Site-directed genotype screening for elimination of antinutritional saponins in quinoa seeds identifies TSARL1 as a master controller of saponin biosynthesis selectively in seeds.

Climate change may result in a drier climate and increased salinization, threatening agricultural productivity worldwide. Quinoa (Chenopodium quinoa) produces highly nutritious seeds and tolerates abiotic stresses such as drought and high salinity, making it a promising future food source. However, the presence of antinutritional saponins in their seeds is an undesirable trait. We mapped genes controlling seed saponin content to a genomic region that includes TSARL1. We isolated desired genetic variation in this gene by producing a large mutant library of a commercial quinoa cultivar and screening the library for specific nucleotide substitutions using droplet digital PCR. We were able to rapidly isolate two independent tsarl1 mutants, which retained saponins in the leaves and roots for defence, but saponins were undetectable in the seed coat. We further could show that TSARL1 specifically controls seed saponin biosynthesis in the committed step after 2,3-oxidosqualene. Our work provides new important knowledge on the function of TSARL1 and represents a breakthrough for quinoa breeding.

Inverse pH Gradient-Assay for Facile Characterization of Proton-Antiporters in Xenopus Oocytes.

Xenopus oocytes represent one of the most versatile model systems for characterizing the properties of membrane transporters. However, for studying proton-coupled antiporters, the use of Xenopus oocytes has so far been limited to so-called injection-based transport assays. In such assays, where the compound is injected directly into the oocytes' cytosol and transport is detected by monitoring substrate efflux, poor control over internal diffusion and concentration are incompatible with mechanistic characterizations. In this study, we present an inverse pH-gradient transport assay. Herein, an outward-facing proton gradient enables the characterization of proton antiporters via facile import-based transport assays. We describe two approaches for establishing sustained outward-facing proton gradients across the oocyte membrane, namely by applying alkaline external conditions or through surprisingly stable carbonyl cyanide m-chlorophenyl-hydrazone (CCCP)-mediated acidification of the cytosol. Previously, genetic evidence has shown that DTX18 from Arabidopsis thaliana is essential for the deposition of the hydroxycinnamic acid amide p-coumaroylagmatine (coumaroylagmatine) defence compound on the leaf surface. However, direct evidence for its ability to transport coumarol-agmatine has not been provided. Here, using Xenopus oocytes as expression hosts, we demonstrate DTX18's ability to transport coumaroyl-agmatine via both injection-based and inverse pH-gradient transport assays. Notably, by showing that DTX18 is capable of accumulating its substrate against its concentration gradient, we showcase the compatibility of the latter with mechanistic investigations.

An UMAMIT-GTR transporter cascade controls glucosinolate seed loading in Arabidopsis.

Many plant species translocate maternally synthesized specialized metabolites to the seed to protect the developing embryo and later the germinating seedling before it initiates its own de novo synthesis. While the transport route into the seed is well established for primary metabolites, no model exists for any class of specialized metabolites that move from maternal source tissue(s) to embryo. Glucosinolate seed loading in Arabidopsis depends on plasma membrane localized exporters (USUALLY MULTIPLE AMINO ACIDS MOVE IN AND OUT TRANSPORTERs, UMAMITs) and importers (GLUCOSINOLATE TRANSPORTERs, GTRs), but the critical barriers in the seed loading process remain unknown. Here we dissect the transport route of glucosinolates from their source in the reproductive organ to the embryo by re-introducing the transporters at specific apoplastic barriers in their respective mutant backgrounds. We find that UMAMIT exporters and GTR importers form a transporter cascade that is both essential and sufficient for moving glucosinolates across at least four plasma membrane barriers along the route. We propose a model in which UMAMITs export glucosinolates out of the biosynthetic cells to the apoplast, from where GTRs import them into the phloem stream, which moves them to the unloading zone in the chalazal seed coat. From here, the UMAMITs export them out of maternal tissue and ultimately, the GTRs import them into the embryo symplasm, where the seed-specific glucosinolate profile is established by enzymatic modifications. Moreover, we propose that methylsulfinylalkyl glucosinolates are the predominant mobile form in seed loading. Elucidation of the seed loading process of glucosinolates identifies barrier-specific targets for transport engineering strategies to eliminate or over-accumulate a specialized metabolite in seeds with minimal interruption of other cellular processes.

