Taraxacum kok-saghyz(TKS)is rich in natural rubber(NR),a natural organic macromolecular compound composed of cis-1,4-polyisoprene,and may become the second NR-bearing plant for biochemical engineering development.In t...Taraxacum kok-saghyz(TKS)is rich in natural rubber(NR),a natural organic macromolecular compound composed of cis-1,4-polyisoprene,and may become the second NR-bearing plant for biochemical engineering development.In this paper,a rapid and quantitative ultra-high performance liquid chromatography tandem mass spectrometry(UHPLCMS/MS)method was established for determination of macromolecular biosynthesis substrate(dimethylallyl pyrophosphate,DMAPP)and initiator(farnesyl pyrophosphate,FPP)contained in TKS.A Kromasil C18 chromatographic column was used for separation,and the multi-reaction monitoring mode(MRM)of triple quadrupole mass spectrometry was used for detection.Quantification was performed by external calibration method.The results showed that the limit of detection(LOD)and the limit of quantitation(LOQ)of DMAPP were 2.42μg/L and 7.26μg/L,respectively,and the LOQ and the LOD of FPP were 1.02μg/L and 3.05μg/L,respectively.At a concentration of 1—1000μg/L,both analytes had good determination coefficients(>0.999)of calibration curve.The recoveries of DMAPP and FPP were between 99.0%and 117.1%.In real samples detection,the contents of DMAPP and FPP in TKS samples were between 23.32—82.77μg/L and 12.03—85.67μg/L,respectively.Thus,this approach is a reliable method to quantify DMAPP and FPP in TKS.展开更多
Isoprenoids are a functionally and structurally diverse class of natural organic chemicals. The universal precursors of all isoprenoids, isopentenyl diphosphate and dimethylallyl diphosphate are synthesized through th...Isoprenoids are a functionally and structurally diverse class of natural organic chemicals. The universal precursors of all isoprenoids, isopentenyl diphosphate and dimethylallyl diphosphate are synthesized through the mevalonate and 2C-methyl- D-erythritol 4-phosphate (MEP) pathways, respectively. Many isoprenoids produced through the MEP pathway play an important role in plant acclimation to different light environments. Eupatorium adenophorum, an invasive weed in China, presents a remarkable capacity to acclimate to various light environments, which constitutes its solid foundation of being a successful invasive species. Thus we aimed at gaining a deeper insight into the regulation of MEP pathway in E. adenophorum to further understand the invasive mechanism. 2C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF or MCS) is an essential enzyme in the MEP pathway. In this paper, a novel IspF gene was cloned and characterized from E. adenophorum. Tissue-specific expression assays revealed a higher expression of EalspF1 in leaves than in stems and roots. The expression of EalspF1 was responsive to different light conditions. Some up-regulation of EalspF1 expression was also found after the treatments with signal compounds and after wounding stress. Interestingly, the over-expression of EalspF1 in Arabidopsis led to increase carotenoids contents, resulting in an enhanced tolerance to high light. Taken together, these results indicate that the EalspFl-derived enzyme participates in isoprenoid metabolism and among others, the expression of this gene in E. adenophorum is involved in the regulation of plastidial isoprenoids, which play an important role in acclimation to various light environments.展开更多
While large quantities of latex can be handled either by standard extraction techniques such as Soxhlet extraction or accelerated solvent extraction (ASE), smaller samples on the order of 0.3 - 0.5 g require handling ...While large quantities of latex can be handled either by standard extraction techniques such as Soxhlet extraction or accelerated solvent extraction (ASE), smaller samples on the order of 0.3 - 0.5 g require handling on a microscale. We collected latex from lettuce plants in microcentrifuge tubes and, after drying under vacuum, resuspended the dried sample in acetone by holding in an ultrasonic cleaner. The resulting fine suspension was readily extracted with acetone and toluene to provide fractions representing the resin and rubber content of the latex. Using this approach, we compared latex from stems of bolting lettuce and from the floral stem of lettuce plants. While both types of stems contained a similar percentage of resin, the rubber content of the bolting stems exceeded that of the floral stems.展开更多
The effects of temperature on net photosynthesis and stomatal conductance, emission of foliar volatile organic compounds (VOCs), and phenolics were investigated after exposing Cordeauxia edulis seedlings to control (2...The effects of temperature on net photosynthesis and stomatal conductance, emission of foliar volatile organic compounds (VOCs), and phenolics were investigated after exposing Cordeauxia edulis seedlings to control (27/19°C) and three levels of elevated (32/23, 37/27, or 42/31°C) day/night temperature regimes in controlled growth chambers. Emissions of foliar VOC were measured on 7th and 14th day (d) of exposures, whereas net photosynthesis and stomatal conductance were measured on the 8th and 15th d. Net photosynthesis and stomatal conductance were not significantly affected by elevated temperatures. Emission rate of isoprene increased by 4-fold with 10°C rise from the control on 7th d of exposure. Emission rates of monoterpenes, sesquiterpenes and total isoprenoids increased to 2-5-fold higher than that of control plants with 5°C rise. Foliar isoprene emission peaked at daytime maximum of 37°C and the mono- and sesquiterpenes at 32°C. Few individual foliar phenolics, and total foliar phenolics showed significant concentration differences between treatments. Although high VOC emissions under warming appeared to help plants to sustain abiotic stresses, arid/semi-arid species might substantially release highly reactive compounds that affect atmospheric chemistry. Hence, more studies are required on plant species of arid/semi-arid ecosystems of Africa to estimate the emission patterns and their role in atmospheric chemistry under the predicted future atmospheric warming.展开更多
Recognition of the biological properties of numerous “natural products” has fueled the current focus of this field, namely, the search for new drugs, antibiotics, insecticides, and herbicides. Based on their biosynt...Recognition of the biological properties of numerous “natural products” has fueled the current focus of this field, namely, the search for new drugs, antibiotics, insecticides, and herbicides. Based on their biosynthetic origins, natural products can be divided into three major groups: the isoprenoids, alkaloids, and phenolic compounds. Isoprenoids are structurally the most diverse group of secondary natural metabolites with different roles in the growth, development, and reproduction of a diverse range of prokaryotic and eukaryotes cells. Mevalonate and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways are known to be responsible for biosynthesis of numerous isoprenoids. HMG-CoA reductase is a rate-determining enzyme in mevalonate pathway, producing intermediates such as farnesyl and geranylgeranyl pyrophosphates, which lead to by-products such as cholesterol. Earlier studies have demonstrated that the inhibition of HMG-CoA reductase is one of the most effective approaches for treating hypercholesterolemia and eventually cardiovascular disease (CVD). Statins are HMG-CoA reductase inhibitors and the most prescribed group of drugs worldwide in treating hypercholesterolemia;however the application of this group of drugs may be expensive and has side effects including rashes and gastrointestinal symptoms. For these reasons, there is an important need to examine the viability of natural products as an alternative to statin treatment. This article is a review of different aforementioned areas with a focus on isoprenoids that can be used for the regulation of HMG-CoA reductase.展开更多
