Strain-level fitness in the gut microbiome is an emergent property of glycans and a single metabolite.

The human gut microbiota resides within a diverse chemical environment challenging our ability to understand the forces shaping this ecosystem. Here, we reveal that fitness of the Bacteroidales, the dominant order of bacteria in the human gut, is an emergent property of glycans and one specific metabolite, butyrate. Distinct sugars serve as strain-variable fitness switches activating context-dependent inhibitory functions of butyrate. Differential fitness effects of butyrate within the Bacteroides are mediated by species-level variation in Acyl-CoA thioesterase activity and nucleotide polymorphisms regulating an Acyl-CoA transferase. Using in vivo multi-omic profiles, we demonstrate Bacteroides fitness in the human gut is associated together, but not independently, with Acyl-CoA transferase expression and butyrate. Our data reveal that each strain of the Bacteroides exists within a unique fitness landscape based on the interaction of chemical components unpredictable by the effect of each part alone mediated by flexibility in the core genome.

Co-evolution of Hormone Metabolism and Signaling Networks Expands Plant Adaptive Plasticity.

Classically, hormones elicit specific cellular responses by activating dedicated receptors. Nevertheless, the biosynthesis and turnover of many of these hormone molecules also produce chemically related metabolites. These molecules may also possess hormonal activities; therefore, one or more may contribute to the adaptive plasticity of signaling outcomes in host organisms. Here, we show that a catabolite of the plant hormone abscisic acid (ABA), namely phaseic acid (PA), likely emerged in seed plants as a signaling molecule that fine-tunes plant physiology, environmental adaptation, and development. This trait was facilitated by both the emergence-selection of a PA reductase that modulates PA concentrations and by the functional diversification of the ABA receptor family to perceive and respond to PA. Our results suggest that PA serves as a hormone in seed plants through activation of a subset of ABA receptors. This study demonstrates that the co-evolution of hormone metabolism and signaling networks can expand organismal resilience.

A molecular gauge for nitrogen economy in plants.

Understanding nitrogen metabolism in plants holds promise for future agricultural improvements. Chellamuthu et al. now identify a feedback regulation in plant nitrogen metabolism through glutamine sensing. This mechanism appears to be conserved from algae to flowering plants with a few surprising exceptions.

Genome-wide association identifies a BAHD acyltransferase activity that assembles an ester of glucuronosylglycerol and phenylacetic acid.

Genome-wide association studies (GWAS) are an effective approach to identify new specialized metabolites and the genes involved in their biosynthesis and regulation. In this study, GWAS of Arabidopsis thaliana soluble leaf and stem metabolites identified alleles of an uncharacterized BAHD-family acyltransferase (AT5G57840) associated with natural variation in three structurally related metabolites. These metabolites were esters of glucuronosylglycerol, with one metabolite containing phenylacetic acid as the acyl component of the ester. Knockout and overexpression of AT5G57840 in Arabidopsis and heterologous overexpression in Nicotiana benthamiana and Escherichia coli demonstrated that it is capable of utilizing phenylacetyl-CoA as an acyl donor and glucuronosylglycerol as an acyl acceptor. We, thus, named the protein Glucuronosylglycerol Ester Synthase (GGES). Additionally, phenylacetyl glucuronosylglycerol increased in Arabidopsis CYP79A2 mutants that overproduce phenylacetic acid and was lost in knockout mutants of UDP-sulfoquinovosyl: diacylglycerol sulfoquinovosyl transferase, an enzyme required for glucuronosylglycerol biosynthesis and associated with glycerolipid metabolism under phosphate-starvation stress. GGES is a member of a well-supported clade of BAHD family acyltransferases that arose by duplication and neofunctionalized during the evolution of the Brassicales within a larger clade that includes HCT as well as enzymes that synthesize other plant-specialized metabolites. Together, this work extends our understanding of the catalytic diversity of BAHD acyltransferases and uncovers a pathway that involves contributions from both phenylalanine and lipid metabolism.

transXpress: a Snakemake pipeline for streamlined de novo transcriptome assembly and annotation.