Systematic engineering pinpoints a versatile strategy for the expression of functional cytochrome P450 enzymes in Escherichia coli cell factories.

Production of plant secondary metabolites in engineered microorganisms provides a scalable and sustainable alternative to their sourcing from nature or through chemical synthesis. However, the biosynthesis of many valuable plant-derived products relies on cytochromes P450 - enzymes notoriously difficult to express in microbes. To improve their expression in Escherichia coli, an arsenal of engineering strategies was developed, often paired with an extensive screening of enzyme variants. Here, attempting to identify a broadly applicable strategy, we systematically evaluated six common cytochrome P450 N-terminal modifications and their effect on in vivo activity of enzymes from the CYP79 and CYP83 families. We found that transmembrane domain truncation was the only modification with a significantly positive effect for all seven tested enzymes, increasing their product titres by 2- to 170-fold. Furthermore, when comparing the changes in the protein titre and product generation, we show that higher protein expression does not directly translate to higher in vivo activity, thus making the protein titre an unreliable screening target in the context of cell factories. We propose the transmembrane domain truncation as a first-line approach that enables the expression of wide range of highly active P450 enzymes in E. coli and circumvents the time-consuming screening process. Our results challenge the notion that the engineering strategy must be tailored for each individual cytochrome P450 enzyme and have the potential to simplify and accelerate the future design of E. coli cell factories.

Epidermal bladder cells as a herbivore defense mechanism.

The aerial surfaces of quinoa (Chenopodium quinoa) and common ice plant (Mesembryanthemum crystallinum) are covered with a layer of epidermal bladder cells (EBCs), which are modified non-glandular trichomes previously considered to be key to the extreme salt and drought tolerance of these plants. Here, however, we find that EBCs of these plants play only minor roles, if any, in abiotic stress tolerance and in fact are detrimental under conditions of water deficit. We report that EBCs instead function as deterrents to a broad range of generalist arthropod herbivores, through their combined function of forming both a chemical and a physical barrier, and they also serve a protective function against a phytopathogen. Our study overturns current models that link EBCs to salt and drought tolerance and assigns new functions to these structures that might provide novel possibilities for protecting crops from arthropod pests.

Identification of key amino acid residues in AtUMAMIT29 for transport of glucosinolates.

Glucosinolates are key defense compounds of plants in Brassicales order, and their accumulation in seeds is essential for the protection of the next generation. Recently, members of the Usually Multiple Amino acids Move In and Out Transporter (UMAMIT) family were shown to be essential for facilitating transport of seed-bound glucosinolates from site of synthesis within the reproductive organ to seeds. Here, we set out to identify amino acid residues responsible for glucosinolate transport activity of the main seed glucosinolate exporter UMAMIT29 in Arabidopsis thaliana. Based on a predicted model of UMAMIT29, we propose that the substrate transporting cavity consists of 51 residues, of which four are highly conserved residues across all the analyzed homologs of UMAMIT29. A comparison of the putative substrate binding site of homologs within the brassicaceous-specific, glucosinolate-transporting clade with the non-brassicaceous-specific, non-glucosinolate-transporting UMAMIT32 clade identified 11 differentially conserved sites. When each of the 11 residues of UMAMIT29 was individually mutated into the corresponding residue in UMAMIT32, five mutant variants (UMAMIT29#V27F, UMAMIT29#M86V, UMAMIT29#L109V, UMAMIT29#Q263S, and UMAMIT29#T267Y) reduced glucosinolate transport activity over 75% compared to wild-type UMAMIT29. This suggests that these residues are key for UMAMIT29-mediated glucosinolate transport activity and thus potential targets for blocking the transport of glucosinolates to the seeds.

Phytoalexins of the crucifer Barbarea vulgaris: Structural profile and correlation with glucosinolate turnover.

