IPP Isomerase Research Articles

Study of heterologous efficient synthesis of β-amyrin and high-density fermentation

In this study, the synthetic pathway of β-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing β-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced β-amyrin up to 5.97 mg·L~(-1). Then, the mevalonate pyrophosphate decarboxylase gene(ERG19), mevalonate kinase gene(ERG12), 3-hydroxy-3-methylglutaryl-CoA synthase gene(ERG13), phosphomevalonate kinase gene(ERG8) and IPP isomerase gene(IDI1)were overexpressed to promoted the metabolic fluxto the direction of β-amyrin synthesis for further improving β-amyrin production, resulting the strain Y2-C2-4 which produced β-amyrin of 10.3 mg·L~(-1)under the shake flask fermentation condition. This is 100% higher than that of strain Y1-C20-6, illustrating the positive effect of the metabolic engineering strategy applied in this study. The titer of β-amyrin was further improved up to 157.4 mg·L~(-1) in the fed-batch fermentation, which was almost 26 fold of that produced by strain Y1-C20-6. This study not only laid the foundation for the biosynthesis of β-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of β-amyrin-based triterpenoids.

Refactoring β‐amyrin synthesis in Saccharomyces cerevisiae

Triterpenoids are a highly diverse group of natural products and used particularly as medicine. Here, a strategy combining stepwise metabolic engineering and transcriptional control was developed to strengthen triterpenoid biosynthesis in Saccharomyces cerevisiae. Consequently, an efficient biosynthetic pathway for producing β‐amyrin, a commercially valuable compound and precursor of triterpenoids, was constructed through expressing a plant‐derived β‐amyrin synthase. Introducing a heterologous squalene monooxygenase greatly dragged intermediate metabolite squalene toward β‐amyrin. Increasing squalene pool by overexpressing IPP isomerase, FPP, and squalene synthase further enhanced β‐amyrin synthesis of 49‐folds. Through reconstructing the promoters with the binding site of transcription factor UPC2, directed transcriptional regulation on engineered pathway was availably achieved, resulting in β‐amyrin titer increased by 65‐folds. Using ethanol fed‐batch fermentation, β‐amyrin titer was finally improved up to 138.80 mg/L with a yield of 16.30 mg/g dry cell, almost 185 and 232 and folds of the initially engineered strain, respectively. © 2015 American Institute of Chemical Engineers AIChE J, 61: 3172–3179, 2015

Comparative Analysis of Fruit Transcriptome in Tomato (Solanum lycopersicum) Genotypes with Contrasting Lycopene Contents

Carotenoid metabolism is regulated by several genes encoding carotenoid biosynthetic pathway enzymes. In the present study, a fruit transcriptome in tomato (Solanum lycopersicum) was compared between high lycopene accumulating genotype EC-521086 and low lycopene accumulating genotype VRT-32-1 at three different stages (green, breaker and red) of fruit ripening. This analysis led to the identification of 2,558 differentially expressed genes at three stages of fruit ripening. Among these genes, 123 were carotenoid-correlated genes. Quantitative RT-PCR analysis revealed high expression of genes encoding enzymes involved in lycopene biosynthesis like IPP isomerase, phytoene synthase, phytoene desaturase, z-carotene desaturase; and comparatively lower expression of genes encoding enzymes involved in lycopene catabolism like lycopene cyclase, carotenoid e-ring hydroxylase, zeaxanthin epoxidase, violaxanthin de-epoxidase and neoxanthin synthase in EC-521086, thereby possibly explaining the high lycopene content in EC-521086 as compared with the low lycopene genotype VRT-32-1. Further, the EC-521086 genotype exhibited high expression of the TOMATO AGAMOUS-LIKE 1 (TAGL1) MADS box gene—a positive regulator of lycopene accumulation—at breaker stage, and low expression of the ethylene receptor LeETR4 gene—a negative regulator of trans-lycopene and β-carotene accumulation, at the red stage of fruit ripening. Our results clearly demonstrate the role of specific genes in accumulation of high lycopene in the EC-521086 tomato genotype during the fruit ripening processes.