1-deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the 2-C-methyl-D- erythritol 4-phosphate (MEP) pathway consisting in the condensation of (hydroxiethyl)thiamin derived from pyruvate with D-g...1-deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the 2-C-methyl-D- erythritol 4-phosphate (MEP) pathway consisting in the condensation of (hydroxiethyl)thiamin derived from pyruvate with D-glyceraldehyde 3-phosphate (GAP) to yield 1-deoxy-D-xylulose 5-phosphate (DXP). The role of the conserved residues H49, E370, D427 and H431 of E. coli DXS was examined by site-directed mutagenesis and kinetic analysis of the purified recombinant enzyme mutants. Mutants at position H49 showed a severe reduction in their specific activities with a decrease of the kcat/KM ratio by two orders of magnitude lower than the wild-type DXS. According to available structural data residue H49 is perfectly positioned to abstract a proton from the donor substrate. Mutations in DXS E370 showed that this residue is also essential for catalytic activity. Three-dimensional structure supports its involvement in cofactor deprotonation, the first step in enzymatic thiamin catalysis. Results obtained with H431 mutant enzymes indicate that this residue plays a role contributing to transition state stabilization. Finally, mutants at position D427 also showed a severe specific activity decrease with a reduction of the kcat/KM ratio. A role in binding the substrate and selecting the stereoisomer is proposed for D427.展开更多
The emergence of malaria parasite strains resistant to practically all the antimalarial drugs in clinical use is now making itnecessary to discover and develop both new antimalarial drugs and treatments. Recent advanc...The emergence of malaria parasite strains resistant to practically all the antimalarial drugs in clinical use is now making itnecessary to discover and develop both new antimalarial drugs and treatments. Recent advances in molecular techniques along withthe availability of genome sequence ofPlasmodiumfalciparum may provide a wide range of novel targets in metabolic pathways likeisoprenoid biosynthesis, fatty acid biosynthesis and heme biosynthesis in the apicoplast of Plasmodiurn. On the other hand, thecombination therapy approach (currently used to retard the selection of parasite strains resistant to individual components of acombination of drugs) has proved to be a success in the combination of sulphadoxine and pyrimethamine, which targets two differentsteps in the folate pathway of malaria parasite. However, after the success of this therapeutic combination, the efficacy of othercombinations of drugs which target different enzymes in a particular metabolic pathway has, apparently, not been reported. Therefore,herein, we review various drug targets so far discovered in apicoplast-related anabolic pathways, especially, with a sharper focus onthe possibility to target more than one enzyme at a time in a particular metabolic pathway of malaria parasites.展开更多
Mevalonate pathway for isoprenoid biosynthesis was constructed in Escherichia coli cells by the transformation with a gene cluster isolated from Streptomyces sp., and farnesyl diphosphate synthase and δ-guaiene synth...Mevalonate pathway for isoprenoid biosynthesis was constructed in Escherichia coli cells by the transformation with a gene cluster isolated from Streptomyces sp., and farnesyl diphosphate synthase and δ-guaiene synthase genes were coexpressed in this strain. This transformant was capable of liberating an appreciable amount of δ-guaiene, an aroma sesquiterpene compound accumulated in agarwood, and its concentration was elevated to more than 30 μg/ml culture by the incubation with mevalonolactone as an isoprene precursor in a nutrient-enriched Terrific broth. Coexpression of type 1 isopentenyl diphosphate isomerase plus acetoacetyl-CoA ligase genes also enhanced δ-guaiene production, and the concentration of the compound was approximately 38 - 42 μg/ml culture in the presence of mevalonolactone or lithium acetoacetate. These results clearly indicate that mevalonate pathway-engineered E. coli cells showed an appreciable δ-guaiene producing activity in the en- riched medium in the presence of appropriate isoprene precursors.展开更多
Plant carotenoids are plastidial isoprenoids that function as photoprotectants,pigments,and precursors of apocarotenoids such as the hormones abscisic acid and strigolactones.Humans do not produce carotenoids but need...Plant carotenoids are plastidial isoprenoids that function as photoprotectants,pigments,and precursors of apocarotenoids such as the hormones abscisic acid and strigolactones.Humans do not produce carotenoids but need to obtain them from their diet as precursors of retinoids,including vitamin A.Carotenoids also provide numerous other health benefits.Multiple attempts to improve the carotenoid profile of different crops have been carried out by manipulating carotenoid biosynthesis,degradation,and/or storage.Here,we will focus on open questions and emerging subjects related to the use of biotechnology for carotenoid biofortification.After impressive achievements,new efforts should be directed to extend the use of genome-editing technologies to overcome regulatory constraints and improve consumer acceptance of the carotenoid-enriched products.Another challenge is to prevent off-target effects like those resulting from altered hormone levels and metabolic homeostasis.Research on biofortification of green tissues should also look for new ways to deal with the negative impact that altered carotenoid contents may have on photosynthesis.Once a carotenoid-enriched product has been obtained,additional effort should be devoted to confirming that carotenoid intake from the engineered food is also improved.Thiswork involves ensuring post-harvest stability and assessing bioaccessibility of the biofortified product to confirm that release of carotenoids from the food matrix has not been negatively affected.Successfully addressing these challenges will ensure new milestones in carotenoid biotechnology and biofortification.展开更多
Isoprenoids are a very large and diverse family of metabolites required by all living organisms.All isoprenoids derive fromthe double-bond isomers isopentenyl diphosphate(IPP)and dimethylallyl diphosphate(DMAPP),which...Isoprenoids are a very large and diverse family of metabolites required by all living organisms.All isoprenoids derive fromthe double-bond isomers isopentenyl diphosphate(IPP)and dimethylallyl diphosphate(DMAPP),which are produced by the methylerythritol 4-phosphate(MEP)pathway in bacteria and plant plastids.It has been reported that IPP and DMAPP feedback-regulate the activity of deoxyxylulose 5-phosphate synthase(DXS),a dimeric enzyme that catalyzes the main flux-controlling step of the MEP pathway.Here we provide experimental insights intotheunderlyingmechanism.Isothermal titration calorimetry and dynamic light scattering approaches showed that IPP and DMAPP can allosterically bind to DXS in vitro,causing a size shift.In silico ligand binding site analysis and docking calculations identified a potential allosteric site in the contact region between the two monomers of the active DXS dimer.Modulation of IPP and DMAPP contents in vivo followed by immunoblot analyses confirmed that high IPP/DMAPP levels resulted in monomerization and eventual aggregation of the enzyme in bacterial and plant cells.Loss of the enzymatically active dimeric conformation allows a fast and reversible reduction of DXS activity in response to a sudden increase or decrease in IPP/DMAPP supply,whereas aggregation and subsequent removal of monomers that would otherwise be available for dimerization appears to be a more drastic response in the case of persistent IPP/DMAPP overabundance(e.g.,by a blockage in their conversion to downstream isoprenoids).Our results represent an important step toward understanding the regulation of the MEP pathway and rational design of biotechnological endeavors aimed at increasing isoprenoid contents in microbial and plant systems.展开更多
