Isoprenoid Biosynthetic Genes Research Articles

Isoprenoid Metabolism and Engineering in Glandular Trichomes of Lamiaceae.

The isoprenoids play important ecological and physiological roles in plants. They also have a tremendous impact on human lives as food additives, medicines, and industrial raw materials, among others. Though some isoprenoids are highly abundant in nature, plants produce many at extremely low levels. Glandular trichomes (GT), which cover the aerial parts of more than 25% of vascular plants, have been considered as natural biofactories for the mass production of rare industrially important isoprenoids. In several plant genera (e.g., Lavandula and Mentha), GTs produce and store large quantities of the low molecular weight isoprenoids, in particular mono- and sesquiterpenes, as essential oil constituents. Within each trichome, a group of secretory cells is specialized to strongly and specifically express isoprenoid biosynthetic genes, and to synthesize and deposit copious amounts of terpenoids into the trichome’s storage reservoir. Despite the abundance of certain metabolites in essential oils and defensive resins, plants, particularly those lacking glandular trichomes, accumulate small quantities of many of the biologically active and industrially important isoprenoids. Therefore, there is a pressing need for technologies to enable the mass production of such metabolites, and to help meet the ever-increasing demand for plant-based bioproducts, including medicines and renewable materials. Considerable contemporary research has focused on engineering isoprenoid metabolism in GTs, with the goal of utilizing them as natural biofactories for the production of valuable phytochemicals. In this review, we summarize recent advances related to the engineering of isoprenoid biosynthetic pathways in glandular trichomes.

In silico prediction and characterization of secondary metabolite biosynthetic gene clusters in the wheat pathogen Zymoseptoria tritici

BackgroundFungal pathogens of plants produce diverse repertoires of secondary metabolites, which have functions ranging from iron acquisition, defense against immune perturbation, to toxic assaults on the host. The wheat pathogen Zymoseptoria tritici causes Septoria tritici blotch, a foliar disease which is a significant threat to global food security. Currently, there is limited knowledge of the secondary metabolite arsenal produced by Z. tritici, which significantly restricts mechanistic understanding of infection. In this study, we analyzed the genome of Z. tritici isolate IP0323 to identify putative secondary metabolite biosynthetic gene clusters, and used comparative genomics to predict their encoded products.ResultsWe identified 32 putative secondary metabolite clusters. These were physically enriched at subtelomeric regions, which may facilitate diversification of cognate products by rapid gene rearrangement or mutations. Comparative genomics revealed a four gene cluster with significant similarity to the ferrichrome-A biosynthetic locus of the maize pathogen Ustilago maydis, suggesting this siderophore is deployed by Z. tritici to acquire iron. The Z. tritici genome also contains several isoprenoid biosynthetic gene clusters, including one with high similarity to a carotenoid/opsin producing locus in several fungi. Furthermore, we identify putative phytotoxin biosynthetic clusters, suggesting Z. tritici can produce an epipolythiodioxopiperazine, and a polyketide and non-ribosomal peptide with predicted structural similarities to fumonisin and the Alternaria alternata AM-toxin, respectively. Interrogation of an existing transcriptional dataset suggests stage specific deployment of numerous predicted loci during infection, indicating an important role of these secondary metabolites in Z. tritici disease.ConclusionsWe were able to assign putative biosynthetic products to numerous clusters based on conservation amongst other fungi. However, analysis of the majority of secondary metabolite loci did not enable prediction of a cluster product, and consequently the capacity of these loci to play as yet undetermined roles in disease or other stages of the Z. tritici lifecycle is significant. These data will drive future experimentation for determining the role of these clusters and cognate secondary metabolite products in Z. tritici virulence, and may lead to discovery of novel bioactive molecules.

A high-quality carrot genome assembly provides new insights into carotenoid accumulation and asterid genome evolution.

