenous promiscuous RDH activity in Y. lipolytica. The introduction of Mb.Blh led to a considerable reduction in β-carotene level, but less than 5% of the consumed β-carotene could be detected in the form of retinal or retinol, implying severe degradation of the produced retinoids. However, addition of the antioxidant butylated hydroxytoluene (BHT) led to a >20-fold increase in retinol production, suggesting oxidative damage is the main cause of intracellular retinol degradation. Overexpression of GSH2 encoding glutathione synthetase further improved retinol production. Raman imaging revealed co-localization of retinol with lipid droplets, and extraction of retinol using Tween 80 was effective in improving retinol production. By combining BHT treatment and extraction using Tween 80, the final strain CJ2104 produced 4.86 g/L retinol and 0.26 g/L retinal in fed-batch fermentation in a 5-L bioreactor, which is the highest retinol production titer ever reported. This study demonstrates that Y. lipolytica is a suitable host for the industrial production of bio-based retinol.     

Sequence variation in 3′UTR region of crtRB1 gene and its effect on β-carotene accumulation in maize kernel

β-carotene fortification of maize has emerged as a potential, long-term and sustainable approach to alleviate vitamin A deficiency in humans. Among the several genes involved in the carotenoid biosynthetic pathway, the 543 bp allele at crtRB1 3′TE (Transposable Element) gene (allele 1, without insertion) is associated with higher β-carotene accumulation. Estimation of β-carotene through high performance liquid chromatography showed that the CIMMYT genotypes with allele 1 had high kernel β-carotene content whereas the Indian inbreds with the same allele had low β-carotene content. To know the reason for this variation, allele 1 of crtRB1 3′TE gene was sequenced from a set of 11 diverse maize inbreds collected from CIMMYT and Indian germplasm. The sequence data of the allele 1 revealed the presence of 13 single nucleotide polymorphisms (SNPs) and 7 insertions and deletions (InDels). Exonic region had two SNPs, intronic region had one SNP and one InDel, whereas 3′-untranslated region (UTR) region of the gene showed 10 SNPs and 6 InDels. Among the several SNPs and InDels, SNP4, SNP13, InDel6 and InDel7 identified in the 3′-UTR region clearly differentiated the high and the low β-carotene genotypes. These 3′-UTR polymorphisms in allele 1 of the crtRB1 3′TE gene could be associated with the variation in kernel β-carotene accumulation by regulating the translation and stability of the mRNA. The SNPs and the InDels associated with higher level of β-carotene will be used as a gene-based marker(s) in selection of genotypes and to develop biofortified maize hybrids to alleviate vitamin A deficiency in humans.     

fldA is an essential gene required in the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis

Although flavodoxin I is indispensable for Escherichia coli growth, the exact pathway(s) where flavodoxin I is essential has not been identified. We performed transposon mutagenesis of the flavodoxin I gene, fldA, in an E. coli strain that expressed mevalonate pathway enzymes and that had a point mutation in the lytB gene of the MEP pathway resulting in the accumulation of (E)-4-hydroxy-3-methylbutyl-2-enyl pyrophosphate (HMBPP). Disruption of fldA abrogated mevalonate-independent growth and dramatically decreased HMBPP levels. The fldA- mutant grew with mevalonate indicating that the essential role of flavodoxin I under aerobic conditions is in the MEP pathway. Growth was restored by fldA complementation. Since GcpE (which synthesizes HMBPP) and LytB are iron-sulfur enzymes that require a reducing system for their activity, we propose that flavodoxin is essential for GcpE and possibly LytB activity. Thus, the essential role for flavodoxin I in E. coli is in the MEP pathway for isoprenoid biosynthesis.     

Enhancement of carotenoid biosynthesis in the green microalga Dunaliella salina with light-emitting diodes and adaptive laboratory evolution

There is a particularly high interest to derive carotenoids such as β-carotene and lutein from higher plants and algae for the global market. It is well known that β-carotene can be overproduced in the green microalga Dunaliella salina in response to stressful light conditions. However, little is known about the effects of light quality on carotenoid metabolism, e.g., narrow spectrum red light. In this study, we present UPLC-UV-MS data from D. salina consistent with the pathway proposed for carotenoid metabolism in the green microalga Chlamydomonas reinhardtii. We have studied the effect of red light-emitting diode (LED) lighting on growth rate and biomass yield and identified the optimal photon flux for D. salina growth. We found that the major carotenoids changed in parallel to the chlorophyll b content and that red light photon stress alone at high level was not capable of upregulating carotenoid accumulation presumably due to serious photodamage. We have found that combining red LED (75 %) with blue LED (25 %) allowed growth at a higher total photon flux. Additional blue light instead of red light led to increased β-carotene and lutein accumulation, and the application of long-term iterative stress (adaptive laboratory evolution) yielded strains of D. salina with increased accumulation of carotenoids under combined blue and red light.     

