A new carbohydrate-active oligosaccharide dehydratase is involved in the degradation of ulvan.

Marine algae catalyze half of all global photosynthetic production of carbohydrates. Owing to their fast growth rates, Ulva spp. rapidly produce substantial amounts of carbohydrate-rich biomass and represent an emerging renewable energy and carbon resource. Their major cell wall polysaccharide is the anionic carbohydrate ulvan. Here, we describe a new enzymatic degradation pathway of the marine bacterium Formosa agariphila for ulvan oligosaccharides involving unsaturated uronic acid at the nonreducing end linked to rhamnose-3-sulfate and glucuronic or iduronic acid (Δ-Rha3S-GlcA/IdoA-Rha3S). Notably, we discovered a new dehydratase (P29_PDnc) acting on the nonreducing end of ulvan oligosaccharides, i.e., GlcA/IdoA-Rha3S, forming the aforementioned unsaturated uronic acid residue. This residue represents the substrate for GH105 glycoside hydrolases, which complements the enzymatic degradation pathway including one ulvan lyase, one multimodular sulfatase, three glycoside hydrolases, and the dehydratase P29_PDnc, the latter being described for the first time. Our research thus shows that the oligosaccharide dehydratase is involved in the degradation of carboxylated polysaccharides into monosaccharides.

Natural combinatorial genetics and prolific polyamine production enable siderophore diversification in Serratia plymuthica.

BACKGROUND: Iron is essential for bacterial survival. Bacterial siderophores are small molecules with unmatched capacity to scavenge iron from proteins and the extracellular milieu, where it mostly occurs as insoluble Fe3+. Siderophores chelate Fe3+ for uptake into the cell, where it is reduced to soluble Fe2+. Siderophores are key molecules in low soluble iron conditions. The ability of bacteria to synthesize proprietary siderophores may have increased bacterial evolutionary fitness; one way that bacteria diversify siderophore structure is by incorporating different polyamine backbones while maintaining the catechol moieties. RESULTS: We report that Serratia plymuthica V4 produces a variety of siderophores, which we term the siderome, and which are assembled by the concerted action of enzymes encoded in two independent gene clusters. Besides assembling serratiochelin A and B with diaminopropane, S. plymuthica utilizes putrescine and the same set of enzymes to assemble photobactin, a siderophore found in the bacterium Photorhabdus luminescens. The enzymes encoded by one of the gene clusters can independently assemble enterobactin. A third, independent operon is responsible for biosynthesis of the hydroxamate siderophore aerobactin, initially described in Enterobacter aerogenes. Mutant strains not synthesizing polyamine-siderophores significantly increased enterobactin production levels, though lack of enterobactin did not impact the production of serratiochelins. Knocking out SchF0, an enzyme involved in the assembly of enterobactin alone, significantly reduced bacterial fitness. CONCLUSIONS: This study shows the natural occurrence of serratiochelins, photobactin, enterobactin, and aerobactin in a single bacterial species and illuminates the interplay between siderophore biosynthetic pathways and polyamine production, indicating routes of molecular diversification. Given its natural yields of diaminopropane (97.75 µmol/g DW) and putrescine (30.83 µmol/g DW), S. plymuthica can be exploited for the industrial production of these compounds.

Core Steps to the Azaphilone Family of Fungal Natural Products.

Azaphilones are a family of polyketide-based fungal natural products that exhibit interesting and useful bioactivities. This minireview explores the literature on various characterised azaphilone biosynthetic pathways, which allows for a proposed consensus scheme for the production of the core azaphilone structure, as well as identifying early diversification steps during azaphilone biosynthesis. A consensus understanding of the core enzymatic steps towards a particular family of fungal natural products can aid in genome-mining experiments. Genome mining for novel fungal natural products is a powerful technique for both exploring chemical space and providing new insights into fungal natural product pathways.

Metabolite-based genome-wide association study enables dissection of the flavonoid decoration pathway of wheat kernels.

