The expression of AcIDI1 reveals diterpenoid alkaloids' allocation strategies in the roots of Aconitum carmichaelii Debx.

Isopentenyl diphosphate isomerase (IDI), a key enzyme in the biosynthetic pathway of diterpenoid alkaloids (DAs), plays an essential regulatory role in the synthesis and accumulation of DAs. In this study, the coding sequence (CDS) of AcIDI1 was isolated from the mother roots of Aconitum carmichaelii Debx. (GeneBank accession number OR915879). Bioinformatics analysis showed that the CDS of AcIDI1 was 894 bp, encoding a protein with 297 amino acids and the putative protein localized in the chloroplast. AcIDI1 exhibited significant homology with sequences encoding IDI in other species, and was most closely related to Aconitum vilmorinianum. Furthermore, the fusion protein has been successfully expressed in Escherichia coli (E. coli), providing a basis for future functional studies of AcIDI1. The expression pattern of AcIDI1 was analyzed by real-time quantitative PCR (qPCR), which demonstrates that AcIDI1 is a tissue-specific gene in the roots of A. carmichaelii and exhibits high expression in both daughter and mother roots. By comparing the expression levels of AcIDI1 in three tissues of the roots of A. carmichaelii at different growth stages, we propose that the mother roots (MRs) are the centers of resources allocation. The roots of A. carmichaelii continuously absorb the energy from external environment, while resources transfer behavior from MRs to both daughter roots (DRs) and axillary buds (ABs) occurs as the plant grows. This study establishes a foundation for applying the IDI gene to regulate the biosynthesis and accumulation of DAs in A. carmichaelii.

YGL3 Encoding an IPP and DMAPP Synthase Interacts with OsPIL11 to Regulate Chloroplast Development in Rice.

As the source of isoprenoid precursors, the plastidial methylerythritol phosphate (MEP) pathway plays an essential role in plant development. Here, we report a novel rice (Oryza sativa L.) mutant ygl3 (yellow-green leaf3) that exhibits yellow-green leaves and lower photosynthetic efficiency compared to the wild type due to abnormal chloroplast ultrastructure and reduced chlorophyll content. Map-based cloning showed that YGL3, one of the major genes involved in the MEP pathway, encodes 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, which is localized in the thylakoid membrane. A single base substitution in ygl3 plants resulted in lower 4-hydroxy-3-methylbut-2-enyl diphosphate reductase activity and lower contents of isopentenyl diphosphate (IPP) compared to the wild type. The transcript levels of genes involved in the syntheses of chlorophyll and thylakoid membrane proteins were significantly reduced in the ygl3 mutant compared to the wild type. The phytochrome interacting factor-like gene OsPIL11 regulated chlorophyll synthesis during the de-etiolation process by directly binding to the promoter of YGL3 to activate its expression. The findings provides a theoretical basis for understanding the molecular mechanisms by which the MEP pathway regulate chloroplast development in rice.

Identification and functional characterization of two trans-isopentenyl diphosphate synthases and one squalene synthase involved in triterpenoid biosynthesis in Platycodon grandiflorus.

MAIN CONCLUSION: Two trans-isopentenyl diphosphate synthase and one squalene synthase genes were identified and proved to be involved in the triterpenoid biosynthesis in Platycodon grandiflorus. Platycodon grandiflorus is a commonly used traditional Chinese medicine. The main bioactive compounds of P. grandiflorus are triterpenoid saponins. The biosynthetic pathway of triterpenoid saponins in P. grandiflorus has been preliminarily explored. However, limited functional information on related genes has been reported. A total of three trans-isopentenyl diphosphate synthases (trans-IDSs) genes (PgFPPS, PgGGPPS1 and PgGGPPS2) and one squalene synthase (SQS) gene (PgSQS) in P. grandiflorus were screened and identified from transcriptome dataset. Subcellular localization of the proteins was defined based on the analysis of GFP-tagged. The activity of genes was verified in Escherichia coli, demonstrating that recombinant PgFPPS catalysed the production of farnesyl diphosphate. PgGGPPS1 produced geranylgeranyl diphosphate, whereas PgGGPPS2 did not exhibit catalytic activity. By structural identification of encoding genes, a transmembrane region was found at the C-terminus of the PgSQS gene, which produced an insoluble protein when expressed in E. coli but showed no apparent effect on the enzyme function. Furthermore, some triterpenoid saponin synthesis-related genes were discovered by combining the component content and the gene expression assays at the five growth stages of P. grandiflorus seedlings. The accumulation of active components in P. grandiflorus was closely associated with the expression level of genes related to the synthesis pathway.