Phytoalexins are antimicrobial plant metabolites elicited by microbial attack or abiotic stress. We investigated phytoalexin profiles after foliar abiotic elicitation in the crucifer Barbarea vulgaris and interactions with the glucosinolate-myrosinase system. The treatment for abiotic elicitation was a foliar spray with CuCl2 solution, a usual eliciting agent, and three independent experiments were carried out. Two genotypes of B. vulgaris (G-type and P-type) accumulated the same three major phytoalexins in rosette leaves after treatment: phenyl-containing nasturlexin D and indole-containing cyclonasturlexin and cyclobrassinin. Phytoalexin levels were investigated daily by UHPLC-QToF MS and tended to differ among plant types and individual phytoalexins. In roots, phytoalexins were low or not detected. In treated leaves, typical total phytoalexin levels were in the range 1-10 nmol/g fresh wt. during three days after treatment while typical total glucosinolate (GSL) levels were three orders of magnitude higher. Levels of some minor GSLs responded to the treatment: phenethylGSL (PE) and 4-substituted indole GSLs. Levels of PE, a suggested nasturlexin D precursor, were lower in treated plants than controls. Another suggested precursor GSL, 3-hydroxyPE, was not detected, suggesting PE hydrolysis to be a key biosynthetic step. Levels of 4-substituted indole GSLs differed markedly between treated and control plants in most experiments, but not in a consistent way. The dominant GSLs, glucobarbarins, are not believed to be phytoalexin precursors. We observed statistically significant linear correlations between total major phytoalexins and the glucobarbarin products barbarin and resedine, suggesting that GSL turnover for phytoalexin biosynthesis was unspecific. In contrast, we did not find correlations between total major phytoalexins and raphanusamic acid or total glucobarbarins and barbarin. In conclusion, two groups of phytoalexins were detected in B. vulgaris, apparently derived from the GSLs PE and indol-3-ylmethylGSL. Phytoalexin biosynthesis was accompanied by depletion of the precursor PE and by turnover of major non-precursor GSLs to resedine. This work paves the way for identifying and characterizing genes and enzymes in the biosyntheses of phytoalexins and resedine.

Export of defensive glucosinolates is key for their accumulation in seeds.

Plant membrane transporters controlling metabolite distribution contribute key agronomic traits1-6. To eliminate anti-nutritional factors in edible parts of crops, the mutation of importers can block the accumulation of these factors in sink tissues7. However, this often results in a substantially altered distribution pattern within the plant8-12, whereas engineering of exporters may prevent such changes in distribution. In brassicaceous oilseed crops, anti-nutritional glucosinolate defence compounds are translocated to the seeds. However, the molecular targets for export engineering of glucosinolates remain unclear. Here we identify and characterize members of the USUALLY MULTIPLE AMINO ACIDS MOVE IN AND OUT TRANSPORTER (UMAMIT) family-UMAMIT29, UMAMIT30 and UMAMIT31-in Arabidopsis thaliana as glucosinolate exporters with a uniport mechanism. Loss-of-function umamit29 umamit30 umamit31 triple mutants have a very low level of seed glucosinolates, demonstrating a key role for these transporters in translocating glucosinolates into seeds. We propose a model in which the UMAMIT uniporters facilitate glucosinolate efflux from biosynthetic cells along the electrochemical gradient into the apoplast, where the high-affinity H+-coupled glucosinolate importers GLUCOSINOLATE TRANSPORTERS (GTRs) load them into the phloem for translocation to the seeds. Our findings validate the theory that two differently energized transporter types are required for cellular nutrient homeostasis13. The UMAMIT exporters are new molecular targets to improve nutritional value of seeds of brassicaceous oilseed crops without altering the distribution of the defence compounds in the whole plant.

Gibberellin and abscisic acid transporters facilitate endodermal suberin formation in Arabidopsis.

The plant hormone gibberellin (GA) regulates multiple developmental processes. It accumulates in the root elongating endodermis, but how it moves into this cell file and the significance of this accumulation are unclear. Here we identify three NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER (NPF) transporters required for GA and abscisic acid (ABA) translocation. We demonstrate that NPF2.14 is a subcellular GA/ABA transporter, presumably the first to be identified in plants, facilitating GA and ABA accumulation in the root endodermis to regulate suberization. Further, NPF2.12 and NPF2.13, closely related proteins, are plasma membrane-localized GA and ABA importers that facilitate shoot-to-root GA12 translocation, regulating endodermal hormone accumulation. This work reveals that GA is required for root suberization and that GA and ABA can act non-antagonistically. We demonstrate how the clade of transporters mediates hormone flow with cell-file-specific vacuolar storage at the phloem unloading zone, and slow release of hormone to induce suberin formation in the maturation zone.

SWEET11b transports both sugar and cytokinin in developing barley grains.

