A Novel Anti-diabetic Metabolite from Plants: Biosynthesis, Gene Discovery, and Metabolic Engineering of Montbretin A

Plant specialized metabolites (i.e. secondary metabolites) have been employed by humans for centuries in traditional and modern medicine. They remain an important source for the discovery of new pharmaceuticals and nutraceuticals. Montbretin A (MbA) is a complex acylated flavonoid glycoside discovered in the below-ground storage organs (corms) of the ornamental plant montbretia (Crocosmia x crocosmiiflora). MbA a highly potent and selective inhibitor of the human pancreatic α-amylase (HPA), a key enzyme in starch degradation. MbA is being tested for the treatment of type-2 diabetes. However, due to low abundance of MbA in montbretia plants and due the complex chemical structure of MbA, natural product extraction and chemical synthesis are insufficient for MbA production. Our goal is to develop a heterologous plant production system or a microbial production system for MbA. This requires knowledge of the genes, enzymes and regulating factors of the MbA biosynthetic system in montbretia. We achieved the discovery of the complete biosynthetic pathway of MbA using an approach that combined knowledge of montbretia biology, metabolite profiling, differential transcriptome analysis, cDNA cloning, heterologous gene expression in E. coli, yeast and tobacco, and enzyme biochemistry. This includes the discovery of five new UDP-sugar dependent glycosyltransferases (UGTs) and a BAHD-acyltransferases (AT) which together catalyze the complete assembly of MbA from its different building blocks. To reconstruct MbA production in tobacco (Nicotiana benthamiana) we enhanced the biosynthesis of flavonol precursors using genes for myricetin biosynthesis and transcription factors from montbtretia, which were stacked with genes of the MbA assembly pathway. We will highlight both challenges and opportunities of exploring novel biosynthetic systems of plant specialized metabolites for the development of new drugs, and bioproducts in general

CYP79D enzymes contribute to jasmonic acid-induced formation of aldoximes and other nitrogenous volatiles in two Erythroxylum species

Background: Amino acid-derived aldoximes and nitriles play important roles in plant defence. They are well-known as precursors for constitutive defence compounds such as cyanogenic glucosides and glucosinolates, but are also released as volatiles after insect feeding. Cytochrome P450 monooxygenases (CYP) of the CYP79 family catalyze the formation of aldoximes from the corresponding amino acids. However, the majority of CYP79s characterized so far are involved in cyanogenic glucoside or glucosinolate biosynthesis and only a few have been reported to be responsible for nitrogenous volatile production. Results: In this study we analysed and compared the jasmonic acid-induced volatile blends of two Erythroxylum species, the cultivated South American crop species E. coca and the African wild species E. fischeri. Both species produced different nitrogenous compounds including aliphatic aldoximes and an aromatic nitrile. Four isolated CYP79 genes (two from each species) were heterologously expressed in yeast and biochemically characterized. CYP79D62 from E. coca and CYP79D61 and CYP79D60 from E. fischeri showed broad substrate specificity in vitro and converted L-phenylalanine, L-isoleucine, L-leucine, L-tryptophan, and L-tyrosine into the respective aldoximes. In contrast, recombinant CYP79D63 from E. coca exclusively accepted L-tryptophan as substrate. Quantitative real-time PCR revealed that CYP79D60, CYP79D61, and CYP79D62 were significantly upregulated in jasmonic acid-treated Erythroxylum leaves. Conclusions: The kinetic parameters of the enzymes expressed in vitro coupled with the expression patterns of the corresponding genes and the accumulation and emission of (E/Z)-phenylacetaldoxime, (E/Z)-indole-3-acetaldoxime, (E/Z)-3-methylbutyraldoxime, and (E/Z)-2-methylbutyraldoxime in jasmonic acid-treated leaves suggest that CYP79D60, CYP79D61, and CYP79D62 accept L-phenylalanine, L-leucine, L-isoleucine, and L-tryptophan as substrates in vivo and contribute to the production of volatile and semi-volatile nitrogenous defence compounds in E. coca and E. fischeri.Other UBCNon UBCReviewedFacult

The nitrilase PtNIT1 catabolizes herbivore-induced nitriles in Populus trichocarpa