Development of a Transformation System for the Medicinal Fungus Sanghuangporus baumii and Acquisition of High-Value Strain.

To further explore the molecular mechanism of triterpenoid biosynthesis and acquire high-value strain of Sanghuangporus baumii, the Agrobacterium tumefaciens-mediated transformation (ATMT) system was studied. The key triterpenoid biosynthesis-associated gene isopentenyl diphosphate isomerase (IDI) was transformed into S. baumii by ATMT system. Then, the qRT-PCR technique was used to analyze gene transcript level, and the widely targeted metabolomics was used to investigate individual triterpenoid content. Total triterpenoid content and anti-oxidant activity were determined by spectrophotometer. In this study, we for the first time established an efficient ATMT system and transferred the IDI gene into S. baumii. Relative to the wild-type (WT) strain, the IDI-transformant (IT) strain showed significantly higher transcript levels of IDI and total triterpenoid content. We then investigated individual triterpenoids in S. baumii, which led to the identification of 10 distinct triterpenoids. The contents of individual triterpenoids produced by the IT2 strain were 1.76-10.03 times higher than those produced by the WT strain. The triterpenoid production showed a significant positive correlation with the IDI gene expression. Besides, IT2 strain showed better anti-oxidant activity. The findings provide valuable information about the biosynthetic pathway of triterpenoids and provide a strategy for cultivating high-value S. baumii strains.

Emergence of terpene chemical communication in insects: Evolutionary recruitment of isoprenoid metabolism.

Insects have evolved a chemical communication system using terpenoids, a structurally diverse class of specialized metabolites, previously thought to be exclusively produced by plants and microbes. Gene discovery, bioinformatics, and biochemical characterization of multiple insect terpene synthases (TPSs) revealed that isopentenyl diphosphate synthases (IDS), enzymes from primary isoprenoid metabolism, are their likely evolutionary progenitors. However, the mutations underlying the emergence of the TPS function remain a mystery. To address this gap, we present the first structural and mechanistic model for the evolutionary emergence of TPS function in insects. Through identifying key mechanistic differences between IDS and TPS enzymes, we hypothesize that the loss of isopentenyl diphosphate (IPP) binding motifs strongly correlates with the gain of the TPS function. Based on this premise, we have elaborated the first explicit structural definition of isopentenyl diphosphate-binding motifs (IBMs) and used the IBM definitions to examine previously characterized insect IDSs and TPSs and to predict the functions of as yet uncharacterized insect IDSs. Consistent with our hypothesis, we observed a clear pattern of disruptive substitutions to IBMs in characterized insect TPSs. In contrast, insect IDSs maintain essential consensus residues for binding IPP. Extending our analysis, we constructed the most comprehensive phylogeny of insect IDS sequences (430 full length sequences from eight insect orders) and used IBMs to predict the function of TPSs. Based on our analysis, we infer multiple, independent TPS emergence events across the class of insects, paving the way for future gene discovery efforts.