Even though Sugars Will Eventually be Exported Transporters (SWEETs) have been found in every sequenced plant genome, a comprehensive understanding of their functionality is lacking. In this study, we focused on the SWEET family of barley (Hordeum vulgare). A radiotracer assay revealed that expressing HvSWEET11b in African clawed frog (Xenopus laevis) oocytes facilitated the bidirectional transfer of not only just sucrose and glucose, but also cytokinin. Barley plants harboring a loss-of-function mutation of HvSWEET11b could not set viable grains, while the distribution of sucrose and cytokinin was altered in developing grains of plants in which the gene was knocked down. Sucrose allocation within transgenic grains was disrupted, which is consistent with the changes to the cytokinin gradient across grains, as visualized by magnetic resonance imaging and Fourier transform infrared spectroscopy microimaging. Decreasing HvSWEET11b expression in developing grains reduced overall grain size, sink strength, the number of endopolyploid endosperm cells, and the contents of starch and protein. The control exerted by HvSWEET11b over sugars and cytokinins likely predetermines their synergy, resulting in adjustments to the grain's biochemistry and transcriptome.

Species-specific dynamics of specialized metabolism in germinating sorghum grain revealed by temporal and tissue-resolved transcriptomics and metabolomics.

Seed germination is crucial for plant productivity, and the biochemical changes during germination affect seedling survival, plant health and yield. While the general metabolism of germination is extensively studied, the role of specialized metabolism is less investigated. We therefore analyzed the metabolism of the defense compound dhurrin during sorghum (Sorghum bicolor) grain germination and early seedling development. Dhurrin is a cyanogenic glucoside, which is catabolized into different bioactive compounds at other stages of plant development, but its fate and role during germination is unknown. We dissected sorghum grain into three different tissues and investigated dhurrin biosynthesis and catabolism at the transcriptomic, metabolomic and biochemical level. We further analyzed transcriptional signature differences of cyanogenic glucoside metabolism between sorghum and barley (Hordeum vulgare), which produces similar specialized metabolites. We found that dhurrin is de novo biosynthesized and catabolized in the growing embryonic axis as well as the scutellum and aleurone layer, two tissues otherwise mainly acknowledged for their involvement in release and transport of general metabolites from the endosperm to the embryonic axis. In contrast, genes encoding cyanogenic glucoside biosynthesis in barley are exclusively expressed in the embryonic axis. Glutathione transferase enzymes (GSTs) are involved in dhurrin catabolism and the tissue-resolved analysis of GST expression identified new pathway candidate genes and conserved GSTs as potentially important in cereal germination. Our study demonstrates a highly dynamic tissue- and species-specific specialized metabolism during cereal grain germination, highlighting the importance of tissue-resolved analyses and identification of specific roles of specialized metabolites in fundamental plant processes.

Multi-Knock-a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants.

Plant genomes are characterized by large and complex gene families that often result in similar and partially overlapping functions. This genetic redundancy severely hampers current efforts to uncover novel phenotypes, delaying basic genetic research and breeding programmes. Here we describe the development and validation of Multi-Knock, a genome-scale clustered regularly interspaced short palindromic repeat toolbox that overcomes functional redundancy in Arabidopsis by simultaneously targeting multiple gene-family members, thus identifying genetically hidden components. We computationally designed 59,129 optimal single-guide RNAs that each target two to ten genes within a family at once. Furthermore, partitioning the library into ten sublibraries directed towards a different functional group allows flexible and targeted genetic screens. From the 5,635 single-guide RNAs targeting the plant transportome, we generated over 3,500 independent Arabidopsis lines that allowed us to identify and characterize the first known cytokinin tonoplast-localized transporters in plants. With the ability to overcome functional redundancy in plants at the genome-scale level, the developed strategy can be readily deployed by scientists and breeders for basic research and to expedite breeding efforts.

Artificial Fluorescent Glucosinolates (F-GSLs) Are Transported by the Glucosinolate Transporters GTR1/2/3.