BACKGROUND: RNA-seq followed by de novo transcriptome assembly has been a transformative technique in biological research of non-model organisms, but the computational processing of RNA-seq data entails many different software tools. The complexity of these de novo transcriptomics workflows therefore presents a major barrier for researchers to adopt best-practice methods and up-to-date versions of software. RESULTS: Here we present a streamlined and universal de novo transcriptome assembly and annotation pipeline, transXpress, implemented in Snakemake. transXpress supports two popular assembly programs, Trinity and rnaSPAdes, and allows parallel execution on heterogeneous cluster computing hardware. CONCLUSIONS: transXpress simplifies the use of best-practice methods and up-to-date software for de novo transcriptome assembly, and produces standardized output files that can be mined using SequenceServer to facilitate rapid discovery of new genes and proteins in non-model organisms.

Adaptive mechanisms of plant specialized metabolism connecting chemistry to function.

As sessile organisms, plants evolved elaborate metabolic systems that produce a plethora of specialized metabolites as a means to survive challenging terrestrial environments. Decades of research have revealed the genetic and biochemical basis for a multitude of plant specialized metabolic pathways. Nevertheless, knowledge is still limited concerning the selective advantages provided by individual and collective specialized metabolites to the reproductive success of diverse host plants. Here we review the biological functions conferred by various classes of plant specialized metabolites in the context of the interaction of plants with their surrounding environment. To achieve optimal multifunctionality of diverse specialized metabolic processes, plants use various adaptive mechanisms at subcellular, cellular, tissue, organ and interspecies levels. Understanding these mechanisms and the evolutionary trajectories underlying their occurrence in nature will ultimately enable efficient bioengineering of desirable metabolic traits in chassis organisms.

Structural basis for divergent and convergent evolution of catalytic machineries in plant aromatic amino acid decarboxylase proteins.

Radiation of the plant pyridoxal 5'-phosphate (PLP)-dependent aromatic l-amino acid decarboxylase (AAAD) family has yielded an array of paralogous enzymes exhibiting divergent substrate preferences and catalytic mechanisms. Plant AAADs catalyze either the decarboxylation or decarboxylation-dependent oxidative deamination of aromatic l-amino acids to produce aromatic monoamines or aromatic acetaldehydes, respectively. These compounds serve as key precursors for the biosynthesis of several important classes of plant natural products, including indole alkaloids, benzylisoquinoline alkaloids, hydroxycinnamic acid amides, phenylacetaldehyde-derived floral volatiles, and tyrosol derivatives. Here, we present the crystal structures of four functionally distinct plant AAAD paralogs. Through structural and functional analyses, we identify variable structural features of the substrate-binding pocket that underlie the divergent evolution of substrate selectivity toward indole, phenyl, or hydroxyphenyl amino acids in plant AAADs. Moreover, we describe two mechanistic classes of independently arising mutations in AAAD paralogs leading to the convergent evolution of the derived aldehyde synthase activity. Applying knowledge learned from this study, we successfully engineered a shortened benzylisoquinoline alkaloid pathway to produce (S)-norcoclaurine in yeast. This work highlights the pliability of the AAAD fold that allows change of substrate selectivity and access to alternative catalytic mechanisms with only a few mutations.

Gene-Guided Discovery and Ribosomal Biosynthesis of Moroidin Peptides.

Moroidin is a bicyclic plant octapeptide with tryptophan side-chain cross-links, originally isolated as a pain-causing agent from the Australian stinging tree Dendrocnide moroides. Moroidin and its analog celogentin C, derived from Celosia argentea, are inhibitors of tubulin polymerization and, thus, lead structures for cancer therapy. However, low isolation yields from source plants and challenging organic synthesis hinder moroidin-based drug development. Here, we present biosynthesis as an alternative route to moroidin-type bicyclic peptides and report that they are ribosomally synthesized and posttranslationally modified peptides (RiPPs) derived from BURP-domain peptide cyclases in plants. By mining 793 plant transcriptomes for moroidin core peptide motifs within BURP-domain precursor peptides, we identified a moroidin cyclase in Japanese kerria, which catalyzes the installation of the tryptophan-indole-centered macrocyclic bonds of the moroidin bicyclic motif in the presence of cupric ions. Based on the kerria moroidin cyclase, we demonstrate the feasibility of producing diverse moroidins including celogentin C in transgenic tobacco plants and report specific cytotoxicity of celogentin C against a lung adenocarcinoma cancer cell line. Our study sets the stage for future biosynthetic development of moroidin-based therapeutics and highlights that mining plant transcriptomes can reveal bioactive cyclic peptides and their underlying cyclases from new source plants.