Cloning and functional analysis of IPI gene from Fritillaria unibracteata Hsiao et K. C. Hsia / 药学学报

An open reading frame (ORF) of isopentenyl-diphosphate delta isomerase gene (FuIPI) was cloned from Fritillaria unibracteata Hsiao et K. C. Hsia. (F. unibracteata). Furthermore, the bioinformatics and functional analyses of FuIPI were performed in this study. The result showed that, the ORF of FuIPI gene was 825 bp, encoding a polypeptide of 274 amino acids in length, with a relative molecular mass of about 31 kD and a theoretical isoelectric point of 5.61. Sequence analysis showed that FuIPI contained conserved structural domains and key residues involved in the catalyzing process. The phylogenetic analysis exhibited that FuIPI was closely related to IPIs of Dendrobium officinale and Musa acuminate. Real-time PCR analysis showed that FuIPI was distributed in different tissues of F. unibracteata, but had the highest transcriptional level in leaves, followed by stems, bulbs, and flowers. Furthermore, the FuIPI protein was successfully expressed in Escherichia coli BL21(DE3). The purified FuIPI protein successfully catalyzed the conversion from isopentenyl diphosphate (IPP) to dimethylallyl pyrophosphate (DMAPP). The above results provided a theoretical basis for further investigation of the molecular role of FuIPI in the biosynthesis of alkaloids.

Metabolome and Transcriptome Profiling Reveal That Four Terpenoid Hormones Dominate the Growth and Development of Sanghuangporus baumii

Sanghuangporus baumii is a traditional medicinal fungus that produces pharmacological terpenoids, but natural resources are insufficient for applications, and its growth and development mechanisms are poorly understood. Combining metabolomic and transcriptomic analyses, we found four terpenoid hormones and a central gene, isopentenyl diphosphate isomerase (IDI), involved in growth and development. Additionally, an exogenous hormone test was used to further confirm the importance of the four terpenoid hormones. Finally, hormone content determination and qRT−PCR were performed to explore the growth and development mechanism; we found thatcis-zeatin (CZ) plays a major role in the mycelia stage, trans-zeatin (TZ) and gibberellin A4 (GA4) are important in the primordia stage, GA4 is crucial for the fruiting bodies stage, and abscisic acid (ABA) may be a marker of maturity. The IDI gene was also found to affectterpenoid hormone content by regulating the relative gene transcript levels, thereby controlling morphological changes in S. baumii. Our results revealthe growth and development mechanisms of S. baumii and may promote the breeding and utilisation of high-quality varieties.

Genome-Wide Identification and Functional Characterization of the Trans-Isopentenyl Diphosphate Synthases Gene Family in Cinnamomum camphora.

Trans-isopentenyl diphosphate synthases (TIDSs) genes are known to be important determinants for terpene diversity and the accumulation of terpenoids. The essential oil of Cinnamomum camphora, which is rich in monoterpenes, sesquiterpenes, and other aromatic compounds, has a wide range of pharmacological activities and has therefore attracted considerable interest. However, the TIDS gene family, and its relationship to the camphor tree (C. camphora L. Presl.), has not yet been characterized. In this study, we identified 10 TIDS genes in the genome of the C. camphora borneol chemotype that were unevenly distributed on chromosomes. Synteny analysis revealed that the TIDS gene family in this species likely expanded through segmental duplication events. Furthermore, cis-element analyses demonstrated that C. camphora TIDS (CcTIDS) genes can respond to multiple abiotic stresses. Finally, functional characterization of eight putative short-chain TIDS proteins revealed that CcTIDS3 and CcTIDS9 exhibit farnesyl diphosphate synthase (FPPS) activity, while CcTIDS1 and CcTIDS2 encode geranylgeranyl diphosphate synthases (GGPPS). Although, CcTIDS8 and CcTIDS10 were found to be catalytically inactive alone, they were able to bind to each other to form a heterodimeric functional geranyl diphosphate synthase (GPPS) in vitro, and this interaction was confirmed using a yeast two-hybrid assay. Furthermore, transcriptome analysis revealed that the CcTIDS3, CcTIDS8, CcTIDS9, and CcTIDS10 genes were found to be more active in C. camphora roots as compared to stems and leaves, which were verified by quantitative real-time PCR (qRT-PCR). These novel results provide a foundation for further exploration of the role of the TIDS gene family in camphor trees, and also provide a potential mechanism by which the production of camphor tree essential oil could be increased for pharmacological purposes through metabolic engineering.