We report a high-quality chromosome-scale assembly and analysis of the carrot (Daucus carota) genome, the first sequenced genome to include a comparative evolutionary analysis among members of the euasterid II clade. We characterized two new polyploidization events, both occurring after the divergence of carrot from members of the Asterales order, clarifying the evolutionary scenario before and after radiation of the two main asterid clades. Large- and small-scale lineage-specific duplications have contributed to the expansion of gene families, including those with roles in flowering time, defense response, flavor, and pigment accumulation. We identified a candidate gene, DCAR_032551, that conditions carotenoid accumulation (Y) in carrot taproot and is coexpressed with several isoprenoid biosynthetic genes. The primary mechanism regulating carotenoid accumulation in carrot taproot is not at the biosynthetic level. We hypothesize that DCAR_032551 regulates upstream photosystem development and functional processes, including photomorphogenesis and root de-etiolation.

Genomic insights into the evolution of hybrid isoprenoid biosynthetic gene clusters in the MAR4 marine streptomycete clade.

BackgroundConsiderable advances have been made in our understanding of the molecular genetics of secondary metabolite biosynthesis. Coupled with increased access to genome sequence data, new insight can be gained into the diversity and distributions of secondary metabolite biosynthetic gene clusters and the evolutionary processes that generate them. Here we examine the distribution of gene clusters predicted to encode the biosynthesis of a structurally diverse class of molecules called hybrid isoprenoids (HIs) in the genus Streptomyces. These compounds are derived from a mixed biosynthetic origin that is characterized by the incorporation of a terpene moiety onto a variety of chemical scaffolds and include many potent antibiotic and cytotoxic agents.ResultsOne hundred and twenty Streptomyces genomes were searched for HI biosynthetic gene clusters using ABBA prenyltransferases (PTases) as queries. These enzymes are responsible for a key step in HI biosynthesis. The strains included 12 that belong to the ‘MAR4’ clade, a largely marine-derived lineage linked to the production of diverse HI secondary metabolites. We found ABBA PTase homologs in all of the MAR4 genomes, which averaged five copies per strain, compared with 21 % of the non-MAR4 genomes, which averaged one copy per strain. Phylogenetic analyses suggest that MAR4 PTase diversity has arisen by a combination of horizontal gene transfer and gene duplication. Furthermore, there is evidence that HI gene cluster diversity is generated by the horizontal exchange of orthologous PTases among clusters. Many putative HI gene clusters have not been linked to their secondary metabolic products, suggesting that MAR4 strains will yield additional new compounds in this structure class. Finally, we confirm that the mevalonate pathway is not always present in genomes that contain HI gene clusters and thus is not a reliable query for identifying strains with the potential to produce HI secondary metabolites.ConclusionsWe found that marine-derived MAR4 streptomycetes possess a relatively high genetic potential for HI biosynthesis. The combination of horizontal gene transfer, duplication, and rearrangement indicate that complex evolutionary processes account for the high level of HI gene cluster diversity in these bacteria, the products of which may provide a yet to be defined adaptation to the marine environment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2110-3) contains supplementary material, which is available to authorized users.

Distribution of the 3-hydroxyl-3-methylglutaryl coenzyme A reductase gene and isoprenoid production in marine-derived Actinobacteria

During the course of our screening program to isolate isoprenoids from marine Actinobacteria, 523 actinobacterial strains were isolated from 18 marine sponges, a tunicate, and two marine sediments. These strains belonged to 21 different genera, but most were members of Streptomyces, Nocardia, Rhodococcus, and Micromonospora. Some Actinobacteria have been reported to use the mevalonate pathway for the production of isoprenoids as secondary metabolites. Therefore, we investigated whether these strains possessed the 3-hydroxyl-3-methylglutaryl coenzyme A reductase (hmgr) gene, which indicates the presence of the mevalonate pathway. As a result, six strains belonging to the genera Streptomyces (SpC080624SC-11, SpA080624GE-02, and Sp080513GE-23), Nocardia (Sp080513SC-18), and Micromonospora (Se080624GE-07 and SpC080624GE-05) were found to possess the hmgr gene, and these genes were highly similar to hmgr genes in isoprenoid biosynthetic gene clusters. Among the six strains, the two strains SpC080624SC-11 and SpA080624GE-02 produced the novel isoprenoids, JBIR-46, -47, and -48, which consisted of phenazine chromophores, and Sp080513GE-23 produced a known isoprenoid, fumaquinone. Furthermore, these compounds showed cytotoxic activity against human acute myelogenous leukemia HL-60 cells.