Background: Nitrilases are nitrile-converting enzymes commonly found within the plant kingdom that play diverse roles in nitrile detoxification, nitrogen recycling, and phytohormone biosynthesis. Although nitrilases are present in all higher plants, little is known about their function in trees. Upon herbivory, poplars produce considerable amounts of toxic nitriles such as benzyl cyanide, 2-methylbutyronitrile, and 3-methylbutyronitrile. In addition, as byproduct of the ethylene biosynthetic pathway upregulated in many plant species after herbivory, toxic β-cyanoalanine may accumulate in damaged poplar leaves. In this work, we studied the nitrilase gene family in Populus trichocarpa and investigated the potential role of the nitrilase PtNIT1 in the catabolism of herbivore-induced nitriles. Results: A BLAST analysis revealed three putative nitrilase genes (PtNIT1, PtNIT2, PtNIT3) in the genome of P. trichocarpa. While PtNIT1 was expressed in poplar leaves and showed increased transcript accumulation after leaf herbivory, PtNIT2 and PtNIT3 appeared not to be expressed in undamaged or herbivore-damaged leaves. Recombinant PtNIT1 produced in Escherichia coli accepted biogenic nitriles such as β-cyanoalanine, benzyl cyanide, and indole-3-acetonitrile as substrates in vitro and converted them into the corresponding acids. In addition to this nitrilase activity, PtNIT1 showed nitrile hydratase activity towards β-cyanoalanine, resulting in the formation of the amino acid asparagine. The kinetic parameters of PtNIT1 suggest that the enzyme utilizes β-cyanoalanine and benzyl cyanide as substrates in vivo. Indeed, β-cyanoalanine and benzyl cyanide were found to accumulate in herbivore-damaged poplar leaves. The upregulation of ethylene biosynthesis genes after leaf herbivory indicates that herbivore-induced β-cyanoalanine accumulation is likely caused by ethylene formation. Conclusions: Our data suggest a role for PtNIT1 in the catabolism of herbivore-induced β-cyanoalanine and benzyl cyanide in poplar leaves.Other UBCNon UBCReviewedFacult

Additional file 1: of One amino acid makes the difference: the formation of ent-kaurene and 16α-hydroxy-ent-kaurane by diterpene synthases in poplar

Figure S1. KS(L) genes located on chromosome 8. Figure S2. GC-MS analysis of ent-CPS, syn-CPS, n-CPS and PtTPS17 products. Table S1. Cq values of poplar CPS and KS(L). Table S2. Oligonucleotides used in this study. Table S3. Signalpeptide prediction using different prediction algorithms. (PPTX 147 kb

A beta-glucosidase of an insect herbivore determines both toxicity and deterrence of a dandelion defense metabolite

Gut enzymes can metabolize plant defense compounds and thereby affect the growth and fitness of insect herbivores. Whether these enzymes also influence feeding preference is largely unknown. We studied the metabolization of taraxinic acid beta-D-glucopyranosyl ester (TA-G), a sesquiterpene lactone of the common dandelion (Taraxacum officinale) that deters its major root herbivore, the common cockchafer larva (Melolontha melolontha). We have demonstrated that TA-G is rapidly deglucosylated and conjugated to glutathione in the insect gut. A broad-spectrum M. melolontha beta-glucosidase, Mm_bGlc17, is sufficient and necessary for TA-G deglucosylation. Using cross-species RNA interference, we have shown that Mm_bGlc17 reduces TA-G toxicity. Furthermore, Mm_bGlc17 is required for the preference of M. melolontha larvae for TA-G-deficient plants. Thus, herbivore metabolism modulates both the toxicity and deterrence of a plant defense compound. Our work illustrates the multifaceted roles of insect digestive enzymes as mediators of plant-herbivore interactions. eLife digest Plants produce certain substances to fend off attackers like plant-feeding insects. To stop these compounds from damaging their own cells, plants often attach sugar molecules to them. When an insect tries to eat the plant, the plant removes the stabilizing sugar, `activating' the compounds and making them toxic or foul-tasting. Curiously, some insects remove the sugar themselves, but it is unclear what consequences this has, especially for insect behavior. Dandelions, Taraxacum officinale, make high concentrations of a sugar-containing defense compound in their roots called taraxinic acid beta-D-glucopyranosyl ester, or TA-G for short. TA-G deters the larvae of the Maybug - a pest also known as the common cockchafer or the doodlebug - from eating dandelion roots. When Maybug larvae do eat TA-G, it is found in their systems without its sugar. However, it is unclear whether it is the plant or the larva that removes the sugar. A second open question is how the sugar removal process affects the behavior of the Maybug larvae. Using chemical analysis and genetic manipulation, Huber et al. investigated what happens when Maybug larvae eat TA-G. This revealed that the acidity levels in the larvae's digestive system deactivate the proteins from the dandelion that would normally remove the sugar from TA-G. However, rather than leaving the compound intact, larvae remove the sugar from TA-G themselves. They do this using a digestive enzyme, known as a beta-glucosidase, that cuts through sugar. Removing the sugar from TA-G made the compound less toxic, allowing the larvae to grow bigger, but it also increased TA-G's deterrent effects, making the larvae less likely to eat the roots. Any organism that eats plants, including humans, must deal with chemicals like TA-G in their food. Once inside the body, enzymes can change these chemicals, altering their effects. This happens with many medicines, too. In the future, it might be possible to design compounds that activate only in certain species, or under certain conditions. Further studies in different systems may aid the development of new methods of pest control, or new drug treatments