Ginsenosides in Panax genus and their biosynthesis.

Ginsenosides are a series of glycosylated triterpenoids which belong to protopanaxadiol (PPD)-, protopanaxatriol (PPT)-, ocotillol (OCT)- and oleanane (OA)-type saponins known as active compounds of Panax genus. They are accumulated in plant roots, stems, leaves, and flowers. The content and composition of ginsenosides are varied in different ginseng species, and in different parts of a certain plant. In this review, we summarized the representative saponins structures, their distributions and the contents in nearly 20 Panax species, and updated the biosynthetic pathways of ginsenosides focusing on enzymes responsible for structural diversified ginsenoside biosynthesis. We also emphasized the transcription factors in ginsenoside biosynthesis and non-coding RNAs in the growth of Panax genus plants, and highlighted the current three major biotechnological applications for ginsenosides production. This review covered advances in the past four decades, providing more clues for chemical discrimination and assessment on certain ginseng plants, new perspectives for rational evaluation and utilization of ginseng resource, and potential strategies for production of specific ginsenosides.

Signs of the plastid: Enzymes involved in plastid-localized metabolic pathways in a eugregarine species.

Apicomplexa mainly comprises parasitic species and some of them, which infect and cause severe diseases to humans and livestock, have been extensively studied due to the clinical and industrial importance. Besides, apicomplexans are a popular subject of the studies focusing on the evolution initiated by a secondary loss of photosynthesis. By interpreting the position in the tree of eukaryotes and lifestyles of the phylogenetic relatives parsimoniously, the extant apicomplexans are predicted to be the descendants of a parasite bearing a non-photosynthetic (cryptic) plastid. The plastid-bearing characteristic for the ancestral apicomplexan is further strengthened by non-photosynthetic plastids found in the extant apicomplexans. The research on apicomplexan members infecting invertebrates is much less advanced than that on the pathogens to humans and livestock. Gregarines are apicomplexans that infect diverse invertebrates and recent studies based on transcriptome data revealed the presence of cryptic plastids in a subset of the species investigated. In this study, we isolated gregarine-like organisms (GLOs) from three arthropod species and conducted transcriptome analyses on the isolated cells. A transcriptome-based, multi-gene phylogenetic analysis clearly indicated that all of the three GLOs are eugregarines. Significantly, the transcriptome data from the GLO in a centipede appeared to contain the transcripts encoding enzymes involved in the non-mevalonate pathway for isopentenyl diphosphate biosynthesis and C5 pathway for heme biosynthesis. The enzymes involved in the two plastid-localized metabolic pathways circumstantially but strongly suggest that the particular GLO possesses a cryptic plastid. The evolution of cryptic plastids in eugregarines is revised by incorporating the new data obtained from the three GLOs in this study.

Molecular Mechanism of Salvia miltiorrhiza Quality Formation Based on Single Nucleotide Polymorphism of Functional Genes / 中国实验方剂学杂志