Targeting pathway expression to subcellular organelles improves astaxanthin synthesis in Yarrowia lipolytica

Metabolic engineering approaches for the production of high-value chemicals in microorganisms mostly use the cytosol as general reaction vessel. However, sequestration of enzymes and substrates, and metabolic cross-talk frequently prevent efficient synthesis of target compounds in the cytosol. Organelle compartmentalization in eukaryotic cells suggests ways for overcoming these challenges. Here we have explored this strategy by expressing the astaxanthin biosynthesis pathway in sub-organelles of the oleaginous yeast Yarrowia lipolytica. We first showed that fusion of the two enzymes converting β-carotene to astaxanthin, β-carotene ketolase and hydroxylase, performs better than the expression of individual enzymes. We next evaluated the pathway when expressed in compartments of lipid body, endoplasmic reticulum or peroxisome, individually and in combination. Targeting the astaxanthin pathway to subcellular organelles not only accelerated the conversion of β-carotene to astaxanthin, but also significantly decreased accumulation of the ketocarotenoid intermediates. Anchoring enzymes simultaneously to all three organelles yielded the largest increase of astaxanthin synthesis, and ultimately produced 858 mg/L of astaxanthin in fed-batch fermentation (a 141-fold improvement over the initial strain). Our study is expected to help unlock the full potential of subcellular compartments and advance LB-based compartmentalized isoprenoid biosynthesis in Y. lipolytica.     

Unlocking the biosynthesis of sesquiterpenoids from methane via the methylerythritol phosphate pathway in methanotrophic bacteria,using α-humulene as a model compound

Isoprenoids are an abundant and diverse class of natural products with various applications in the pharmaceutical, cosmetics and biofuel industries. A methanotroph-based biorefinery is an attractive scenario for the production of a variety of value-added compounds from methane, because methane is a promising alternative feedstock for industrial biomanufacturing. In this study, we metabolically engineered Methylotuvimicrobium alcaliphilum 20Z for de novo synthesis of a sesquiterpenoid from methane, using α-humulene as a model compound, via optimization of the native methylerythritol phosphate (MEP) pathway. Expression of codon-optimized α-humulene synthase from Zingiber zerumbet in M. alcaliphilum 20Z resulted in an initial yield of 0.04 mg/g dry cell weight. Overexpressing key enzymes (IspA, IspG, and Dxs) for debottlenecking of the MEP pathway increased α-humulene production 5.2-fold compared with the initial strain. Subsequently, redirecting the carbon flux through the Embden–Meyerhof–Parnas pathway resulted in an additional 3-fold increase in α-humulene production. Additionally, a genome-scale model using flux scanning based on enforced objective flux method was used to identify potential overexpression targets to increase flux towards isoprenoid production. Several target reactions from cofactor synthesis pathways were probed and evaluated for their effects on α-humulene synthesis, resulting in α-humulene yield up to 0.75 mg/g DCW with 18.8-fold enhancement from initial yield. This study first demonstrates production of a sesquiterpenoid from methane using methanotrophs as the biocatalyst and proposes potential strategies to enhance production of sesquiterpenoid and related isoprenoid products in engineered methanotrophic bacteria.     

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

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.     

Over-expression of Arabidopsis thaliana carotenoid hydroxylases individually and in combination with a β-carotene ketolase provides insight into in vivo functions

Carotenoids represent a group of widely distributed pigments derived from the general isoprenoid biosynthetic pathway that possess diverse functions in plant primary and secondary metabolism. Modification of α- and β-carotene backbones depends in part on ring hydroxylation. Two ferredoxin-dependent non-heme di-iron monooxygenases (AtB1 and AtB2) that mainly catalyze in vivo β-carotene hydroxylations of β,β-carotenoids, and two heme-containing cytochrome P450 (CYP) monooxygenases (CYP97A3 and CYP97C1) that preferentially hydroxylate the ε-ring of α-carotene or the β-ring of β,ε-carotenoids, have been characterized in Arabidopsis by analysis of loss-of-function mutant phenotypes. We further investigated functional roles of both hydroxylase classes in modification of the β- and ε-rings of α-carotene and β-carotene through over-expression of AtB1, CYP97A3, CYP97C1, and the hydroxylase candidate CYP97B3. Since carotenoid hydroxylation is required for generation of ketocarotenoids by the bkt1(CrtO) β-carotene ketolase, all hydroxylase constructs were also introduced into an Arabidopsis line expressing the Haematococcus pluvalis bkt1 β-carotene ketolase. Analysis of foliar carotenoid profiles in lines overexpressing the individual hydroxylases indicate a role for CYP97B3 in carotenoid biosynthesis, confirm and extend previous findings of hydroxylase activities based on knock-out mutants, and suggest functions of the multifunctional enzymes in carotenoid biosynthesis. Hydroxylase over-expression in combination with bkt1 did not result in ketocarotenoid accumulation, but instead unexpected patterns of α-carotene derivatives, accompanied by a reduction of α-carotene, were observed. These data suggest possible interactions between the β-carotene ketolase bkt1 and the hydroxylases that impact partitioning of carbon flux into different carotenoid branch pathways.