The marriage of metabolomic approaches with genetic design has proven a powerful tool in dissecting diversity in the metabolome and has additionally enhanced our understanding of complex traits. That said, such studies have rarely been carried out in wheat. In this study, we detected 805 metabolites from wheat kernels and profiled their relative contents among 182 wheat accessions, conducting a metabolite-based genome-wide association study (mGWAS) utilizing 14 646 previously described polymorphic SNP markers. A total of 1098 mGWAS associations were detected with large effects, within which 26 candidate genes were tentatively designated for 42 loci. Enzymatic assay of two candidates indicated they could catalyse glucosylation and subsequent malonylation of various flavonoids and thereby the major flavonoid decoration pathway of wheat kernel was dissected. Moreover, numerous high-confidence genes associated with metabolite contents have been provided, as well as more subdivided metabolite networks which are yet to be explored within our data. These combined efforts presented the first step towards realizing metabolomics-associated breeding of wheat.

The cytochrome P450 CYP72A552 is key to production of hederagenin-based saponins that mediate plant defense against herbivores.

Plants continuously evolve new defense compounds. One class of such compounds is triterpenoid saponins. A few species in the Barbarea genus produce saponins as the only ones in the large crucifer family. However, the molecular mechanism behind saponin biosynthesis and their role in plant defense remains unclear. We used pathway reconstitution in planta, enzymatic production of saponins in vitro, insect feeding assays, and bioinformatics to identify a missing gene involved in saponin biosynthesis and saponin-based herbivore defense. A tandem repeat of eight CYP72A cytochromes P450 colocalise with a quantitative trait locus (QTL) for saponin accumulation and flea beetle resistance in Barbarea vulgaris. We found that CYP72A552 oxidises oleanolic acid at position C-23 to hederagenin. In vitro-produced hederagenin monoglucosides reduced larval feeding by up to 90% and caused 75% larval mortality of the major crucifer pest diamondback moth and the tobacco hornworm. Sequence analysis indicated that CYP72A552 evolved through gene duplication and has been under strong selection pressure. In conclusion, CYP72A552 has evolved to catalyse the formation of hederagenin-based saponins that mediate plant defense against herbivores. Our study highlights the evolution of chemical novelties by gene duplication and selection for enzyme innovations, and the importance of chemical modification in plant defense evolution.

Evolution of the biochemistry underpinning purine alkaloid metabolism in plants.

Purine alkaloids are naturally occurring nitrogenous methylated derivatives of purine nucleotide degradation products, having essential roles in medicine, food and various other aspects of our daily lives. They are generated through convergent evolution in different plant species. The pivotal reaction steps within the purine alkaloid metabolic pathways have been largely elucidated, and the convergent evolution of purine alkaloids has been substantiated through bioinformatic, biochemical and other research perspectives within S-adenosyl-ʟ-methionine-dependent N-methyltransferases. Currently, the biological and ecological roles of purine alkaloids, further refinement of the purine alkaloid metabolic pathways and the investigation of purine alkaloid adaptive evolutionary mechanisms continue to attract widespread research interest. The exploration of the purine alkaloid metabolic pathways also enhances our comprehension of the biochemical mechanism, providing insights into inter-species interactions and adaptive evolution and offering potential value in drug development and agricultural applications. Here, we review the progress of research in the distribution, metabolic pathway elucidation and regulation, evolutionary mechanism and ecological roles of purine alkaloids in plants. The opportunities and challenges involved in elucidating the biochemical basis and evolutionary mechanisms of the purine alkaloid metabolic pathways, as well as other research aspects, are also discussed. This article is part of the theme issue 'The evolution of plant meta-bolism'.

Leveraging synthetic biology and metabolic engineering to overcome obstacles in plant pathway elucidation.

Major hurdles in plant biosynthetic pathway elucidation and engineering include the need for rapid testing of enzyme candidates and the lack of complex substrates that are often not accumulated in the plant, amenable to synthesis, or commercially available. Linking metabolic engineering with gene discovery in both yeast and plant holds great promise to expedite the elucidation process and, at the same time, provide a platform for the sustainable production of plant metabolites. In this review, we highlight how synthetic biology and metabolic engineering alleviated longstanding obstacles in plant pathway elucidation. Recent advances in developing these chassis that showcase established and emerging strategies in accelerating biosynthetic gene discovery will also be discussed.

Recent advances in triterpenoid pathway elucidation and engineering.