Objective:To investigate the relationship between the single nucleotide polymorphism（SNP）of function genes and effective components of <italic>Salvia miltiorrhiza</italic> and the molecular mechanism of specific quality formation of <italic>S. miltiorrhiza</italic>. Method:The fingerprints of components in <italic>S. miltiorrhiza</italic> from eight different habitats and varieties were obtained by high-performance liquid chromatography （HPLC）. The full-length cDNA of three functional genes<italic> </italic>acetyl-CoA C-acetyltransferase（<italic>SmAACT</italic>），4-diphosphocytidyl-2-C-methyl-<italic>D</italic>-erythritol kinase（<italic>SmCMK</italic>） and isopentenyl diphosphate isomerase（<italic>SmIPPI</italic>） in tanshinone metabolic pathway were amplified by polymerase chain reaction（PCR），cloned， and sequenced，followed by bioinformatics analysis. Result:The full-length cDNA sequences of three functional genes <italic>SmAACT</italic>，<italic>SmCMK</italic>， and <italic>SmIPPI</italic> in tanshinone metabolic pathway were obtained from 23 strains of <italic>S. miltiorrhiza</italic> from eight different habitats and varieties. As revealed by the analysis of SNP and amino acid polymorphisms of three functional genes，18，16， and 14 SNP sites were found respectively. HPLC results showed the samples from Beijing，Hubei，Shandong （No. SDB），Shanxi，Henan， and Shandong （No. SDZ） were clustered into one branch，and those from Hebei and Inner Mongolia were clustered into another branch， which suggested that the variation trend of <italic>S. miltiorrhiza</italic> components had little correlation with geographical distance，but the variety was a critical factor for the quality. Conclusion:There was an obvious genetic differentiation trend in <italic>S. miltiorrhiza</italic> from different habitats，and different origin-specific genotypes were formed. The molecular mechanism of the formation of the specific quality of <italic>S. miltiorrhiza</italic> from different habitats was discussed，which laid a foundation for the stability and effectiveness of clinical medication，and guided the breeding of excellent varieties of <italic>S. miltiorrhiza</italic>.

Cloning and functional characterization of isopentenyl diphosphate isomerase genes from Panax vietnamensis var. fuscidiscus / 药学学报

Isopentenyl diphosphate isomerase (IDI) is a key enzyme in the regulation of triterpenes biosynthesis and plays an important role in ginsenoside biosynthesis. In this study, two IDI genes, PvfIDI1 (GenBank No. MZ736417) and PvfIDI2 (GenBank No. MZ736418) were cloned from Panax vietnamensis var. fuscidiscus. The open reading frame of both PvfIDI1 and PvfIDI2 was 924 bp encoding 307 amino acids. The molecular weights of PvfIDI1 and PvfIDI2 were 34.84 kDa and 34.66 kDa, respectively, with theoretical pIs of 6.01 and 5.66. Bioinformatic analysis indicated that PvfIDI1 and PvfIDI2 contained two conserved sequences: TNTCCSHPL and WGEHELDY. Phylogenetic analysis showed that PvfIDI1 and PvfIDI2 were closely related to Panax notoginseng IDI. Expression analysis showed that both PvfIDI1 and PvfIDI2 genes are expressed in root, rhizome, stem and leaf of P. vietnamensis var. fuscidiscus. However, PvfIDI1 is highly expressed in the rhizome and PvfIDI2 is highly expressed in the stem. PvfIDI1 and PvfIDI2 recombinant proteins were expressed in E. coli; a functional coloration experiment showed that PvfIDI1 and PvfIDI2 could promote the accumulation of lycopene, indicating that both PvfIDI1 and PvfIDI2 encode functional IDI enzymes. The cloning and functional studies on PvfIDI1 and PvfIDI2 provide a foundation for the further study of IDI and the regulation of ginsenoside biosynthesis in P. vietnamensis var. fuscidiscus.

Analysis of the isoprenoid pathway intermediates, dimethylallyl diphosphate and isopentenyl diphosphate, from crude plant extracts by liquid chromatography tandem mass spectrometry.