Metabolic engineering has been widely used for production of natural medicinal molecules.However, engineering high-yield platforms is hindered in large part by limited knowledge of complex regulatory machinery of meta...Metabolic engineering has been widely used for production of natural medicinal molecules.However, engineering high-yield platforms is hindered in large part by limited knowledge of complex regulatory machinery of metabolic network. N~6-Methyladenosine(m^(6)A) modification of RNA plays critical roles in regulation of gene expression. Herein, we identify 1470 putatively m^(6)A peaks within 1151 genes from the haploid Saccharomyces cerevisiae strain. Among them, the transcript levels of 94 genes falling into the pathways which are frequently optimized for chemical production, are remarkably altered upon overexpression of IME4(the yeast m^(6)A methyltransferase). In particular, IME4 overexpression elevates the mRNA levels of the methylated genes in the glycolysis, acetyl-CoA synthesis and shikimate/aromatic amino acid synthesis modules. Furthermore, ACS1 and ADH2, two key genes responsible for acetyl-CoA synthesis, are induced by IME4 overexpression in a transcription factor-mediated manner.Finally, we show IME4 overexpression can significantly increase the titers of isoprenoids and aromatic compounds. Manipulation of m^(6)A therefore adds a new layer of metabolic regulatory machinery and may be broadly used in bioproduction of various medicinal molecules of terpenoid and phenol classes.展开更多
Isoprenoid and branched glycerol dialkyl glycerol tetraethers (GDGTs) lipids were studied in flooded and non-flooded paddy soil in Wuhan, central China, to examine the response of the GDGTs distribution to the soil ...Isoprenoid and branched glycerol dialkyl glycerol tetraethers (GDGTs) lipids were studied in flooded and non-flooded paddy soil in Wuhan, central China, to examine the response of the GDGTs distribution to the soil flooding. Samples were collected before and after the soil flooding in four specific months. Both core (CL) and intact polar (IPL) GDGTs were quantified. Increase in the abundance of archaeol and caldarchaeol may be indicative of the occurrence of methanogens in the flooded soil. A negative correlation was observed between the ratio of IPL branched GDGT-IIa to GDGT-Ia and the soil pH. The rise of the soil pH in the acid soil is known to be controlled by the redox conditions resulting from flooding. Thus, the branched GDGTs distribution may be controlled by the water content in the paddy soil. In addition, we suggest that the anoxic conditions resulting from flooding may also control the abundance of branched GDGTs relative to crenarchaeol, which in turn results in the increase of branched and isoprenoidal tetraethers (BIT) values, the index for the terrestrial input to the marine sediments.展开更多
Carotenoids are indispensable to plants and play a critical role in human nutrition and health. Significant progress has been made in our understanding of carotenoid metabolism in plants. The biosynthetic pathway has ...Carotenoids are indispensable to plants and play a critical role in human nutrition and health. Significant progress has been made in our understanding of carotenoid metabolism in plants. The biosynthetic pathway has been extensively studied. Nearly all the genes encoding the biosynthetic enzymes have been isolated and characterized from various organisms. In recent years, there is an increasing body of work on the signaling pathways and plastid development, which might provide global control of carotenoid biosynthesis and accumulation. Herein, we will highlight recent progress on the biosynthesis, regulation, and metabolic engineering of carotenoids in plants, as well as the future research towards elucidating the regulatory mechanisms and metabolic network that control carotenoid metabolism.展开更多
Isoprenoids are functionally and structurally the most diverse group of plant metabolites reported to date. They can function as primary metabolites, participating in essential plant cellular processes, and as seconda...Isoprenoids are functionally and structurally the most diverse group of plant metabolites reported to date. They can function as primary metabolites, participating in essential plant cellular processes, and as secondary metabolites, of which many have substantial commercial, pharmacological, and agricultural value. Isoprenoid end products participate in plants in a wide range of physiological processes acting in them both synergistically, such as chlorophyll and carotenoids during photosynthesis, or antagonistically, such as gibberellic acid and abscisic acid during seed germination. It is therefore expected that fluxes via isoprenoid metabolic network are tightly controlled both temporally and spatially, and that this control occurs at different levels of regulation and in an orchestrated manner over the entire isoprenoid metabolic network. In this review, we summarize our current knowledge of the topology of the plant isoprenoid pathway network and its regulation at the gene expression level following diverse stimuli. We conclude by discussing agronomical and biotechnological applications emerging from the plant isoprenoid metabolism and provide an outlook on future directions in the systems analysis of the plant isoprenoid pathway network.展开更多
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...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.展开更多
Carotenoids are pigments required for photosynthesis, photoprotection and the production of carotenoid- derived hormones such as ABA and strigolactones. The carotenoid biosynthetic pathway bifurcates after lycopene to...Carotenoids are pigments required for photosynthesis, photoprotection and the production of carotenoid- derived hormones such as ABA and strigolactones. The carotenoid biosynthetic pathway bifurcates after lycopene to produce epsilon- and beta-carotenoids and this branch is critical for determining carotenoid composition. Here, we show how the branch point can be regulated by the chromatin-modifying histone methyltransferase, Set Domain Group 8 (SDG8) targeting the carotenoid isomerase (CRTISO). SDG8 is required to maintain permissive expression of CRTISO during seedling development, in leaves, shoot apex, and some floral organs. The CRTISO and SDG8 promoters show overlapping tissue-specific patterns of reporter gene activity. Interestingly, CRTISO showed atypical reporter gene expression in terms of greater variability between different lines compared to the Cauliflower Mosaic Virus 35S promoter (CaMV35s) and ~LCY promoters, potentially due to chromosomal position effects. Regulation of the CRTISO promoter was dependent in part upon the presence or absence of SDG8. Knockouts of SDG8 (carotenoid and chloroplast regulation (ccrl)) and CRTISO (ccr2) result in altered carotenoid composition and this could be restored in ccr2 using the CaMV35s or CRTISO promoters. In contrast, varying degrees of GUS expression and carotenoid complementation by CRTISO overexpression using CaMV35S or CRTISO promoters in the ccrl background demonstrated that both the CRTISO promoter and open reading frame are necessary for SDG8-mediated expression of CRTISO.展开更多