대사 제어된 스피아민트와 야생 스피아민트 기내배양배지의 터페노이드 성분 비교 분석

IPP isomerase (Iso) and Limonene synthase (Limo) are important enzymes in terpenoids biosynthesis pathway. The wild type and each metabolically engineered (Iso and Limo) transgenic spearmint (Mentha spicata Linne) plants were compared for their growth patterns and the contents of essential oil in in vitro culture media. The profile of terpenoid metabolites was obtained from the essential oil of the metabolically engineered transgenic spearmint, which was extracted using a modified SDE method, by GC-MS analysis. The growth of wild spearmint was more profuse in B5 culture medium than in other media. Significant differences in leaf and root growth patterns were observed between metabolically engineered transgenic and wild type spearmint plants. The leaves of the transgenic spearmint plants were slightly elongated but were dramatically narrower than those of wild type spearmints. The content of essential oil of transgenic spearmint was different slightly depending on the target terpenoid genes. The content of essential oils in Limo transgenic plants was higher than that of Iso, except for transgenic plant in B5 medium. The transgenic spearmint produced more terpenoids than the wild type. Iso spearmint extracts showed eleven terpenoids and a phenylpropane, while Limo spearmint extracts contained nine terpenoids. However, extracts from the wild type showed the presence of only four terpenoids.

A genome-wide survey for prion-regulated miRNAs associated with cholesterol homeostasis

BackgroundPrion diseases are neurodegenerative diseases that are characterized by the conversion of the cellular prion protein (PrPc) into a pathogenic isoform (PrPSc). It is known that neurodegeneration is often accompanied by the disturbance of cholesterol homeostasis. We have recently identified a set of genes that were upregulated after prion infection of N2a neuronal cells (Bach et al., 2009).ResultsWe have now used ultra-deep sequencing technology to profile all microRNAs (miRNA) that could be associated with this effect in these N2a cells. Using stringent filters and normalization strategies we identified a small set of miRNAs that were up- or downregulated upon prion infection. Using bioinformatic tools we predicted whether the downregulated miRNAs could target mRNAs that have been previously identified to enhance cholesterol synthesis in these cells. Application of this joint profiling approach revealed that nine miRNAs potentially target cholesterol-related genes. Four of those miRNAs are localized in a miRNA-dense cluster on the mouse X-chromosome. Among these, twofold downregulation of mmu-miR-351 and mmu-miR-542-5p was confirmed by qRT-PCR. The same miRNAs were predicted as putative regulators of the sterol regulatory element-binding factor 2 (Srebf2), the low-density lipoprotein receptor (Ldlr) or the IPP isomerase.ConclusionsThe results demonstrate that joined profiling by ultra-deep sequencing is highly valuable to identify candidate miRNAs involved in prion-induced dysregulation of cholesterol homeostasis.

Asymmetric somatic hybridization between Bupleurum scorzonerifolium Willd. and Taxus chinensis var. mairei

In an attempt to transfer the genetic components needed for taxol synthesis into a more tractable plant, protoplasts of Taxus chinensis var. mairei were UV irradiated for various times prior to their fusion with protoplasts of Bupleurum scorzonerifolium. Of the 60 presumptive hybrid calli obtained, selections of the 9 most rapidly growing were recognized as hybrid clones based on a combination of their callus morphology, esterase profile, chromosome number, and RAPD genotype. The RAPD data showed that 82.4-96.8% of the hybrid genome was inherited from the recipient (B. scorzonerifolium) and 4.6-13.9% from the donor (T. chinensis), with 1.6-6.9% of the RAPD fragments being not present in either parent. None of the hybrid clones expressed a detectable quantity of taxol, but four produced more of the triterpenoid oleanolic acid than did calli of the recipient. With the use of semi-quantitative reverse transcriptase PCR, it was possible to show variation in the expression of several triterpenoid biosynthetic pathway related genes between B. scorzonerifolium and the hybrids. Increasing the concentration of oleanolic acid requires that the expression levels of the genes encoding IPP isomerase, squalene synthase, squalene epoxidase and β-amyrin synthase are increased, while at the same time, those of the genes encoding the branching enzymes cycloartenol synthase and lupeol synthase need to be reduced.