PRX9 and PRX40 Are Extensin Peroxidases Essential for Maintaining Tapetum and Microspore Cell Wall Integrity during Arabidopsis Anther Development.

Pollen and microspore development are essential steps in the life cycle of all land plants that generate male gametes. Within flowering plants, pollen development occurs inside of the anther. Here, we report the identification of two class III peroxidase-encoding genes, PEROXIDASE9 (PRX9) and PRX40, that are genetically redundant and essential for proper anther and pollen development in Arabidopsis (Arabidopsis thaliana). Arabidopsis double mutants devoid of functional PRX9 and PRX40 are male sterile. The mutant anthers display swollen, hypertrophic tapetal cells and pollen grains, suggesting disrupted cell wall integrity. These phenotypes lead to nearly 100%-penetrant pollen degeneration upon anther maturation. Using immunochemical and biochemical approaches, we show that PRX9 and PRX40 likely cross-link extensins to contribute to tapetal cell wall integrity during anther development. This work suggests that PRX9 and PRX40 encode Arabidopsis extensin peroxidases and highlights the importance of extensin cross-linking during pollen development.

New developments in RiPP discovery, enzymology and engineering.

Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.

Gene-guided discovery and engineering of branched cyclic peptides in plants.

The plant kingdom contains vastly untapped natural product chemistry, which has been traditionally explored through the activity-guided approach. Here, we describe a gene-guided approach to discover and engineer a class of plant ribosomal peptides, the branched cyclic lyciumins. Initially isolated from the Chinese wolfberry Lycium barbarum, lyciumins are protease-inhibiting peptides featuring an N-terminal pyroglutamate and a macrocyclic bond between a tryptophan-indole nitrogen and a glycine α-carbon. We report the identification of a lyciumin precursor gene from L. barbarum, which encodes a BURP domain and repetitive lyciumin precursor peptide motifs. Genome mining enabled by this initial finding revealed rich lyciumin genotypes and chemotypes widespread in flowering plants. We establish a biosynthetic framework of lyciumins and demonstrate the feasibility of producing diverse natural and unnatural lyciumins in transgenic tobacco. With rapidly expanding plant genome resources, our approach will complement bioactivity-guided approaches to unlock and engineer hidden plant peptide chemistry for pharmaceutical and agrochemical applications.

Noncatalytic chalcone isomerase-fold proteins in Humulus lupulus are auxiliary components in prenylated flavonoid biosynthesis.

Xanthohumol (XN) and demethylxanthohumol (DMX) are specialized prenylated chalconoids with multiple pharmaceutical applications that accumulate to high levels in the glandular trichomes of hops (Humulus lupulus L.). Although all structural enzymes in the XN pathway have been functionally identified, biochemical mechanisms underlying highly efficient production of XN have not been fully resolved. In this study, we characterized two noncatalytic chalcone isomerase (CHI)-like proteins (designated as HlCHIL1 and HlCHIL2) using engineered yeast harboring all genes required for DMX production. HlCHIL2 increased DMX production by 2.3-fold, whereas HlCHIL1 significantly decreased DMX production by 30%. We show that CHIL2 is part of an active DMX biosynthetic metabolon in hop glandular trichomes that encompasses a chalcone synthase (CHS) and a membrane-bound prenyltransferase, and that type IV CHI-fold proteins of representative land plants contain conserved function to bind with CHS and enhance its activity. Binding assays and structural docking uncover a function of HlCHIL1 to bind DMX and naringenin chalcone to stabilize the ring-open configuration of these chalconoids. This study reveals the role of two HlCHILs in DMX biosynthesis in hops, and provides insight into their evolutionary development from the ancestral fatty acid-binding CHI-fold proteins to specialized auxiliary proteins supporting flavonoid biosynthesis in plants.