Plant isoprenoids are formed from precursors synthesized by the mevalonate (MVA) pathway in the cytosol or by the methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. Although some exchange of precursors occ...Plant isoprenoids are formed from precursors synthesized by the mevalonate (MVA) pathway in the cytosol or by the methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. Although some exchange of precursors occurs, cytosolic sesquiterpenes are assumed to derive mainly from MVA, while plastidial monoterpenes are produced preferentially from MEP precursors. Additional complexity arises in the first step of the MEP pathway, which is typically catalyzed by two divergent 1-deoxy-D-xylulose 5-phosphate synthase isoforms (DXS1, DXS2). In tomato (Solanum lycopersicum), the SIDXS1 gene is ubiquitously expressed with highest levels during fruit ripening, whereas SIDXS2 transcripts are abundant in only few tissues, including young leaves, petals, and isolated trichomes. Specific down-regulation of SIDXS2 expression was performed by RNA interference in transgenic plants to investigate feedback mechanisms. SIDXS2 down-regulation led to a decrease in the monoterpene β-phellandrene and an increase in two sesquiterpenes in trichomes. Moreover, incorporation of MVA-derived precursors into residual monoterpenes and into sesquiterpenes was elevated as determined by comparison of ^13C to ^12C natural isotope ratios. A compensatory up-regulation of SIDXS1 was not observed. Down-regulated lines also exhibited increased trichome density and showed less damage by leaf-feeding Spodoptera littoralis caterpillars. The results reveal novel, non-redundant roles of DXS2 in modulating isoprenoid metabolism and a pronounced plasticity in isoprenoid precursor allocation.展开更多
Astaxanthin is a red-colored carotenoid,used as food and feed additive.Astaxanthin is mainly produced by chemical synthesis,however,the process is expensive and synthetic astaxanthin is not approved for human consumpt...Astaxanthin is a red-colored carotenoid,used as food and feed additive.Astaxanthin is mainly produced by chemical synthesis,however,the process is expensive and synthetic astaxanthin is not approved for human consumption.In this study,we engineered the oleaginous yeast Yarrowia lipolytica for de novo production of astaxanthin by fermentation.First,we screened 12 different Y.lipolytica isolates for β-carotene production by introducing two genes for β-carotene biosynthesis:bi-functional phytoene synthase/lycopene cyclase(crtYB)and phytoene desaturase(crtI)from the red yeast Xanthophyllomyces dendrorhous.The best strain produced 31.1±0.5 mg/L β-carotene.Next,we optimized the activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMG1)and geranylgeranyl diphosphate synthase(GGS1/crtE)in the best producing strain and obtained 453.9±20.2 mg/L β-carotene.Additional downregulation of the competing squalene synthase SQS1 increased the β-carotene titer to 797.1±57.2 mg/L.Then we introduced β-carotene ketolase(crtW)from Paracoccus sp.N81106 and hydroxylase(crtZ)from Pantoea ananatis to convert β-carotene into astaxanthin.The constructed strain accumulated 10.4±0.5 mg/L of astaxanthin but also accumulated astaxanthin biosynthesis intermediates,5.7±0.5 mg/L canthaxanthin,and 35.3±1.8 mg/L echinenone.Finally,we optimized the copy numbers of crtZ and crtW to obtain 3.5 mg/g DCW(54.6 mg/L)of astaxanthin in a microtiter plate cultivation.Our study for the first time reports engineering of Y.lipolytica for the production of astaxanthin.The high astaxanthin content and titer obtained even in a small-scale cultivation demonstrates a strong potential for Y.lipolytica-based fermentation process for astaxanthin production.展开更多
The biosynthesis of isoprenoids in plant cells occurs from precursors produced in the cytosol by the mevalonate (MVA) pathway and in the plastid by the methylerythritol 4-phosphate (MEP) pathway, but little is kno...The biosynthesis of isoprenoids in plant cells occurs from precursors produced in the cytosol by the mevalonate (MVA) pathway and in the plastid by the methylerythritol 4-phosphate (MEP) pathway, but little is known about the mechanisms coordinating both pathways. Evidence of the importance of sugar signaling for such coordination in Arabi- dopsis thaliana is provided here by the characterization of a mutant showing an increased accumulation of MEP-derived isoprenoid products (chlorophylls and carotenoids) without changes in the levels of relevant MEP pathway transcripts, proteins, or enzyme activities. This mutant was found to be a new loss-of-function allele of PRL1 (Pleiotropic Regulatory Locus 1), a gene encoding a conserved WD-protein that functions as a global regulator of sugar, stress, and hormone responses, in part by inhibition of SNFl-related protein kinases (SnRK1). Consistent with the reported role of SnRK1 kinases in the phosphorylation and inactivation of the main regulatory enzyme of the MVA pathway (hydroxymethylglutaryl coenzyme-A reductase), its activity but not transcript or protein levels was reduced in prll seedlings. However, the accumulation of MVA-derived end products (sterols) was unaltered in mutant seedlings. Sucrose supplementation to wild- type seedlings phenocopied the prll mutation in terms of isoprenoid metabolism, suggesting that the observed isoprenoid phenotypes result from the increased sugar accumulation in the prll mutant. In summary, PRL1 appears to coordinate isoprenoid metabolism with sugar, hormone, and stress responses.展开更多
Current yeast metabolic engineering in isoprenoids production mainly focuses on rewiring of cytosolic metabolic pathway.However,the precursors,cofactors and the enzymes are distributed in various sub-cellular compartm...Current yeast metabolic engineering in isoprenoids production mainly focuses on rewiring of cytosolic metabolic pathway.However,the precursors,cofactors and the enzymes are distributed in various sub-cellular compartments,which may hamper isoprenoid biosynthesis.On the other side,pathway compartmentalization provides several advantages for improving metabolic flux toward target products.We here summarize the recent advances on harnessing sub-organelle for isoprenoids biosynthesis in yeast,and analyze the knowledge about the localization of enzymes,cofactors and metabolites for guiding the rewiring of the sub-organelle metabolism.This review may provide some insights for constructing efficient yeast cell factories for production of isoprenoids and even other natural products.展开更多
基金the supports of the National Key Research and Development of BioBased Rubber(2017YFB0306900&2017YFB0306901)the National Natural Science Foundation of China(51673012)+1 种基金the Fundamental Research Funds for the Central Universities(PYBZ1828)the Beijing Technology and Business Universtiy Youth Scholoars Funds(PXM2019014213000007)。
文摘Taraxacum kok-saghyz(TKS)is rich in natural rubber(NR),a natural organic macromolecular compound composed of cis-1,4-polyisoprene,and may become the second NR-bearing plant for biochemical engineering development.In this paper,a rapid and quantitative ultra-high performance liquid chromatography tandem mass spectrometry(UHPLCMS/MS)method was established for determination of macromolecular biosynthesis substrate(dimethylallyl pyrophosphate,DMAPP)and initiator(farnesyl pyrophosphate,FPP)contained in TKS.A Kromasil C18 chromatographic column was used for separation,and the multi-reaction monitoring mode(MRM)of triple quadrupole mass spectrometry was used for detection.Quantification was performed by external calibration method.The results showed that the limit of detection(LOD)and the limit of quantitation(LOQ)of DMAPP were 2.42μg/L and 7.26μg/L,respectively,and the LOQ and the LOD of FPP were 1.02μg/L and 3.05μg/L,respectively.At a concentration of 1—1000μg/L,both analytes had good determination coefficients(>0.999)of calibration curve.The recoveries of DMAPP and FPP were between 99.0%and 117.1%.In real samples detection,the contents of DMAPP and FPP in TKS samples were between 23.32—82.77μg/L and 12.03—85.67μg/L,respectively.Thus,this approach is a reliable method to quantify DMAPP and FPP in TKS.