OBJECTIVE: We sought to develop a sensitive and accurate analytical method for the detection and quantification of IDP and DMADP as well as their monophosphate derivatives in crude plant extracts. METHODS: A liquid chromatography method coupled to tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) was established to measure the amounts of IDP and DMADP down to low picogram levels, which was linear over at least three orders of magnitude. Extracts were enriched using an anion exchanger, and chromatographic separation was achieved using a ß-cyclodextrin column. A S-thiolodiphosphate analog of DMADP was employed as an internal standard. RESULTS: Dilution series of authentic compounds were used to determine the limits of detection and quantification for IDP, DMADP and their corresponding monophosphates. A survey of plant species producing varying amounts of isoprenoids showed a corresponding variation in IDP and DMADP with the ratio of DMADP/IDP ranging from 4:1 to 2:1. Trace levels of isopentenyl monophosphate (IP) and dimethylallyl monophosphate (DMAP) were also detected. CONCLUSION: The LC-MS/MS method described enables absolute quantification of in planta levels of IDP and DMADP for the first time. The method is also suitable for analysing bacterial and animal samples as well as enzyme assays.

Metabolic flux analysis of secondary metabolism in plants.

Numerous secondary metabolites from plants are important for their medicinal, nutraceutical or sensory properties. Recently, significant progress has been made in the identification of the genes and enzymes of plant secondary metabolic pathways. Hence, there is interest in using synthetic biology to enhance the production of targeted valuable metabolites in plants. In this article, we examine the contribution that metabolic flux analysis will have on informing the rational selection of metabolic engineering targets as well as analysis of carbon and energy efficiency. Compared to microbes, plants have more complex tissue, cellular and subcellular organization, making precise metabolite concentration measurements more challenging. We review different techniques involved in quantifying flux and provide examples illustrating the application of the techniques. For linear and branched pathways that lead to end products with low turnover, flux quantification is straightforward and doesn't require isotopic labeling. However, for metabolites synthesized via parallel pathways, there is a requirement for isotopic labeling experiments. If the fed isotopically labeled carbons don't scramble, one needs to apply transient label balancing methods. In the transient case, it is also necessary to measure metabolite concentrations. While flux analysis is not able to directly identify mechanisms of regulation, it is a powerful tool to examine flux distribution at key metabolic nodes in intermediary metabolism, detect flux to wasteful side pathways, and show how parallel pathways handle flux in wild-type and engineered plants under a variety of physiological conditions.

Tanshinone and salvianolic acid biosynthesis are regulated by SmMYB98 in Salvia miltiorrhiza hairy roots.

Salvia miltiorrhiza Bunge is an herb rich in bioactive tanshinone and salvianolic acid compounds. It is primarily used as an effective medicine for treating cardiovascular and cerebrovascular diseases. Liposoluble tanshinones and water-soluble phenolic acids are a series of terpenoids and phenolic compounds, respectively. However, the regulation mechanism for the simultaneous promotion of tanshinone and salvianolic acid biosynthesis remains unclear. This study identified a R2R3-MYB subgroup 20 transcription factor (TF), SmMYB98, which was predominantly expressed in S. miltiorrhiza lateral roots. The accumulation of major bioactive metabolites, tanshinones, and salvianolic acids, was improved in SmMYB98 overexpression (OE) hairy root lines, but reduced in SmMYB98 knockout (KO) lines. The qRT-PCR analysis revealed that the transcriptional expression levels of tanshinone and salvianolic acid biosynthesis genes were upregulated by SmMYB98-OE and downregulated by SmMYB98-KO. Dual-Luciferase (Dual-LUC) assays demonstrated that SmMYB98 significantly activated the transcription of SmGGPPS1, SmPAL1, and SmRAS1. These results suggest that SmMYB98-OE can promote tanshinone and salvianolic acid production. The present findings illustrate the exploitation of R2R3-MYB in terpenoid and phenolic biosynthesis, as well as provide a feasible strategy for improving tanshinone and salvianolic acid contents by MYB proteins in S. miltiorrhiza.

The subcellular localization of two isopentenyl diphosphate isomerases in rice suggests a role for the endoplasmic reticulum in isoprenoid biosynthesis.