Triterpenoids are among the most assorted class of specialized metabolites found in all the taxa of living organisms. Triterpenoids are the leading active ingredients sourced from plant species and are utilized in pharmaceutical and cosmetic industries. The triterpenoid precursor 2,3-oxidosqualene, which is biosynthesized via the mevalonate (MVA) pathway is structurally diversified by the oxidosqualene cyclases (OSCs) and other scaffold-decorating enzymes such as cytochrome P450 monooxygenases (P450s), UDP-glycosyltransferases (UGTs) and acyltransferases (ATs). A majority of the bioactive triterpenoids are harvested from the native hosts using the traditional methods of extraction and occasionally semi-synthesized. These methods of supply are time-consuming and do not often align with sustainability goals. Recent advancements in metabolic engineering and synthetic biology have shown prospects for the green routes of triterpenoid pathway reconstruction in heterologous hosts such as Escherichia coli, Saccharomyces cerevisiae and Nicotiana benthamiana, which appear to be quite promising and might lead to the development of alternative source of triterpenoids. The present review describes the biotechnological strategies used to elucidate complex biosynthetic pathways and to understand their regulation and also discusses how the advances in triterpenoid pathway engineering might aid in the scale-up of triterpenoid production in engineered hosts.

Exploring the genic resources underlying metabolites through mGWAS and mQTL in wheat: From large-scale gene identification and pathway elucidation to crop improvement.

Common wheat (Triticum aestivum L.) is a leading cereal crop, but has lagged behind with respect to the interpretation of the molecular mechanisms of phenotypes compared with other major cereal crops such as rice and maize. The recently available genome sequence of wheat affords the pre-requisite information for efficiently exploiting the potential molecular resources for decoding the genetic architecture of complex traits and identifying valuable breeding targets. Meanwhile, the successful application of metabolomics as an emergent large-scale profiling methodology in several species has demonstrated this approach to be accessible for reaching the above goals. One such productive avenue is combining metabolomics approaches with genetic designs. However, this trial is not as widespread as that for sequencing technologies, especially when the acquisition, understanding, and application of metabolic approaches in wheat populations remain more difficult and even arguably underutilized. In this review, we briefly introduce the techniques used in the acquisition of metabolomics data and their utility in large-scale identification of functional candidate genes. Considerable progress has been made in delivering improved varieties, suggesting that the inclusion of information concerning these metabolites and genes and metabolic pathways enables a more explicit understanding of phenotypic traits and, as such, this procedure could serve as an -omics-informed roadmap for executing similar improvement strategies in wheat and other species.

An Improved CRISPR Interference Tool to Engineer Rhodococcus opacus.

Rhodococcus opacus is a nonmodel bacterium that is well suited for valorizing lignin. Despite recent advances in our systems-level understanding of its versatile metabolism, studies of its gene functions at a single gene level are still lagging. Elucidating gene functions in nonmodel organisms is challenging due to limited genetic engineering tools that are convenient to use. To address this issue, we developed a simple gene repression system based on CRISPR interference (CRISPRi). This gene repression system uses a T7 RNA polymerase system to express a small guide RNA, demonstrating improved repression compared to the previously demonstrated CRISPRi system (i.e., the maximum repression efficiency improved from 58% to 85%). Additionally, our cloning strategy allows for building multiple CRISPRi plasmids in parallel without any PCR step, facilitating the engineering of this GC-rich organism. Using the improved CRISPRi system, we confirmed the annotated roles of four metabolic pathway genes, which had been identified by our previous transcriptomic analysis to be related to the consumption of benzoate, vanillate, catechol, and acetate. Furthermore, we showed our tool's utility by demonstrating the inducible accumulation of muconate that is a precursor of adipic acid, an important monomer for nylon production. While the maximum muconate yield obtained using our tool was 30% of the yield obtained using gene knockout, our tool showed its inducibility and partial repressibility. Our CRISPRi tool will be useful to facilitate functional studies of this nonmodel organism and engineer this promising microbial chassis for lignin valorization.

Current State and Future Directions of Genetics and Genomics of Endophytic Fungi for Bioprospecting Efforts.