基金supported by a Public-Agricultural Research Project, Ministry of Agriculture, China (201103027)the Genetically Modifi ed Organism Breeding Major Project, Ministry of Agriculture, China (2013ZX08005002)
文摘Isoprenoids are a functionally and structurally diverse class of natural organic chemicals. The universal precursors of all isoprenoids, isopentenyl diphosphate and dimethylallyl diphosphate are synthesized through the mevalonate and 2C-methyl- D-erythritol 4-phosphate (MEP) pathways, respectively. Many isoprenoids produced through the MEP pathway play an important role in plant acclimation to different light environments. Eupatorium adenophorum, an invasive weed in China, presents a remarkable capacity to acclimate to various light environments, which constitutes its solid foundation of being a successful invasive species. Thus we aimed at gaining a deeper insight into the regulation of MEP pathway in E. adenophorum to further understand the invasive mechanism. 2C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF or MCS) is an essential enzyme in the MEP pathway. In this paper, a novel IspF gene was cloned and characterized from E. adenophorum. Tissue-specific expression assays revealed a higher expression of EalspF1 in leaves than in stems and roots. The expression of EalspF1 was responsive to different light conditions. Some up-regulation of EalspF1 expression was also found after the treatments with signal compounds and after wounding stress. Interestingly, the over-expression of EalspF1 in Arabidopsis led to increase carotenoids contents, resulting in an enhanced tolerance to high light. Taken together, these results indicate that the EalspFl-derived enzyme participates in isoprenoid metabolism and among others, the expression of this gene in E. adenophorum is involved in the regulation of plastidial isoprenoids, which play an important role in acclimation to various light environments.
文摘While large quantities of latex can be handled either by standard extraction techniques such as Soxhlet extraction or accelerated solvent extraction (ASE), smaller samples on the order of 0.3 - 0.5 g require handling on a microscale. We collected latex from lettuce plants in microcentrifuge tubes and, after drying under vacuum, resuspended the dried sample in acetone by holding in an ultrasonic cleaner. The resulting fine suspension was readily extracted with acetone and toluene to provide fractions representing the resin and rubber content of the latex. Using this approach, we compared latex from stems of bolting lettuce and from the floral stem of lettuce plants. While both types of stems contained a similar percentage of resin, the rubber content of the bolting stems exceeded that of the floral stems.
文摘The effects of temperature on net photosynthesis and stomatal conductance, emission of foliar volatile organic compounds (VOCs), and phenolics were investigated after exposing Cordeauxia edulis seedlings to control (27/19°C) and three levels of elevated (32/23, 37/27, or 42/31°C) day/night temperature regimes in controlled growth chambers. Emissions of foliar VOC were measured on 7th and 14th day (d) of exposures, whereas net photosynthesis and stomatal conductance were measured on the 8th and 15th d. Net photosynthesis and stomatal conductance were not significantly affected by elevated temperatures. Emission rate of isoprene increased by 4-fold with 10°C rise from the control on 7th d of exposure. Emission rates of monoterpenes, sesquiterpenes and total isoprenoids increased to 2-5-fold higher than that of control plants with 5°C rise. Foliar isoprene emission peaked at daytime maximum of 37°C and the mono- and sesquiterpenes at 32°C. Few individual foliar phenolics, and total foliar phenolics showed significant concentration differences between treatments. Although high VOC emissions under warming appeared to help plants to sustain abiotic stresses, arid/semi-arid species might substantially release highly reactive compounds that affect atmospheric chemistry. Hence, more studies are required on plant species of arid/semi-arid ecosystems of Africa to estimate the emission patterns and their role in atmospheric chemistry under the predicted future atmospheric warming.
文摘Recognition of the biological properties of numerous “natural products” has fueled the current focus of this field, namely, the search for new drugs, antibiotics, insecticides, and herbicides. Based on their biosynthetic origins, natural products can be divided into three major groups: the isoprenoids, alkaloids, and phenolic compounds. Isoprenoids are structurally the most diverse group of secondary natural metabolites with different roles in the growth, development, and reproduction of a diverse range of prokaryotic and eukaryotes cells. Mevalonate and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways are known to be responsible for biosynthesis of numerous isoprenoids. HMG-CoA reductase is a rate-determining enzyme in mevalonate pathway, producing intermediates such as farnesyl and geranylgeranyl pyrophosphates, which lead to by-products such as cholesterol. Earlier studies have demonstrated that the inhibition of HMG-CoA reductase is one of the most effective approaches for treating hypercholesterolemia and eventually cardiovascular disease (CVD). Statins are HMG-CoA reductase inhibitors and the most prescribed group of drugs worldwide in treating hypercholesterolemia;however the application of this group of drugs may be expensive and has side effects including rashes and gastrointestinal symptoms. For these reasons, there is an important need to examine the viability of natural products as an alternative to statin treatment. This article is a review of different aforementioned areas with a focus on isoprenoids that can be used for the regulation of HMG-CoA reductase.