Studies on Biosynthetic Genes and Enzymes of Isoprenoids Produced by Actinomycetes

For Abstract see ChemInform Abstract in Full Text.

Studies on Biosynthetic Genes and Enzymes of Isoprenoids Produced by Actinomycetes

Most Streptomyces strains are equipped with only the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the formation of isopentenyl diphosphate, a common precursor of isoprenoids. In addition to this pathway, some Streptomyces strains possess the mevalonate (MV) pathway via which isoprenoid antibiotics are produced. We have recently cloned and analyzed the MV pathway gene clusters and their flanking regions from terpentecin, BE-40644, and furaquinocin A producers. All these clusters contained genes coding for mevalonate kinase, mevalonate diphosphate decarboxylase, phosphomevalonate kinase, type 2 IPP isomerase, HMG-CoA reductase, and HMG-CoA synthase. The order of each of the open reading frames (ORFs) is also the same, and the respective homologous ORFs show more than 70% amino acid identity with each other. In contrast to these conservative gene organizations, the biosynthetic genes of terpentecin, BE-40644, and furaquinocin A were located just upstream and/or downstream of the MV pathway gene cluster. These facts suggested that all the actinomycete strains possessing both the MV and MEP pathways produce isoprenoid compounds and the biosynthetic genes of one of these isoprenoids usually exist adjacent to the MV pathway gene cluster. Therefore, when the presence of the MV cluster is detected by molecular genetic techniques, isoprenoids may be produced by the cultivation of these actinomycete strains. During the course of these studies, we identified diterpene cyclases possessing unique primary structures that differ from those of eukaryotes and catalyze unique reactions.

Mono and diterpene production in Escherichia coli.

Mono- and diterpenoids are of great industrial and medical value as specialty chemicals and pharmaceuticals. Production of these compounds in microbial hosts, such as Escherichia coli, can be limited by intracellular levels of the polyprenyl diphosphate precursors, geranyl diphosphate (GPP), and geranylgeranyl diphosphate (GGPP). To alleviate this limitation, we constructed synthetic operons that express three key enzymes for biosynthesis of these precursors: (1). DXS,1-deoxy-d-xylulose-5-phosphate synthase; (2). IPPHp, IPP isomerase from Haematococcus pluvialis; and (3). one of two variants of IspA, FPP synthase that produces either GPP or GGPP. The reporter plasmids pAC-LYC and pACYC-IB, which encode enzymes that convert either FPP or GGPP, respectively, to the pigment lycopene, were used to demonstrate that at full induction, the operon encoding the wild-type FPP synthase and mutant GGPP synthase produced similar levels of lycopene. To synthesize di- or monoterpenes in E. coli using the GGPP and GPP encoding operons either a diterpene cyclase [casbene cyclase (Ricinus communis L) and ent-kaurene cyclase (Phaeosphaeria sp. L487)] or a monoterpene cyclase [3-carene cyclase (Picea abies)] was coexpressed with their respective precursor production operon. Analysis of culture extracts or headspace by gas chromatography-mass spectrometry confirmed the in vivo production of the diterpenes casbene, kaur-15-ene, and kaur-16-ene and the monoterpenes alpha-pinene, myrcene, sabinene, 3-carene, alpha-terpinene, limonene, beta-phellandrene, alpha-terpinene, and terpinolene. Construction and functional expression of GGPP and GPP operons provides an in vivo precursor platform host for the future engineering of di- and monoterpene cyclases and the overproduction of terpenes in bacteria.

Identification of an Archaeal type II isopentenyl diphosphate isomerase in methanothermobacter thermautotrophicus.

Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks required for biosynthesis of isoprenoid compounds. Two different isomerases have been reported. The type I enzyme, first characterized in the late 1950s, is widely distributed in eukaryota and eubacteria. The type II enzyme was recently discovered in Streptomyces sp. strain CL190. Open reading frame 48 (ORF48) in the archaeon Methanothermobacter thermautotrophicus encodes a putative type II IPP isomerase. A plasmid-encoded copy of the ORF complemented IPP isomerase activity in vivo in Salmonella enterica serovar Typhimurium strain RMC29, which contains chromosomal knockouts in the genes for type I IPP isomerase (idi) and 1-deoxy-D-xylulose 5-phosphate (dxs). The dxs gene was interrupted with a synthetic operon containing the Saccharomyces cerevisiae genes erg8, erg12, and erg19 allowing for the conversion of mevalonic acid to IPP by the mevalonate pathway. His6-tagged M. thermautotrophicus type II IPP isomerase was produced in Escherichia coli and purified by Ni2+ chromatography. The purified protein was characterized by matrix-assisted laser desorption ionization mass spectrometry. The enzyme has optimal activity at 70 degrees C and pH 6.5. NADPH, flavin mononucleotide, and Mg2+ are required cofactors. The steady-state kinetic constants for the archaeal type II IPP isomerase from M. thermautotrophicus are as follows: K(m), 64 microM; specific activity, 0.476 micromol mg(-1) min(-1); and k(cat), 1.6 s(-1).

Isopentenyl pyrophosphate isomerases from Melaleuca alternifolia (Cheel) and their role in isoprenoid biosynthesis

SummaryTwo cDNA clones, idil and idi2, representing different isoforms of isopentenyl pyrophosphate isomerase (IPP isomerase) were isolated from Melaleuca alternifolia (Cheel) by functional complementation of carotenoid biosynthesis in E. coli. Excluding the putative transit peptide region, the isoforms share 89.5% predicted protein sequence identity. The high level of conservation between the isoforms indicates that these genes may share a common ancestral origin and supports the proposition that cytosolic and plastid targeted IPP isomerase may be differentially translated from a single gene. This study supports recent evidence suggesting that isopentenyl pyrophosphate and dimethylallylic pyrophosphate are both immediate products of the deoxyxylulose pathway and that IPP isomerase may have a more central role in the biosynthesis of carotenoids than in the biosynthesis of monoterpenes.

Copper induces the expression of cholesterogenic genes in human macrophages

Accumulation of lipids and cholesterol by macrophages and subsequent transformation into foam cells are key features in development of atherosclerosis. Serum copper concentrations have been shown to be associated with cardiovascular disease. However, the mechanism behind the proatherogenic effect of copper is not clear. We used DNA microarrays to define the changes in gene expression profile in response to copper exposure of human macrophages. Expression monitoring by DNA microarray revealed 91 genes that were regulated. Copper increased the expression of seven cholesterogenic genes (3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase, IPP isomerase, squalene synthase, squalene epoxidase, methyl sterol oxidase, H105e3 mRNA and sterol-C5-desaturase) and low-density lipoprotein receptor (LDL-R), and decreased the expression of CD36 and lipid binding proteins. The expression of LDL-R and HMG CoA reductase was also investigated using real time PCR. The expression of both of these genes was increased after copper treatment of macrophages ( P<0.01 and P<0.01, respectively). We conclude that copper activates cholesterogenic genes in macrophages, which may provide a mechanism for the association between copper and atherosclerosis. The effect of copper on cholesterogenic genes may also have implications for liver steatosis in early stages of Wilson's disease.

Crystal structure of isopentenyl diphosphate:dimethylallyl diphosphate isomerase.

Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase catalyses a crucial activation step in the isoprenoid biosynthesis pathway. This enzyme is responsible for the isomerization of the carbon-carbon double bond of IPP to create the potent electrophile DMAPP. DMAPP then alkylates other molecules, including IPP, to initiate the extraordinary variety of isoprenoid compounds found in nature. The crystal structures of free and metal-bound Escherichia coli IPP isomerase reveal critical active site features underlying its catalytic mechanism. The enzyme requires one Mn(2+) or Mg(2+) ion to fold in its active conformation, forming a distorted octahedral metal coordination site composed of three histidines and two glutamates and located in the active site. Two critical residues, C67 and E116, face each other within the active site, close to the metal-binding site. The structures are compatible with a mechanism in which the cysteine initiates the reaction by protonating the carbon-carbon double bond, with the antarafacial rearrangement ultimately achieved by one of the glutamates involved in the metal coordination sphere. W161 may stabilize the highly reactive carbocation generated during the reaction through quadrupole- charge interaction.