The bioprospecting of secondary metabolites from endophytic fungi received great attention in the 1990s and 2000s, when the controversy around taxol production from Taxus spp. endophytes was at its height. Since then, hundreds of reports have described the isolation and characterization of putative secondary metabolites from endophytic fungi. However, only very few studies also report the genetic basis for these phenotypic observations. With low sequencing cost and fast sample turnaround, genetics- and genomics-based approaches have risen to become comprehensive approaches to study natural products from a wide-range of organisms, especially to elucidate underlying biosynthetic pathways. However, in the field of fungal endophyte biology, elucidation of biosynthetic pathways is still a major challenge. As a relatively poorly investigated group of microorganisms, even in the light of recent efforts to sequence more fungal genomes, such as the 1000 Fungal Genomes Project at the Joint Genome Institute (JGI), the basis for bioprospecting of enzymes and pathways from endophytic fungi is still rather slim. In this review we want to discuss the current approaches and tools used to associate phenotype and genotype to elucidate biosynthetic pathways of secondary metabolites in endophytic fungi through the lens of bioprospecting. This review will point out the reported successes and shortcomings, and discuss future directions in sampling, and genetics and genomics of endophytic fungi. Identifying responsible biosynthetic genes for the numerous secondary metabolites isolated from endophytic fungi opens the opportunity to explore the genetic potential of producer strains to discover novel secondary metabolites and enhance secondary metabolite production by metabolic engineering resulting in novel and more affordable medicines and food additives.

Biosynthesis of UDP-2-acetamido-4-formamido-2,4,6-trideoxy-hexose by WekG, WekE, WekF, and WekD: Enzymes in the Wek pathway responsible for O-antigen Assembly in Escherichia coli O119.

Considering the importance of bacterial glycoconjugates on virulence and host mimicry, there is a need to better understand the biosynthetic pathways of these unusual sugars to identify critical targets involved in bacterial pathogenesis. In this report, we describe the cloning, overexpression, purification, and biochemical characterization of the four central enzymes in the biosynthesis pathway for UDP-2-acetamido-4-formamido-2,4,6-trideoxy-hexose, WekG, WekE, WekF, and WekD. Product peaks from enzyme-substrate reactions were detected by using a combination of capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS). Putative enzyme assignments were provided by protein sequence analysis. Combined with the mass spectrometric characterization of pathway intermediates, we propose a biosynthetic pathway for UDP-2-acetamido-4-formamido-2,4,6-trideoxy-hexose. This process involves C-4, C-6 dehydration, C-4 amination, and formylation. CID-ESI-MSn result confirmed that the final product is a 4 formamido derivative too rather than the 3 formamido derivatives as reported earlier.

Exploiting Natural Variation in Tomato to Define Pathway Structure and Metabolic Regulation of Fruit Polyphenolics in the Lycopersicum Complex.

While the structures of plant primary metabolic pathways are generally well defined and highly conserved across species, those defining specialized metabolism are less well characterized and more highly variable across species. In this study, we investigated polyphenolic metabolism in the lycopersicum complex by characterizing the underlying biosynthetic and decorative reactions that constitute the metabolic network of polyphenols across eight different species of tomato. For this purpose, GC-MS- and LC-MS-based metabolomics of different tissues of Solanum lycopersicum and wild tomato species were carried out, in concert with the evaluation of cross-hybridized microarray data for MapMan-based transcriptomic analysis, and publicly available RNA-sequencing data for annotation of biosynthetic genes. The combined data were used to compile species-specific metabolic networks of polyphenolic metabolism, allowing the establishment of an entire pan-species biosynthetic framework as well as annotation of the functions of decoration enzymes involved in the formation of metabolic diversity of the flavonoid pathway. The combined results are discussed in the context of the current understanding of tomato flavonol biosynthesis as well as a global view of metabolic shifts during fruit ripening. Our results provide an example as to how large-scale biology approaches can be used for the definition and refinement of large specialized metabolism pathways.

Kaempferol-3-o-ß-d-glucuronate exhibit potential anti-inflammatory effect in LPS stimulated RAW 264.7 cells and mice model.

Kaempferol-3-O-ß-d-glucuronide (K3G) having various pharmacological effects was explored for its anti-inflammatory effect in LPS induced RAW 264.7 cells and mice model. K3G significantly inhibited various pro-inflammatory mediators like IL-1ß, NO, PGE2, and LTB4. It upregulated the secretion of anti-inflammatory cytokine IL-10. K3G is found to reduce inflammation when studied for parameters like phagocytic index, carrageenan induced paw edema in mice and organ weight. It reduced inflammation in a dose dependent manner both in-vitro and in-vivo. Further molecular insights into the study reveal that K3G blocks the phosphorylation of NF-kB which is key regulator of inflammation, thereby exhibiting anti-inflammatory potential. Hence, this study permits further investigation to develop K3G as anti-inflammatory drug.

Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii.