文摘1-deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the 2-C-methyl-D- erythritol 4-phosphate (MEP) pathway consisting in the condensation of (hydroxiethyl)thiamin derived from pyruvate with D-glyceraldehyde 3-phosphate (GAP) to yield 1-deoxy-D-xylulose 5-phosphate (DXP). The role of the conserved residues H49, E370, D427 and H431 of E. coli DXS was examined by site-directed mutagenesis and kinetic analysis of the purified recombinant enzyme mutants. Mutants at position H49 showed a severe reduction in their specific activities with a decrease of the kcat/KM ratio by two orders of magnitude lower than the wild-type DXS. According to available structural data residue H49 is perfectly positioned to abstract a proton from the donor substrate. Mutations in DXS E370 showed that this residue is also essential for catalytic activity. Three-dimensional structure supports its involvement in cofactor deprotonation, the first step in enzymatic thiamin catalysis. Results obtained with H431 mutant enzymes indicate that this residue plays a role contributing to transition state stabilization. Finally, mutants at position D427 also showed a severe specific activity decrease with a reduction of the kcat/KM ratio. A role in binding the substrate and selecting the stereoisomer is proposed for D427.
文摘The emergence of malaria parasite strains resistant to practically all the antimalarial drugs in clinical use is now making itnecessary to discover and develop both new antimalarial drugs and treatments. Recent advances in molecular techniques along withthe availability of genome sequence ofPlasmodiumfalciparum may provide a wide range of novel targets in metabolic pathways likeisoprenoid biosynthesis, fatty acid biosynthesis and heme biosynthesis in the apicoplast of Plasmodiurn. On the other hand, thecombination therapy approach (currently used to retard the selection of parasite strains resistant to individual components of acombination of drugs) has proved to be a success in the combination of sulphadoxine and pyrimethamine, which targets two differentsteps in the folate pathway of malaria parasite. However, after the success of this therapeutic combination, the efficacy of othercombinations of drugs which target different enzymes in a particular metabolic pathway has, apparently, not been reported. Therefore,herein, we review various drug targets so far discovered in apicoplast-related anabolic pathways, especially, with a sharper focus onthe possibility to target more than one enzyme at a time in a particular metabolic pathway of malaria parasites.
文摘Mevalonate pathway for isoprenoid biosynthesis was constructed in Escherichia coli cells by the transformation with a gene cluster isolated from Streptomyces sp., and farnesyl diphosphate synthase and δ-guaiene synthase genes were coexpressed in this strain. This transformant was capable of liberating an appreciable amount of δ-guaiene, an aroma sesquiterpene compound accumulated in agarwood, and its concentration was elevated to more than 30 μg/ml culture by the incubation with mevalonolactone as an isoprene precursor in a nutrient-enriched Terrific broth. Coexpression of type 1 isopentenyl diphosphate isomerase plus acetoacetyl-CoA ligase genes also enhanced δ-guaiene production, and the concentration of the compound was approximately 38 - 42 μg/ml culture in the presence of mevalonolactone or lithium acetoacetate. These results clearly indicate that mevalonate pathway-engineered E. coli cells showed an appreciable δ-guaiene producing activity in the en- riched medium in the presence of appropriate isoprene precursors.
基金supported by grants from the Spanish MCIN/AEI/10.13039/501100011033European NextGeneration EU/PRTR and PRIMA programs to M.R.-C.(PID2020-115810GB-I00 and UToPIQ-PCI2021-121941)+3 种基金supported by CSIC(202040E299)Generalitat Valenciana(PROMETEU/2021/056)funded by a PhD fellowship from“la Caixa”Foundation(INPhINIT program LCF/BQ/IN18/11660004)an EMBO short-term fellowship(ASTF 8907)for a short stay at the University of Neuchatel(Switzerland).
文摘Plant carotenoids are plastidial isoprenoids that function as photoprotectants,pigments,and precursors of apocarotenoids such as the hormones abscisic acid and strigolactones.Humans do not produce carotenoids but need to obtain them from their diet as precursors of retinoids,including vitamin A.Carotenoids also provide numerous other health benefits.Multiple attempts to improve the carotenoid profile of different crops have been carried out by manipulating carotenoid biosynthesis,degradation,and/or storage.Here,we will focus on open questions and emerging subjects related to the use of biotechnology for carotenoid biofortification.After impressive achievements,new efforts should be directed to extend the use of genome-editing technologies to overcome regulatory constraints and improve consumer acceptance of the carotenoid-enriched products.Another challenge is to prevent off-target effects like those resulting from altered hormone levels and metabolic homeostasis.Research on biofortification of green tissues should also look for new ways to deal with the negative impact that altered carotenoid contents may have on photosynthesis.Once a carotenoid-enriched product has been obtained,additional effort should be devoted to confirming that carotenoid intake from the engineered food is also improved.Thiswork involves ensuring post-harvest stability and assessing bioaccessibility of the biofortified product to confirm that release of carotenoids from the food matrix has not been negatively affected.Successfully addressing these challenges will ensure new milestones in carotenoid biotechnology and biofortification.
基金funded by grants from the Spanish MCIN/AEI/10.13039/501100011033European ERDF/FEDER,NextGeneration EU/PRTR and PRIMA programs(PID2020-115810GB-I00+3 种基金UToPIQ-PCI2021-121941 to M.R.-C.and BFU2016-78232-P to A.V.-C.).M.R.-C.is also supported by CSIC(202040E299)Generalitat Valenciana(PROMETEU/2021/056).R.K.and E.E.K.B.conducted the metabolite analysis at the Joint BioEnergy Institute(http://www.jbei.org),supported by the US Department of Energy,Office of Science,Office of Biological and Environmental Research under contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy.J.P.-Gwas supported by a Marie Curie International Outgoing Fellowship within the EC-FP7 Program(project 627639)X.D.was supported by the China Scholarship Council and D.O.-A.by an MCIN/AEI/fellowship(BES-2017-080739).