Independent evolution of rosmarinic acid biosynthesis in two sister families under the Lamiids clade of flowering plants.

As a means to maintain their sessile lifestyle amid challenging environments, plants produce an enormous diversity of compounds as chemical defenses against biotic and abiotic insults. The underpinning metabolic pathways that support the biosynthesis of these specialized chemicals in divergent plant species provide a rich arena for understanding the molecular evolution of complex metabolic traits. Rosmarinic acid (RA) is a phenolic natural product first discovered in plants of the mint family (Lamiaceae) and is recognized for its wide range of medicinal properties and potential applications in human dietary and medical interventions. Interestingly, the RA chemotype is present sporadically in multiple taxa of flowering plants as well as some hornworts and ferns, prompting the question whether its biosynthesis arose independently across different lineages. Here we report the elucidation of the RA biosynthetic pathway in Phacelia campanularia (desert bells). This species represents the borage family (Boraginaceae), an RA-producing family closely related to the Lamiaceae within the Lamiids clade. Using a multi-omics approach in combination with functional characterization of candidate genes both in vitro and in vivo, we found that RA biosynthesis in P. campanularia involves specific activities of a BAHD acyltransferase and two cytochrome P450 hydroxylases. Further phylogenetic and comparative structure-function analyses of the P. campanularia RA biosynthetic enzymes clearly indicate that RA biosynthesis has evolved independently at least twice in the Lamiids, an exemplary case of chemotypic convergence through disparate evolutionary trajectories.

SG2-Type R2R3-MYB Transcription Factor MYB15 Controls Defense-Induced Lignification and Basal Immunity in Arabidopsis.

Lignification of cell wall appositions is a conserved basal defense mechanism in the plant innate immune response. However, the genetic pathway controlling defense-induced lignification remains unknown. Here, we demonstrate the Arabidopsis thaliana SG2-type R2R3-MYB transcription factor MYB15 as a regulator of defense-induced lignification and basal immunity. Loss of MYB15 reduces the content but not the composition of defense-induced lignin, whereas constitutive expression of MYB15 increases lignin content independently of immune activation. Comparative transcriptional and metabolomics analyses implicate MYB15 as necessary for the defense-induced synthesis of guaiacyl lignin and the basal synthesis of the coumarin metabolite scopoletin. MYB15 directly binds to the secondary wall MYB-responsive element consensus sequence, which encompasses the AC elements, to drive lignification. The myb15 and lignin biosynthetic mutants show increased susceptibility to the bacterial pathogen Pseudomonas syringae, consistent with defense-induced lignin having a major role in basal immunity. A scopoletin biosynthetic mutant also shows increased susceptibility independently of immune activation, consistent with a role in preformed defense. Our results support a role for phenylalanine-derived small molecules in preformed and inducible Arabidopsis defense, a role previously dominated by tryptophan-derived small molecules. Understanding the regulatory network linking lignin biosynthesis to plant growth and defense will help lignin engineering efforts to improve the production of biofuels and aromatic industrial products as well as increase disease resistance in energy and agricultural crops.

Mechanistic basis for the evolution of chalcone synthase catalytic cysteine reactivity in land plants.