The glucosinolate transporters 1/2/3 (GTR1/2/3) from the Nitrate and Peptide transporter Family (NPF) play an essential role in the transport, accumulation, and distribution of the specialized plant metabolite glucosinolates. Due to representing both antinutritional and health-promoting compounds, there is increasing interest in characterizing GTRs from various plant species. We generated seven artificial glucosinolates (either aliphatic or benzenic) bearing different fluorophores (Fluorescein, BODIPY, Rhodamine, Dansylamide, and NBD) and investigated the ability of GTR1/2/3 from Arabidopsis thaliana to import the fluorescent glucosinolates (F-GSLs) into oocytes from Xenopus laevis. Five out of the seven F-GSLs synthesized were imported by at least one of the GTRs. GTR1 and GTR2 were able to import three F-GSLs actively above external concentration, while GTR3 imported only one actively. Competition assays indicate that the F-GSLs are transported by the same mechanism as non-tagged natural glucosinolates. The GTR-mediated F-GSL uptake is detected via a rapid and sensitive assay only requiring simple fluorescence measurements on a standard plate reader. This is highly useful in investigations of glucosinolate transport function and provides a critical prerequisite for elucidating the relationship between structure and function through high-throughput screening of GTR mutant libraries. The F-GSL themselves may also be suitable for future studies on glucosinolate transport in vivo.

Comparison of Genome and Plasmid-Based Engineering of Multigene Benzylglucosinolate Pathway in Saccharomyces cerevisiae.

Intake of brassicaceous vegetables such as cabbage is associated with numerous health benefits. The major defense compounds in the Brassicales order are the amino acid-derived glucosinolates that have been associated with the health-promoting effects. This has primed a desire to build glucosinolate-producing microbial cell factories as a stable and reliable source. Here, we established-for the first time-production of the phenylalanine-derived benzylglucosinolate (BGLS) in Saccharomyces cerevisiae using two different engineering strategies: stable genome integration versus plasmid-based introduction of the biosynthetic genes. Although the plasmid-engineered strain showed a tendency to generate higher expression level of each gene (except CYP83B1) in the biosynthetic pathway, the genome-engineered strain produced 8.4-fold higher BGLS yield compared to the plasmid-engineered strain. Additionally, we optimized the genome-engineered strain by overexpressing the entry point genes CYP79A2 and CYP83B1, resulting in a 2-fold increase in BGLS production but also a 4.8-fold increase in the level of the last intermediate desulfo-benzylglucosinolate (dsBGLS). We applied several approaches to alleviate the metabolic bottleneck in the step where dsBGLS is converted to BGLS by sulfotransferase, SOT16 dependent on 3'-phosphoadenosine-5'-phosphosulfate (PAPS). BGLS production increased 1.7-fold by overexpressing SOT16 and 1.7-fold by introducing APS kinase, APK1, from Arabidopsis thaliana involved in the PAPS regeneration cycle. Modulating the endogenous sulfur assimilatory pathway through overexpression of MET3 and MET14 resulted in 2.4-fold to 12.81 µmol/L (=5.2 mg/L) for BGLS production. IMPORTANCE Intake of brassicaceous vegetables such as cabbage is associated with numerous health benefits. The major defense compounds in the Brassicales order are the amino acid-derived glucosinolates that have been associated with the health-promoting effects. This has primed a desire to build glucosinolate-producing microbial cell factories as a stable and reliable source. In this study, we engineered for the first time the production of phenylalanine-derived benzylglucosinolate in Saccharomyces cerevisiae with two engineering strategies: stable genome integration versus plasmid-based introduction of the biosynthetic genes. Although the plasmid-engineered strain generally showed higher expression level of each gene (except CYP83B1) in the biosynthetic pathway, the genome-engineered strain produced higher production level of benzylglucosinolate. Based on the genome-engineered strain, the benzylglucosinolate level was improved by optimization. Our study compared different approaches to engineer a multigene pathway for production of the plant natural product benzylglucosinolate. This may provide potential application in industrial biotechnology.

Structure-guided engineering of key amino acids in UGT85B1 controlling substrate and stereo-specificity in aromatic cyanogenic glucoside biosynthesis.