文摘Isoprenoids are a very large and diverse family of metabolites required by all living organisms.All isoprenoids derive fromthe double-bond isomers isopentenyl diphosphate(IPP)and dimethylallyl diphosphate(DMAPP),which are produced by the methylerythritol 4-phosphate(MEP)pathway in bacteria and plant plastids.It has been reported that IPP and DMAPP feedback-regulate the activity of deoxyxylulose 5-phosphate synthase(DXS),a dimeric enzyme that catalyzes the main flux-controlling step of the MEP pathway.Here we provide experimental insights intotheunderlyingmechanism.Isothermal titration calorimetry and dynamic light scattering approaches showed that IPP and DMAPP can allosterically bind to DXS in vitro,causing a size shift.In silico ligand binding site analysis and docking calculations identified a potential allosteric site in the contact region between the two monomers of the active DXS dimer.Modulation of IPP and DMAPP contents in vivo followed by immunoblot analyses confirmed that high IPP/DMAPP levels resulted in monomerization and eventual aggregation of the enzyme in bacterial and plant cells.Loss of the enzymatically active dimeric conformation allows a fast and reversible reduction of DXS activity in response to a sudden increase or decrease in IPP/DMAPP supply,whereas aggregation and subsequent removal of monomers that would otherwise be available for dimerization appears to be a more drastic response in the case of persistent IPP/DMAPP overabundance(e.g.,by a blockage in their conversion to downstream isoprenoids).Our results represent an important step toward understanding the regulation of the MEP pathway and rational design of biotechnological endeavors aimed at increasing isoprenoid contents in microbial and plant systems.
基金supported by the National Key Research and Development Program of China [2022YFF1100304 (2022YFF1100300), China]National Natural Science Foundation of China [82293682 (82293680), China]+1 种基金Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2021-RC350-009, China)the CAMS Innovation Fund for Medical Sciences (2021-I2M-1-029, China)。
文摘Metabolic engineering has been widely used for production of natural medicinal molecules.However, engineering high-yield platforms is hindered in large part by limited knowledge of complex regulatory machinery of metabolic network. N~6-Methyladenosine(m^(6)A) modification of RNA plays critical roles in regulation of gene expression. Herein, we identify 1470 putatively m^(6)A peaks within 1151 genes from the haploid Saccharomyces cerevisiae strain. Among them, the transcript levels of 94 genes falling into the pathways which are frequently optimized for chemical production, are remarkably altered upon overexpression of IME4(the yeast m^(6)A methyltransferase). In particular, IME4 overexpression elevates the mRNA levels of the methylated genes in the glycolysis, acetyl-CoA synthesis and shikimate/aromatic amino acid synthesis modules. Furthermore, ACS1 and ADH2, two key genes responsible for acetyl-CoA synthesis, are induced by IME4 overexpression in a transcription factor-mediated manner.Finally, we show IME4 overexpression can significantly increase the titers of isoprenoids and aromatic compounds. Manipulation of m^(6)A therefore adds a new layer of metabolic regulatory machinery and may be broadly used in bioproduction of various medicinal molecules of terpenoid and phenol classes.
基金We would thank Dr. WeiHua Ding for analytical assistance with the HPLC-MS equipment. This work was supported by the National Basic Research Program of China (No. 2011CB808800), the National Natural Science Foundation of China (Grant Nos. 40930210 and 40921062) and the 111 program (B08030) in China.
文摘Isoprenoid and branched glycerol dialkyl glycerol tetraethers (GDGTs) lipids were studied in flooded and non-flooded paddy soil in Wuhan, central China, to examine the response of the GDGTs distribution to the soil flooding. Samples were collected before and after the soil flooding in four specific months. Both core (CL) and intact polar (IPL) GDGTs were quantified. Increase in the abundance of archaeol and caldarchaeol may be indicative of the occurrence of methanogens in the flooded soil. A negative correlation was observed between the ratio of IPL branched GDGT-IIa to GDGT-Ia and the soil pH. The rise of the soil pH in the acid soil is known to be controlled by the redox conditions resulting from flooding. Thus, the branched GDGTs distribution may be controlled by the water content in the paddy soil. In addition, we suggest that the anoxic conditions resulting from flooding may also control the abundance of branched GDGTs relative to crenarchaeol, which in turn results in the increase of branched and isoprenoidal tetraethers (BIT) values, the index for the terrestrial input to the marine sediments.
基金the National Natural Science Foundation of China (30771167)the State Key Basic Research and Development Plan of China(2007CB108802)the USDA National Research Initiative Competitive Grants (2007-35318-17794 and 2001-35318-11136)
文摘Carotenoids are indispensable to plants and play a critical role in human nutrition and health. Significant progress has been made in our understanding of carotenoid metabolism in plants. The biosynthetic pathway has been extensively studied. Nearly all the genes encoding the biosynthetic enzymes have been isolated and characterized from various organisms. In recent years, there is an increasing body of work on the signaling pathways and plastid development, which might provide global control of carotenoid biosynthesis and accumulation. Herein, we will highlight recent progress on the biosynthesis, regulation, and metabolic engineering of carotenoids in plants, as well as the future research towards elucidating the regulatory mechanisms and metabolic network that control carotenoid metabolism.
文摘Isoprenoids are functionally and structurally the most diverse group of plant metabolites reported to date. They can function as primary metabolites, participating in essential plant cellular processes, and as secondary metabolites, of which many have substantial commercial, pharmacological, and agricultural value. Isoprenoid end products participate in plants in a wide range of physiological processes acting in them both synergistically, such as chlorophyll and carotenoids during photosynthesis, or antagonistically, such as gibberellic acid and abscisic acid during seed germination. It is therefore expected that fluxes via isoprenoid metabolic network are tightly controlled both temporally and spatially, and that this control occurs at different levels of regulation and in an orchestrated manner over the entire isoprenoid metabolic network. In this review, we summarize our current knowledge of the topology of the plant isoprenoid pathway network and its regulation at the gene expression level following diverse stimuli. We conclude by discussing agronomical and biotechnological applications emerging from the plant isoprenoid metabolism and provide an outlook on future directions in the systems analysis of the plant isoprenoid pathway network.
文摘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.