KEY MESSAGE: Both OsIPPI1 and OsIPPI2 enzymes are found in the endoplasmic reticulum, providing novel important insights into the role of this compartment in the synthesis of MVA pathway isoprenoids. Isoprenoids are synthesized from the precursor's isopentenyl diphosphate (IPP) and dimethylallyl diphosphosphate (DMAPP), which are interconverted by the enzyme isopentenyl diphosphate isomerase (IPPI). Many plants express multiple isoforms of IPPI, the only enzyme shared by the mevalonate (MVA) and non-mevalonate (MEP) pathways, but little is known about their specific roles. Rice (Oryza sativa) has two IPPI isoforms (OsIPPI1 and OsIPPI2). We, therefore, carried out a comprehensive comparison of IPPI gene expression, protein localization, and isoprenoid biosynthesis in this species. We found that OsIPPI1 mRNA was more abundant than OsIPPI2 mRNA in all tissues, and its expression in de-etiolated leaves mirrored the accumulation of phytosterols, suggesting a key role in the synthesis of MVA pathway isoprenoids. We investigated the subcellular localization of both isoforms by constitutively expressing them as fusions with synthetic green fluorescent protein. Both proteins localized to the endoplasmic reticulum (ER) as well as peroxisomes and mitochondria, whereas only OsIPPI2 was detected in plastids, due to an N-terminal transit peptide which is not present in OsIPPI1. Despite the plastidial location of OsIPPI2, the expression of OsIPPI2 mRNA did not mirror the accumulation of chlorophylls or carotenoids, indicating that OsIPPI2 may be a redundant component of the MEP pathway. The detection of both OsIPPI isoforms in the ER indicates that DMAPP can be synthesized de novo in this compartment. Our work shows that the ER plays an as yet unknown role in the synthesis of MVA-derived isoprenoids, with important implications for the metabolic engineering of isoprenoid biosynthesis in higher plants.

A cryptic activity in the Nudix hydrolase superfamily.

The Nudix hydrolase superfamily is identified by a conserved cassette of 23 amino acids, and it is characterized by its pyrophosphorylytic activity on a wide variety of nucleoside diphosphate derivatives. Of the 13 members of the family in Escherichia coli, only one, Orf180, has not been identified with a substrate, although a host of nucleoside diphosphate compounds has been tested. Several reports have noted a strong similarity in the three-dimensional structure of the unrelated enzyme, isopentenyl diphosphate isomerase (IDI) to the Nudix structure, and the report that a Nudix enzyme was involved in the synthesis of geraniol, a product of the two substrates of IDI, prompted an investigation of whether the IDI substrates, isopentenyl diphosphate (IPP), and dimethylallyl diphosphate (DAPP) could be substrates of Orf180. This article demonstrates that Orf180 does have a very low activity on IPP, DAPP, and geranyl pyrophosphate (GPP). However, several of the other Nudix enzymes with established nucleoside diphosphate substrates hydrolyze these compounds at substantial rates. In fact, some Nudix hydrolases have higher activities on IPP, DAPP, and GPP than on their signature nucleoside diphosphate derivatives.

Engineering metabolic pathways in Amycolatopsis japonicum for the optimization of the precursor supply for heterologous brasilicardin congeners production.

The isoprenoid brasilicardin A is a promising immunosuppressant compound with a unique mode of action, high potency and reduced toxicity compared to today's standard drugs. However, production of brasilicardin has been hampered since the producer strain Nocardia terpenica IFM0406 synthesizes brasilicardin in only low amounts and is a biosafety level 2 organism. Previously, we were able to heterologously express the brasilicardin gene cluster in the nocardioform actinomycete Amycolatopsis japonicum. Four brasilicardin congeners, intermediates of the BraA biosynthesis, were produced. Since chemical synthesis of the brasilicardin core structure has remained elusive we intended to produce high amounts of the brasilicardin backbone for semi synthesis and derivatization. Therefore, we used a metabolic engineering approach to increase heterologous production of brasilicardin in A. japonicum. Simultaneous heterologous expression of genes encoding the MVA pathway and expression of diterpenoid specific prenyltransferases were used to increase the provision of the isoprenoid precursor isopentenyl diphosphate (IPP) and to channel the precursor into the direction of diterpenoid biosynthesis. Both approaches contributed to an elevated heterologous production of the brasilicardin backbone, which can now be used as a starting point for semi synthesis of new brasilicardin congeners with better properties.