Isopentenyl diphosphate isomerase deficiency in Synechocystis sp. strain PCC6803

Isopentenyl diphosphate isomerase (IPP isomerase) in many organisms and in plastids is central to isoprenoid synthesis and involves the conversion between IPP and dimethylallyl diphosphate (DMAPP). It is shown that Synechocystis PCC6803 is deficient in IPP isomerase activity, consistent with the absence in its genome of an obvious homologue for the enzyme. Incorporation of [1- 14C]IPP in cell extracts, primarily into C 20, occurs only upon priming with DMAPP in Synechocystis PCC6803 and in Synechococcus PCC7942. Isoprenoid synthesis in these cyanobacteria does not appear to involve interconversion of IPP and DMAPP, raising the possibility that they are not within the plastid evolutionary lineage.

IPP isomerase, an enzyme of mevalonate pathway, is preferentially expressed in postnatal cortical neurons and induced after nerve transection

To understand the molecular mechanisms underlying the developmental processes of the cerebral cortex, we screened genes whose mRNA expression was up-regulated in neonatal in the rat cortex to a greater extent than in adult by differential display and obtained five genes. Among these genes, we focused on pyrophosphate (isopentenyl diphosphate, dimethylallyl diphosphate: IPP) isomerase gene, the product of which is known as an enzyme of the mevalonate pathway. Rat IPP isomerase was recently cloned and the gene expression was shown to be dependent on the activation of the mevalonate pathway. Its expression and roles in the brain, however, have not been investigated hitherto. In the present study, Northern blots and in situ hybridization analysis showed that at embryonic stage weak signals for IPP mRNA were diffusely detected in the CNS, and the signal in the cortex became intense at postnatal day 1 and maximized in almost all neurons of all layers at postnatal day 7 with a subsequent reduction. At 8 weeks, the expression of IPP isomerase mRNA in neurons decreased, while it was detected in the oligodendrocytes in the regions containing abundant nerve fibers. These findings suggested that IPP isomerase contributes to postnatal neuronal maturation and myelination. We also demonstrated that IPP isomerase mRNA is induced after nerve axotomy, which suggests a relationship between neuronal regeneration and IPP isomerase. Taken together, these results suggest that elevation of IPP isomerase mRNA levels in neurons contributes to construction of nerve fibers both during the postnatal period in the cortex and their regeneration.

Analysis of the isopentenyl diphosphate isomerase gene family from Arabidopsis thaliana.

Two Arabidopsis thaliana cDNAs (IPP1 and IPP2) encoding isopentenyl diphosphate isomerase (IPP isomerase) were isolated by complementation of an IPP isomerase mutant strain of Saccharomyces cerevisiae. Both cDNAs encode enzymes with an amino terminus that may function as a transit peptide for localization in plastids. At least 31 amino acids from the amino terminus of the IPP1 protein and 56 amino acids from the amino terminus of the IPP2 protein are not essential for enzymatic activity. Genomic DNA blot analysis confirmed that IPP1 and IPP2 are derived from a small gene family in A. thaliana. Based on northern analysis expression of both cDNAs occurs predominantly in roots of mature A. thaliana plants grown to the pre-flowering stage.

Terpenoid indole alkaloid biosynthesis and enzyme activities in two cell lines of Tabernaemontana divaricata

The possible limitation of the rate of biosynthesis of terpenoid indole alkaloids by low enzyme levels was investigated in two cell lines of Tabernaemontana divaricata with different terpenoid indole alkaloid biosynthetic capacities. The activities of tryptophan decarboxylase (TDC), strictosidine synthase (SSS), strictosidine glucosidase (SG), isopentenyl pyrophosphate isomerase (IPP isomerase) and geraniol 10-hydroxylase (G10H) of both cell lines were compared. The activities of TDC, SSS and IPP isomerase did not show a direct relationship with the biosynthetic capacity but SG and G10H might be limiting. In order to test whether the availability of the terpenoid precursor limits the biosynthesis of the terpenoid indole alkaloids, loganin was fed to the cultures. Loganin-feeding did not influence any of the measured enzyme activities but increased the terpenoid indole alkaloid accumulation of both cell lines to similar levels. A five-fold increase was observed for the accumulating line and a more than 100-fold increase for the low-accumulating one. Strictosidine accumulated mainly in the low-accumulating cell line which has high TDC and low SG activity; the amounts and types of the other terpenoid indole alkaloids which accumulated were similar in both lines. From this it can be concluded that the biosynthesis of terpenoid indole alkaloids in both cultures is limited by the availability of terpenoid precursors; this pathway is not saturated with substrates under normal culture conditions.