文摘Carotenoids are pigments required for photosynthesis, photoprotection and the production of carotenoid- derived hormones such as ABA and strigolactones. The carotenoid biosynthetic pathway bifurcates after lycopene to produce epsilon- and beta-carotenoids and this branch is critical for determining carotenoid composition. Here, we show how the branch point can be regulated by the chromatin-modifying histone methyltransferase, Set Domain Group 8 (SDG8) targeting the carotenoid isomerase (CRTISO). SDG8 is required to maintain permissive expression of CRTISO during seedling development, in leaves, shoot apex, and some floral organs. The CRTISO and SDG8 promoters show overlapping tissue-specific patterns of reporter gene activity. Interestingly, CRTISO showed atypical reporter gene expression in terms of greater variability between different lines compared to the Cauliflower Mosaic Virus 35S promoter (CaMV35s) and ~LCY promoters, potentially due to chromosomal position effects. Regulation of the CRTISO promoter was dependent in part upon the presence or absence of SDG8. Knockouts of SDG8 (carotenoid and chloroplast regulation (ccrl)) and CRTISO (ccr2) result in altered carotenoid composition and this could be restored in ccr2 using the CaMV35s or CRTISO promoters. In contrast, varying degrees of GUS expression and carotenoid complementation by CRTISO overexpression using CaMV35S or CRTISO promoters in the ccrl background demonstrated that both the CRTISO promoter and open reading frame are necessary for SDG8-mediated expression of CRTISO.
文摘Plant isoprenoids are formed from precursors synthesized by the mevalonate (MVA) pathway in the cytosol or by the methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. Although some exchange of precursors occurs, cytosolic sesquiterpenes are assumed to derive mainly from MVA, while plastidial monoterpenes are produced preferentially from MEP precursors. Additional complexity arises in the first step of the MEP pathway, which is typically catalyzed by two divergent 1-deoxy-D-xylulose 5-phosphate synthase isoforms (DXS1, DXS2). In tomato (Solanum lycopersicum), the SIDXS1 gene is ubiquitously expressed with highest levels during fruit ripening, whereas SIDXS2 transcripts are abundant in only few tissues, including young leaves, petals, and isolated trichomes. Specific down-regulation of SIDXS2 expression was performed by RNA interference in transgenic plants to investigate feedback mechanisms. SIDXS2 down-regulation led to a decrease in the monoterpene β-phellandrene and an increase in two sesquiterpenes in trichomes. Moreover, incorporation of MVA-derived precursors into residual monoterpenes and into sesquiterpenes was elevated as determined by comparison of ^13C to ^12C natural isotope ratios. A compensatory up-regulation of SIDXS1 was not observed. Down-regulated lines also exhibited increased trichome density and showed less damage by leaf-feeding Spodoptera littoralis caterpillars. The results reveal novel, non-redundant roles of DXS2 in modulating isoprenoid metabolism and a pronounced plasticity in isoprenoid precursor allocation.
基金This research was financially supported by the Novo Nordisk Foundation(grant number NNF15OC0016592)BAD was supported by the ERASMUS Traineeship program.We acknowledge Mette Kristensen for technical assistance on HPLC analysis.
文摘Astaxanthin is a red-colored carotenoid,used as food and feed additive.Astaxanthin is mainly produced by chemical synthesis,however,the process is expensive and synthetic astaxanthin is not approved for human consumption.In this study,we engineered the oleaginous yeast Yarrowia lipolytica for de novo production of astaxanthin by fermentation.First,we screened 12 different Y.lipolytica isolates for β-carotene production by introducing two genes for β-carotene biosynthesis:bi-functional phytoene synthase/lycopene cyclase(crtYB)and phytoene desaturase(crtI)from the red yeast Xanthophyllomyces dendrorhous.The best strain produced 31.1±0.5 mg/L β-carotene.Next,we optimized the activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMG1)and geranylgeranyl diphosphate synthase(GGS1/crtE)in the best producing strain and obtained 453.9±20.2 mg/L β-carotene.Additional downregulation of the competing squalene synthase SQS1 increased the β-carotene titer to 797.1±57.2 mg/L.Then we introduced β-carotene ketolase(crtW)from Paracoccus sp.N81106 and hydroxylase(crtZ)from Pantoea ananatis to convert β-carotene into astaxanthin.The constructed strain accumulated 10.4±0.5 mg/L of astaxanthin but also accumulated astaxanthin biosynthesis intermediates,5.7±0.5 mg/L canthaxanthin,and 35.3±1.8 mg/L echinenone.Finally,we optimized the copy numbers of crtZ and crtW to obtain 3.5 mg/g DCW(54.6 mg/L)of astaxanthin in a microtiter plate cultivation.Our study for the first time reports engineering of Y.lipolytica for the production of astaxanthin.The high astaxanthin content and titer obtained even in a small-scale cultivation demonstrates a strong potential for Y.lipolytica-based fermentation process for astaxanthin production.
文摘The biosynthesis of isoprenoids in plant cells occurs from precursors produced in the cytosol by the mevalonate (MVA) pathway and in the plastid by the methylerythritol 4-phosphate (MEP) pathway, but little is known about the mechanisms coordinating both pathways. Evidence of the importance of sugar signaling for such coordination in Arabi- dopsis thaliana is provided here by the characterization of a mutant showing an increased accumulation of MEP-derived isoprenoid products (chlorophylls and carotenoids) without changes in the levels of relevant MEP pathway transcripts, proteins, or enzyme activities. This mutant was found to be a new loss-of-function allele of PRL1 (Pleiotropic Regulatory Locus 1), a gene encoding a conserved WD-protein that functions as a global regulator of sugar, stress, and hormone responses, in part by inhibition of SNFl-related protein kinases (SnRK1). Consistent with the reported role of SnRK1 kinases in the phosphorylation and inactivation of the main regulatory enzyme of the MVA pathway (hydroxymethylglutaryl coenzyme-A reductase), its activity but not transcript or protein levels was reduced in prll seedlings. However, the accumulation of MVA-derived end products (sterols) was unaltered in mutant seedlings. Sucrose supplementation to wild- type seedlings phenocopied the prll mutation in terms of isoprenoid metabolism, suggesting that the observed isoprenoid phenotypes result from the increased sugar accumulation in the prll mutant. In summary, PRL1 appears to coordinate isoprenoid metabolism with sugar, hormone, and stress responses.
基金This study was financially supported by National Natural Science Foundation of China(Grant no.21877111 and no.21922812)China Postdoctoral Science Foundation(199936).
文摘Current yeast metabolic engineering in isoprenoids production mainly focuses on rewiring of cytosolic metabolic pathway.However,the precursors,cofactors and the enzymes are distributed in various sub-cellular compartments,which may hamper isoprenoid biosynthesis.On the other side,pathway compartmentalization provides several advantages for improving metabolic flux toward target products.We here summarize the recent advances on harnessing sub-organelle for isoprenoids biosynthesis in yeast,and analyze the knowledge about the localization of enzymes,cofactors and metabolites for guiding the rewiring of the sub-organelle metabolism.This review may provide some insights for constructing efficient yeast cell factories for production of isoprenoids and even other natural products.