Global gene regulation by Fusarium transcription factors Tri6 and Tri10 reveals adaptations for toxin biosynthesis

Trichothecenes are isoprenoid mycotoxins produced in wheat infected with the filamentous fungus Fusarium graminearum. Some fungal genes for trichothecene biosynthesis (Tri genes) are known to be under control of transcription factors encoded by Tri6 and Tri10. Tri6 and Tri10 deletion mutants were constructed in order to discover additional genes regulated by these factors in planta. Both mutants were greatly reduced in pathogenicity and toxin production and these phenotypes were largely restored by genetic complementation with the wild-type gene. Transcript levels for over 200 genes were altered > or = twofold for Deltatri6 or Deltatri10 mutants including nearly all known Tri genes. Also reduced were transcript levels for enzymes in the isoprenoid biosynthetic pathway leading to farnesyl pyrophosphate, the immediate molecular precursor of trichothecenes. DNA sequences 5' to isoprenoid biosynthetic genes were enriched for the Tri6p DNA binding motif, YNAGGCC, in F. graminearum but not in related species that do not produce trichothecenes. To determine the effect of trichothecene metabolites on gene expression, cultures were treated with trichodiene, the first metabolic intermediate specific to the trichothecene biosynthetic pathway. A total of 153 genes were upregulated by added trichodiene and were significantly enriched for genes likely involved in cellular transport. Differentially regulated genes will be targeted for functional analysis to discover additional factors involved in toxin biosynthesis, toxin resistance and pathogenesis.

The Mevalonate Pathway ofStaphylococcus aureus

Isoprenoids are a class of ubiquitous organic molecules synthesized from the five-carbon starter unit isopentenyl pyrophosphate (IPP). Comprising more than 30,000 known natural products, isoprenoids serve various important biological functions in many organisms. In bacteria, undecaprenyl pyrophosphate is absolutely required for the formation of cell wall peptidoglycan and other cell surface structures, while ubiquinones and menaquinones, both containing an essential prenyl moiety, are key electron carriers in respiratory energy generation. There is scant knowledge on the nature and regulation of bacterial isoprenoid pathways. In order to explore the cellular responses to perturbations in the mevalonate pathway, responsible for producing the isoprenoid precursor IPP in many gram-positive bacteria and eukaryotes, we constructed three strains of Staphylococcus aureus in which each of the mevalonate pathway genes is regulated by an IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible promoter. We used DNA microarrays to profile the transcriptional effects of downregulating the components of the mevalonate pathway in S. aureus and demonstrate that decreased expression of the mevalonate pathway leads to widespread downregulation of primary metabolism genes, an upregulation in virulence factors and cell wall biosynthetic determinants, and surprisingly little compensatory expression in other isoprenoid biosynthetic genes. We subsequently correlate these transcriptional changes with downstream metabolic consequences.

Biosynthesis of a Natural Polyketide-Isoprenoid Hybrid Compound, Furaquinocin A: Identification and Heterologous Expression of the Gene Cluster