Enhancement of the catalytic activity of Isopentenyl diphosphate isomerase (IDI) from Saccharomyces cerevisiae through random and site-directed mutagenesis.

BACKGROUND: Lycopene is a terpenoid pigment that has diverse applications in the food and medicine industries. A prospective approach for lycopene production is by metabolic engineering in microbial hosts, such as Escherichia coli. Isopentenyl diphosphate isomerase (IDI, E.C. 5.3.3.2) is one of the rate-limiting enzymes in the lycopene biosynthetic pathway and one major target during metabolic engineering. The properties of IDIs differ depending on the sources, but under physiological conditions, IDIs are limited by low enzyme activity, short half-life and weak substrate affinity. Therefore, it is important to prepare an excellent IDI by protein engineering. RESULTS: Directed evolution strategy (error-prone PCR) was utilized to optimize the activity of Saccharomyces cerevisiae IDI. Using three rounds of error-prone PCR; screening the development of a lycopene-dependent color reaction; and combinatorial site-specific saturation mutagenesis, three activity-enhancing mutations were identified: L141H, Y195F, and W256C. L141H, located near the active pocket inside the tertiary structure of IDI, formed a hydrogen bond with nearby ß-phosphates of isopentenylpyrophosphate (IPP). Phe-195 and Cys-256 were nonpolar amino acids and located near the hydrophobic group of IPP, enlarging the hydrophobic scope, and the active pocket indirectly. Purified IDI was characterized and the result showed that the Km of mutant IDI decreased by 10% compared with Km of the parent IDI, and Kcat was 28% fold improved compared to that of the original IDI. Results of a fermentation experiment revealed that mutant IDI had a 1.8-fold increased lycopene production and a 2.1-fold increased yield capacity compared to wild-type IDI. CONCLUSION: We prepared an engineered variant of IDI with improved catalytic activity by combining random and site directed mutagenesis. The best mutants produced by this approach enhanced catalytic activity while also displaying improved stability in pH, enhanced thermostability and longer half-life. Importantly, the mutant IDI could play an important role in fed-batch fermentation, being an effective and attractive biocatalyst for the production of biochemicals.

Biotechnology of cyanobacterial isoprene production.

Heterologous cyanobacterial production of isoprene (C5H8) presents an opportunity to develop renewable resources for fuel and industrial chemicals. Isoprene can be generated photosynthetically in these microorganisms from dimethylallyl-diphosphate (DMAPP) by the recombinant enzyme isoprene synthase (ISPS), as a transgenic product of the isoprenoid biosynthetic pathway. The present work sought to combine recent enhancements in the cellular level of reactant (DMAPP) and enzyme (ISPS), as a means in the further development of this technology. This objective was approached upon the heterologous overexpression of fni, an isopentenyl isomerase from Streptococcus pneumoniae, which increased the amount of the DMAPP reactant at the expense of its isomer, isopentenyl-diphosphate (IPP), in the cells. In addition, the cellular concentration of ISPS was substantially enhanced upon expression of the ISPS gene, as a fusion construct with the highly expressed in cyanobacteria cpcB gene, encoding the abundant ß-subunit of phycocyanin. Synergy between these two modifications, i.e., enhancement in DMAPP substrate availability and enhancement in the concentration of the ISPS enzyme, improved the isoprene-to-biomass production ratio in cyanobacteria from 0.2:1 mg g-1 (w:w), attained with the ISPS transgene alone, up to 12.3:1 mg g-1 (w:w), measured when the combined two modifications were applied to the same cell. This is the highest verifiable yield of heterologous photosynthetic isoprene production reported so far. Findings in this work constitute a step forward in the development of the cyanobacterial biotechnology for isoprene production.