Effects of Clomazone on IPP Isomerase and Prenyl Transferase Activities in Cell Suspension Cultures and Cotyledons of Solanaceous Species

Laboratory assays were conducted to determine the sensitivity of tomato and tobacco cell suspension cultures and tomato and pepper cotyledons to clomazone. A comparison of fresh weight and carotenoid content indicated up to a three-fold difference between the clomazone-tolerant tobacco and clomazone-susceptible tomato cell suspension cultures. In contrast, an approximate 60-fold difference between the tolerant pepper and susceptible tomato cotyledons was observed when total chlorophyll and carotenoid contents were measured. The effect of clomazone and its possible metabolites on in vivo and in vitro extractable IPP isomerase (EC 5.3.3.2) and prenyltransferase (EC 2.5.1.29) activity was investigated. There was no clear inhibitory effect of clomazone or possible clomazone metabolites upon enzyme activity in tomato or tobacco cell suspension cultures or on light or dark grown tomato or pepper cotyledons. No specific enzymatic target site of clomazone was identified in correlation with the reduction in total chlorophyll or carotenoid content.

Enzymological aspects of the redirection of terpenoid biosynthesis in elicitor-treated cultures of Tabernaemontana divaricata

The elicitor-mediated induction of pentacyclic triterpenoid phytoalexin accumulation in cells of five-day-old suspension cultures of Tabernaemontana divaricata is accompanied by: a rapid and transient increase in HMG-CoA reductase (EC 1.1.1.34) activity; an increase in IPP isomerase (EC 5.3.3.2), prenyl transferase (EC 2.5.1.1) and squalene synthetase (EC 2.5.1.21) activity; a rapid inhibition of squalene 2,3-oxide:cycloartenol cyclase activity (EC 5.4.99.8), and a rapid and relatively transient appearance of squalene 2,3-oxide:amyrin cyclase (EC 5.4.99.-) activity. These findings are entirely consistent with an elicitor-induced redirection of the cytosolic-microsomal pathway of terpenoid biosynthesis away from phytosterol biosynthesis and towards pentacyclic triterpenoid phytoalexin biosynthesis. The switch being mediated as a direct result of the rapid inhibition of squalene 2,3-oxide:cycloartenol cyclase activity just prior to the de novo synthesis of squalene 2,3-oxide:amyrin cyclase and the other enzymes on the post-squalene 2,3-oxide span of the pentacyclic triterpenoid phytoalexin pathway. The increased activities of the enzymes common to both pathways reflects the fact that the rate of accumulation of pentacyclic triterpenoid phytoalexins in elicited cultures is more rapid than the rate of phytosterol biosynthesis in control cultures. The very rapid and transient increase in HMG-CoA reductase activity points to the microsomal form(s) of this enzyme having a key regulatory role in controlling the flux of carbon into the cytosolic-microsomal pathway of terpenoid biosynthesis.

A Human Promyelocyte mRNA Transiently Induced by TPA Is Homologous to Yeast IPP Isomerase

A cDNA clone was isolated from HL60 human promyelocyte cells on the basis of the induction of its cognate mRNA by the phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate) in the presence of cycloheximide. Northern analysis showed a peak of induction of a 1.9-kb mRNA at 3-6 h after TPA addition. Sequence analysis revealed that this cDNA was likely a human homolog of yeast isopentenyl diphosphate:dimethylallyl diphosphate isomerase, an enzyme important for isoprenoid synthesis and the prenylation of proteins such as ras p21.