Forskolin is a unique structurally complex labdane-type diterpenoid used in the treatment of glaucoma and heart failure based on its activity as a cyclic AMP booster. Commercial production of forskolin relies exclusively on extraction from its only known natural source, the plant Coleus forskohlii, in which forskolin accumulates in the root cork. Here, we report the discovery of five cytochrome P450s and two acetyltransferases which catalyze a cascade of reactions converting the forskolin precursor 13R-manoyl oxide into forskolin and a diverse array of additional labdane-type diterpenoids. A minimal set of three P450s in combination with a single acetyl transferase was identified that catalyzes the conversion of 13R-manoyl oxide into forskolin as demonstrated by transient expression in Nicotiana benthamiana. The entire pathway for forskolin production from glucose encompassing expression of nine genes was stably integrated into Saccharomyces cerevisiae and afforded forskolin titers of 40 mg/L.

Students' perspective on genomics: from sample to sequence using the case study of blueberry.

Advances in genomic sequencing technologies in the past decade have revolutionized the field of genomics, resulting in faster and less expensive sequencing. Holding back the potential for innovation, however, is a widespread lack of understanding of genomics and sequencing by the general public. In an attempt to remedy this problem, this paper presents an introduction to the fields of genomics, bioinformatics, and proteomics using the blueberry genome as a model case study of the plant genomics field. The blueberry (Vaccinium sect. Cyanococcus) is often cited as a "super food" in the media due to its nutritional benefits and global economic importance. There have been a number of related genomic publications in the past 20 years; however, a completed genome and a full analysis into the health-related pathways are still needed. As exemplified by this blueberry case study, there are opportunities for future genomic research into numerous beneficial plant species. The solid background presented in this paper provides future researchers the foundation to explore these uncharted areas.

Comprehensive discovery of 13C labeled metabolites in the bacterium Methylobacterium extorquens AM1 using gas chromatography-mass spectrometry.

Herein, we report the identification of isotopically labeled metabolite peaks (or the lack of labeling) between sets of GC-MS data from Methylobacterium extorquens AM1. M. extorquens AM1 is one of the best-characterized model organisms for the study of C1 metabolism in methylotrophic bacteria, a diverse group of microbes that can use reduced one-carbon (C1) sources, such as methanol and methane as a sole source for both energy generation and carbon assimilation. Application of a match value (MV) based metric was used to rank the metabolite peaks in the data from those exhibiting the most mass spectral indications of labeling, to those not exhibiting any indications of labeling. The MV-based ranking corresponded well with analyst interpretation of the mass spectra. The MV-based method was initially demonstrated and validated using a mixture of 21 standards with data sets generated for mixtures at natural abundance, a mixture with 6 of the compounds labeled, and a 1:1 mixture of the natural abundance and labeled mixtures. Experimental data from TMS-derivatized extracts from the bacterium M. extorquens AM1 grown with natural abundance or (13)C-labeled methanol as the carbon source were analyzed. Of 131 peaks considered for the analysis of M. extorquens AM1, the 40 peaks ranked highest for indications of (13)C labeling were all found to be labeled, while those peaks ranked lower progressed from peaks for which labeling was uncertain, to a larger number of peaks that were clearly not labeled. The list of peaks determined to be labeled forms a library of compounds that are known to be labeled following the methanol metabolic pathway in M. extorquens AM1 that can be further investigated in future work, e.g. fluxomic studies.

The use of natural genetic diversity in the understanding of metabolic organization and regulation.

The study of metabolic regulation has traditionally focused on analysis of specific enzymes, emphasizing kinetic properties, and the influence of protein interactions and post-translational modifications. More recently, reverse genetic approaches permit researchers to directly determine the effects of a deficiency or a surplus of a given enzyme on the biochemistry and physiology of a plant. Furthermore, in many model species, gene expression atlases that give important spatial information concerning the quantitative expression level of metabolism-associated genes are being produced. In parallel, "top-down" approaches to understand metabolic regulation have recently been instigated whereby broad genetic diversity is screened for metabolic traits and the genetic basis of this diversity is defined thereafter. In this article we will review recent examples of this latter approach both in the model species Arabidopsis thaliana and the crop species tomato (Solanum lycopersicum). In addition to highlighting examples in which this genetic diversity approach has proven promising, we will discuss the challenges associated with this approach and provide a perspective for its future utility.