Infection of Corn Ears by Fusarium spp. Induces the Emission of Volatile Sesquiterpenes

Infection of corn (Zea mays L.) ears with fungal pathogens of the <i>Fusarium</i> genus might result in yield losses and in the accumulation of mycotoxins. The aim of this study was to investigate whether volatile profiles could be used to identify <i>Fusarium</i>-infected corn ears. The volatiles released by corn ears infected by Fusarium graminearum, Fusarium verticillioides, and Fusarium subglutinans were studied. Volatile emission was recorded at 24 days postinoculation (dpi) and in a time series (from 4 to 24 dpi). Twenty-two volatiles were differentially emitted from <i>Fusarium</i>-infected versus healthy corn ears. These included C₆–C₈ compounds and sesquiterpenoids. All volatiles indicative of <i>Fusarium</i> infection were detectable as early as 4–8 dpi and continued to be produced to the final sampling time (early milk maturity stage). The induced emission of β-macrocarpene and β-bisabolene correlated with an increased transcript accumulation of corn terpene synthase 6/11 (<i>tps6/11</i>). Additionally, the modification of volatile profiles after <i>Fusarium</i> infection was accompanied by the induction of plant defense compounds such as zealexins and oxylipins. Together, these results reveal a broad metabolic response of the plant to pathogen attack. Volatile biomarkers of <i>Fusarium</i> infection are promising indicators for the early detection of fungal infection before disease symptoms become visible

Characterization of Biosynthetic Pathways for the Production of the Volatile Homoterpenes DMNT and TMTT in Zea mays

Plant volatiles not only have multiple defense functions against herbivores, fungi, and bacteria, but also have been implicated in signaling within the plant and toward other organisms. Elucidating the function of individual plant volatiles will require more knowledge of their biosynthesis and regulation in response to external stimuli. By exploiting the variation of herbivore-induced volatiles among 26 maize (Zea mays) inbred lines, we conducted a nested association mapping and genome-wide association study (GWAS) to identify a set of quantitative trait loci (QTLs) for investigating the pathways of volatile terpene production. The most significant identified QTL affects the emission of (E)-nerolidol, linalool, and the two homoterpenes (E)-3,8-dimethyl-1,4,7-nonatriene (DMNT) and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). GWAS associated a single nucleotide polymorphism in the promoter of the gene encoding the terpene synthase TPS2 with this QTL Biochemical characterization of TPS2 verified that this plastid-localized enzyme forms linalool, (E)-nerolidol, and (E,E)-geranyllinalool. The subsequent conversion of (E)-nerolidol into DMNT maps to a P450 monooxygenase, CYP92C5, which is capable of converting nerolidol into DMNT by oxidative degradation. A QTL influencing TMTT accumulation corresponds to a similar monooxygenase, CYP92C6, which is specific for the conversion of (E,E)-geranyllinalool to TMTT The DMNT biosynthetic pathway and both monooxygenases are distinct from those previously characterized for DMNT and TMTT synthesis in Arabidopsis thaliana, suggesting independent evolution of these enzymatic activities