Cyanogenic glucosides are important defense molecules in plants with useful biological activities in animals. Their last biosynthetic step consists of a glycosylation reaction that confers stability and increases structural diversity and is catalyzed by the UDP-dependent glycosyltransferases (UGTs) of glycosyltransferase family 1. These versatile enzymes have large and varied substrate scopes, and the structure-function relationships controlling scope and specificity remain poorly understood. Here, we report substrate-bound crystal structures and rational engineering of substrate and stereo-specificities of UGT85B1 from Sorghum bicolor involved in biosynthesis of the cyanogenic glucoside dhurrin. Substrate specificity was shifted from the natural substrate (S)-p-hydroxymandelonitrile to (S)-mandelonitrile by combining a mutation to abolish hydrogen bonding to the p-hydroxyl group with a mutation to provide steric hindrance at the p-hydroxyl group binding site (V132A/Q225W). Further, stereo-specificity was shifted from (S) to (R) by substituting four rationally chosen residues within 6 Å of the nitrile group (M312T/A313T/H408F/G409A). These activities were compared to two other UGTs involved in the biosynthesis of aromatic cyanogenic glucosides in Prunus dulcis (almond) and Eucalyptus cladocalyx. Together, these studies enabled us to pinpoint factors that drive substrate and stereo-specificities in the cyanogenic glucoside biosynthetic UGTs. The structure-guided engineering of the functional properties of UGT85B1 enhances our understanding of the evolution of UGTs involved in the biosynthesis of cyanogenic glucosides and will enable future engineering efforts towards new biotechnological applications.

Structural Insights into the Substrate Transport Mechanisms in GTR Transporters through Ensemble Docking.

Glucosinolate transporters (GTRs) are part of the nitrate/peptide transporter (NPF) family, members of which also transport specialized secondary metabolites as substrates. Glucosinolates are defense compounds derived from amino acids. We selected 4-methylthiobutyl (4MTB) and indol-3-ylmethyl (I3M) glucosinolates to study how GTR1 from Arabidopsis thaliana transports these substrates in computational simulation approaches. The designed pipeline reported here includes massive docking of 4MTB and I3M in an ensemble of GTR1 conformations (in both inward and outward conformations) extracted from molecular dynamics simulations, followed by clustered and substrate-protein interactions profiling. The identified key residues were mutated, and their role in substrate transport was tested. We were able to identify key residues that integrate a major binding site of these substrates, which is critical for transport activity. In silico approaches employed here represent a breakthrough in the plant transportomics field, as the identification of key residues usually takes a long time if performed from a purely wet-lab experimental perspective. The inclusion of structural bioinformatics in the analyses of plant transporters significantly speeds up the knowledge-gaining process and optimizes valuable time and resources.

Cyanogenesis in the Sorghum Genus: From Genotype to Phenotype.

Domestication has resulted in a loss of genetic diversity in our major food crops, leading to susceptibility to biotic and abiotic stresses linked with climate change. Crop wild relatives (CWR) may provide a source of novel genes potentially important for re-gaining climate resilience. Sorghum bicolor is an important cereal crop with wild relatives that are endemic to Australia. Sorghum bicolor is cyanogenic, but the cyanogenic status of wild Sorghum species is not well known. In this study, leaves of wild species endemic in Australia are screened for the presence of the cyanogenic glucoside dhurrin. The direct measurement of dhurrin content and the potential for dhurrin-derived HCN release (HCNp) showed that all the tested Australian wild species were essentially phenotypically acyanogenic. The unexpected low dhurrin content may reflect the variable and generally nutrient-poor environments in which they are growing in nature. Genome sequencing of six CWR and PCR amplification of the CYP79A1 gene from additional species showed that a high conservation of key amino acids is required for correct protein function and dhurrin synthesis, pointing to the transcriptional regulation of the cyanogenic phenotype in wild sorghum as previously shown in elite sorghum.

Cytosolic phosphofructokinases are important for sugar homeostasis in leaves of Arabidopsis thaliana.

BACKGROUND AND AIMS: ATP-dependent phosphofructokinases (PFKs) catalyse phosphorylation of the carbon-1 position of fructose-6-phosphate, to form fructose-1,6-bisphosphate. In the cytosol, this is considered a key step in channelling carbon into glycolysis. Arabidopsis thaliana has seven genes encoding PFK isoforms, two chloroplastic and five cytosolic. This study focuses on the four major cytosolic isoforms of PFK in vegetative tissues of A. thaliana. METHODS: We isolated homozygous knockout individual mutants (pfk1, pfk3, pfk6 and pfk7) and two double mutants (pfk1/7 and pfk3/6), and characterized their growth and metabolic phenotypes. KEY RESULTS: In contrast to single mutants and the double mutant pfk3/6 for the hypoxia-responsive isoforms, the double mutant pfk1/7 had reduced PFK activity and showed a clear visual and metabolic phenotype with reduced shoot growth, early flowering and elevated hexose levels. This mutant also has an altered ratio of short/long aliphatic glucosinolates and an altered root-shoot distribution. Surprisingly, this mutant does not show any major changes in short-term carbon flux and in levels of hexose-phosphates. CONCLUSIONS: We conclude that the two isoforms PFK1 and PFK7 are important for sugar homeostasis in leaf metabolism and apparently in source-sink relationships in A. thaliana, while PFK3 and PFK6 only play a minor role under normal growth conditions.