Furaquinocin (FQ) A, produced by Streptomyces sp. strain KO-3988, is a natural polyketide-isoprenoid hybrid compound that exhibits a potent antitumor activity. As a first step toward understanding the biosynthetic machinery of this unique and pharmaceutically useful compound, we have cloned an FQ A biosynthetic gene cluster by taking advantage of the fact that an isoprenoid biosynthetic gene cluster generally exists in flanking regions of the mevalonate (MV) pathway gene cluster in actinomycetes. Interestingly, Streptomyces sp. strain KO-3988 was the first example of a microorganism equipped with two distinct mevalonate pathway gene clusters. We were able to localize a 25-kb DNA region that harbored FQ A biosynthetic genes (fur genes) in both the upstream and downstream regions of one of the MV pathway gene clusters (MV2) by using heterologous expression in Streptomyces lividans TK23. This was the first example of a gene cluster responsible for the biosynthesis of a polyketide-isoprenoid hybrid compound. We have also confirmed that four genes responsible for viguiepinol [3-hydroxypimara-9(11),15-diene] biosynthesis exist in the upstream region of the other MV pathway gene cluster (MV1), which had previously been cloned from strain KO-3988. This was the first example of prokaryotic enzymes with these biosynthetic functions. By phylogenetic analysis, these two MV pathway clusters were identified as probably being independently distributed in strain KO-3988 (orthologs), rather than one cluster being generated by the duplication of the other cluster (paralogs).

Identification of new genes positively regulated by Tri10 and a regulatory network for trichothecene mycotoxin production.

Tri10, a regulatory gene in trichothecene mycotoxin-producing Fusarium species, is required for trichothecene biosynthesis and the coordinated expression of four trichothecene pathway-specific genes (Tri4, Tri5, Tri6, and Tri101) and the isoprenoid biosynthetic gene for farnesyl pyrophosphate synthetase (FPPS). We showed that six more trichothecene genes (Tri3, Tri7, Tri8, Tri9, Tri11, and Tri12) are regulated by Tri10. We also constructed a cDNA library from a strain of Fusarium sporotrichioides that overexpresses Tri10 ( upward arrow Tri10) and used cDNA derived from the upward arrow Tri10 strain and a non-Tri10-expressing strain (DeltaTri10) to differentially screen macroarrays prepared from the cDNA library. This screen identified 15 additional Tri10-regulated transcripts. Four of these transcripts represent Tri1, Tri13, and Tri14 and a gene designated Tri15. Three other sequences are putative orthologs of genes for isoprenoid biosynthesis, the primary metabolic pathway preceding trichothecene biosynthesis. The remaining eight sequences have been designated Ibt (influenced by Tri10) genes. Of the 26 transcripts now known to be positively regulated by Tri10, 22 are positively coregulated by Tri6, a gene that encodes a previously characterized trichothecene pathway-specific transcription factor. These 22 Tri10- and Tri6-coregulated sequences include all of the known Tri genes (except for Tri10), the FPPS gene, and the other three putative isoprenoid biosynthetic genes. Tri6 also regulates a transcript that is not regulated by Tri10. Thus, Tri10 and Tri6 regulate overlapping sets of genes that include a common group of multiple genes for both primary and secondary metabolism.

A relationship between the mevalonate pathway and isoprenoid production in actinomycetes.

Most Streptomyces strains are equipped with only the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the formation of isopentenyl diphosphate. In addition to this pathway, some Strepromyces strains have the mevalonate pathway to produce terpenoid antibiotics. We have previously shown that a gene cluster for biosynthesis of terpentecin, a diterpene antibiotic, was located in adjacent the mevalonate pathway gene cluster. In this study, a mevalonate pathway gene cluster was cloned from Actinoplanes sp. strain A40644, an isoprenoid antibiotic BE-40644 producer, to examine whether the mevalonate pathway genes and isoprenoid biosynthetic genes are clustered in genomic DNA. By sequencing flanking regions a probable BE-40644 biosynthetic gene cluster was found in the downstream region of the mevalonate pathway gene cluster. Heterologous expression of a 9-kb fragment confirmed that a set of the BE-40644 biosynthetic genes was involved in the fragment. This result suggested that the presence of the mevalonate pathway might be a good landmark to detect the production of isoprenoid compounds by actinomycetes.

A novel gene mutation that confers abnormal patterns of beta-carotene accumulation in cauliflower (Brassica oleracea var. botrytis).

The Or gene of cauliflower (Brassica oleracea var. botrytis) causes many tissues of the plant to accumulate carotenoids and turn orange, which is suggestive of a perturbation of the normal regulation of carotenogenesis. A series of experiments to explore the cellular basis of the carotenoid accumulation induced by the Or gene was completed. The Or gene causes obvious carotenoid accumulation in weakly or unpigmented tissues such as the curd, pith, leaf bases and shoot meristems, and cryptically in some cells of other organs, including the roots and developing fruits. The dominant carotenoid accumulated is beta-carotene, which can reach levels that are several hundred-fold higher than those in comparable wild-type tissues. The beta-carotene accumulates in plastids mainly as a component of massive, highly ordered sheets. The Or gene does not affect carotenoid composition of leaves, nor does it alter color and chromoplast appearance in flower petals. Interestingly, mRNA from carotenogenic and other isoprenoid biosynthetic genes upstream of the carotenoid pathway was detected both in orange tissues of the mutant, and in comparable unpigmented wild-type tissues. Thus the unpigmented wild-type tissues are likely to be competent to synthesize carotenoids, but this process is suppressed by an unidentified mechanism. Our results suggest that the Or gene may induce carotenoid accumulation by initiating the synthesis of a carotenoid deposition sink in the form of the large carotenoid-sequestering sheets.

Expression of an active phytoene synthase from Erwinia uredovora and biochemical properties of the enzyme

The crtB gene encoding phytoene synthase from the carotenogenic enterobacterium Erwinia uredovora was overexpressed to about 20% of the total cellular protein in Escherichia coli. Formation of the active phytoene synthase had the effect of suppressing the growth of the expressing strain. Presumably inhibition of growth arose from the depletion of the substrate geranylgeranyl pyrophosphate (GGPP) which, in E. coli, is necessary for the synthesis of essential prenylpyrophosphate derivatives. In order to overcome the poor growth characteristics of the phytoene synthase expressing strain, GGPP levels were increased by co-expressing the isoprenoid biosynthetic genes crtE and idi, encoding the Erwinia GGPP synthase and Rhodobacter isopentenyl pyrophosphate isomerase, respectively. The crude enzyme preparation was partially purified 15-fold by chromatography on a DEAE column. A non-radioactive assay was developed that enabled the conversion of GGPP to phytoene. The reaction product was identified by co-chromatography with authentic standards on HPLC systems and comparison of spectral characteristics. The phytoene formed in vitro was present in both a 15- cis and all- trans isomeric configuration. The essential cofactors required were ATP in combinations with either Mn 2+ or Mg 2+. The K m value for GGPP was determined as 41 μM. Phytoene synthesis was inhibited by phosphate ions and squalestatin. The I 50 value for the latter inhibitor was 15 μM. Lineweaver–Burk plots showed constant K m values in the presence or absence of squalestatin.

Developmental and stress regulation of gene expression for plastid and cytosolic isoprenoid pathways in pepper fruits.

Plant cells synthesize a myriad of isoprenoid compounds in different subcellular compartments, which include the plastid, the mitochondria, and the endoplasmic reticulum cytosol. To start the study of the regulation of these parallel pathways, we used pepper (Capsicum annuum) fruit as a model. Using different isoprenoid biosynthetic gene probes from cloned cDNAs, we showed that only genes encoding the plastid enzymes (geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and capasanthin-capsorubin synthase) are specifically triggered during the normal period of development, at the ripening stage. This pattern of expression can be mimicked and precociously induced by a simple wounding stress. Concerning the cytosol-located enzymes, we observed that the expression of the gene encoding farnesyl pyrophosphate synthase is constitutive, whereas that of farnesyl pyrophosphate cyclase (5-epi-aristolochene synthase) is undetectable during the normal development of the fruit. The expression of these later genes are, however, only selectively triggered after elicitor treatment. The results provide evidence for developmental control of isoprenoid biosynthesis occurring in plastids and that cytoplasmic isoprenoid biosynthesis is regulated, in part, by environmental signals.