Exploiting photosynthesis-driven P450 activity to produce indican in tobacco chloroplasts.

Photosynthetic organelles offer attractive features for engineering small molecule bioproduction by their ability to convert solar energy into chemical energy required for metabolism. The possibility to couple biochemical production directly to photosynthetic assimilation as a source of energy and substrates has intrigued metabolic engineers. Specifically, the chemical diversity found in plants often relies on cytochrome P450-mediated hydroxylations that depend on reductant supply for catalysis and which often lead to metabolic bottlenecks for heterologous production of complex molecules. By directing P450 enzymes to plant chloroplasts one can elegantly deal with such redox prerequisites. In this study, we explore the capacity of the plant photosynthetic machinery to drive P450-dependent formation of the indigo precursor indoxyl-ß-D-glucoside (indican) by targeting an engineered indican biosynthetic pathway to tobacco (Nicotiana benthamiana) chloroplasts. We show that both native and engineered variants belonging to the human CYP2 family are catalytically active in chloroplasts when driven by photosynthetic reducing power and optimize construct designs to improve productivity. However, while increasing supply of tryptophan leads to an increase in indole accumulation, it does not improve indican productivity, suggesting that P450 activity limits overall productivity. Co-expression of different redox partners also does not improve productivity, indicating that supply of reducing power is not a bottleneck. Finally, in vitro kinetic measurements showed that the different redox partners were efficiently reduced by photosystem I but plant ferredoxin provided the highest light-dependent P450 activity. This study demonstrates the inherent ability of photosynthesis to support P450-dependent metabolic pathways. Plants and photosynthetic microbes are therefore uniquely suited for engineering P450-dependent metabolic pathways regardless of enzyme origin. Our findings have implications for metabolic engineering in photosynthetic hosts for production of high-value chemicals or drug metabolites for pharmacological studies.

The biosynthesis of thymol, carvacrol, and thymohydroquinone in Lamiaceae proceeds via cytochrome P450s and a short-chain dehydrogenase.

Thymol and carvacrol are phenolic monoterpenes found in thyme, oregano, and several other species of the Lamiaceae. Long valued for their smell and taste, these substances also have antibacterial and anti-spasmolytic properties. They are also suggested to be precursors of thymohydroquinone and thymoquinone, monoterpenes with anti-inflammatory, antioxidant, and antitumor activities. Thymol and carvacrol biosynthesis has been proposed to proceed by the cyclization of geranyl diphosphate to γ-terpinene, followed by a series of oxidations via p-cymene. Here, we show that γ-terpinene is oxidized by cytochrome P450 monooxygenases (P450s) of the CYP71D subfamily to produce unstable cyclohexadienol intermediates, which are then dehydrogenated by a short-chain dehydrogenase/reductase (SDR) to the corresponding ketones. The subsequent formation of the aromatic compounds occurs via keto-enol tautomerisms. Combining these enzymes with γ-terpinene in in vitro assays or in vivo in Nicotiana benthamiana yielded thymol and carvacrol as products. In the absence of the SDRs, only p-cymene was formed by rearrangement of the cyclohexadienol intermediates. The nature of these unstable intermediates was inferred from reactions with the γ-terpinene isomer limonene and by analogy to reactions catalyzed by related enzymes. We also identified and characterized two P450s of the CYP76S and CYP736A subfamilies that catalyze the hydroxylation of thymol and carvacrol to thymohydroquinone when heterologously expressed in yeast and N. benthamiana Our findings alter previous views of thymol and carvacrol formation, identify the enzymes involved in the biosynthesis of these phenolic monoterpenes and thymohydroquinone in the Lamiaceae, and provide targets for metabolic engineering of high-value terpenes in plants.