Flavonoids are important polyphenolic natural products, ubiquitous in land plants, that play diverse functions in plants' survival in their ecological niches, including UV protection, pigmentation for attracting pollinators, symbiotic nitrogen fixation, and defense against herbivores. Chalcone synthase (CHS) catalyzes the first committed step in plant flavonoid biosynthesis and is highly conserved in all land plants. In several previously reported crystal structures of CHSs from flowering plants, the catalytic cysteine is oxidized to sulfinic acid, indicating enhanced nucleophilicity in this residue associated with its increased susceptibility to oxidation. In this study, we report a set of new crystal structures of CHSs representing all five major lineages of land plants (bryophytes, lycophytes, monilophytes, gymnosperms, and angiosperms), spanning 500 million years of evolution. We reveal that the structures of CHS from a lycophyte and a moss species preserve the catalytic cysteine in a reduced state, in contrast to the cysteine sulfinic acid seen in all euphyllophyte CHS structures. In vivo complementation, in vitro biochemical and mutagenesis analyses, and molecular dynamics simulations identified a set of residues that differ between basal-plant and euphyllophyte CHSs and modulate catalytic cysteine reactivity. We propose that the CHS active-site environment has evolved in euphyllophytes to further enhance the nucleophilicity of the catalytic cysteine since the divergence of euphyllophytes from other vascular plant lineages 400 million years ago. These changes in CHS could have contributed to the diversification of flavonoid biosynthesis in euphyllophytes, which in turn contributed to their dominance in terrestrial ecosystems.

Production of nonnatural straight-chain amino acid 6-aminocaproate via an artificial iterative carbon-chain-extension cycle.

Bioplastics produced from microbial source are promising green alternatives to traditional petrochemical-derived plastics. Nonnatural straight-chain amino acids, especially 5-aminovalerate, 6-aminocaproate and 7-aminoheptanoate are potential monomers for the synthesis of polymeric bioplastics as their primary amine and carboxylic acid are ideal functional groups for polymerization. Previous pathways for 5-aminovalerate and 6-aminocaproate biosynthesis in microorganisms are derived from L-lysine catabolism and the citric acid cycle, respectively. Here, we show the construction of an artificial iterative carbon-chain-extension cycle in Escherichia coli for simultaneous production of a series of nonnatural amino acids with varying chain length. Overexpression of L-lysine α-oxidase in E. coli yields 2-keto-6-aminocaproate (2K6AC) as a non-native substrate for the artificial iterative carbon-chain-extension cycle. The chain-extended α-ketoacid products are decarboxylated and oxidized by an α-ketoacid decarboxylase and an aldehyde dehydrogenase, respectively, to yield their corresponding nonnatural straight-chain amino acids. The engineered system demonstrated simultaneous in vitro production of 99.16 mg/L of 5-aminovalerate, 46.96 mg/L of 6-aminocaproate and 4.78 mg/L of 7-aminoheptanoate after 8 h of enzyme catalysis starting from 2K6AC as the substrate. Furthermore, simultaneous production of 2.15 g/L of 5-aminovalerate, 24.12 mg/L of 6-aminocaproate and 4.74 mg/L of 7-aminoheptanoate was achieved in engineered E. coli. This work illustrates a promising metabolic-engineering strategy to access other medium-chain organic acids with -NH2, -SCH3, -SOCH3, -SH, -COOH, -COH, or -OH functional groups through carbon-chain-elongation chemistry.

Structural and dynamic basis of substrate permissiveness in hydroxycinnamoyltransferase (HCT).

Substrate permissiveness has long been regarded as the raw materials for the evolution of new enzymatic functions. In land plants, hydroxycinnamoyltransferase (HCT) is an essential enzyme of the phenylpropanoid metabolism. Although essential enzymes are normally associated with high substrate specificity, HCT can utilize a variety of non-native substrates. To examine the structural and dynamic basis of substrate permissiveness in this enzyme, we report the crystal structure of HCT from Selaginella moellendorffii and molecular dynamics (MD) simulations performed on five orthologous HCTs from several major lineages of land plants. Through altogether 17-µs MD simulations, we demonstrate the prevalent swing motion of an arginine handle on a submicrosecond timescale across all five HCTs, which plays a key role in native substrate recognition by these intrinsically promiscuous enzymes. Our simulations further reveal how a non-native substrate of HCT engages a binding site different from that of the native substrate and diffuses to reach the catalytic center and its co-substrate. By numerically solving the Smoluchowski equation, we show that the presence of such an alternative binding site, even when it is distant from the catalytic center, always increases the reaction rate of a given substrate. However, this increase is only significant for enzyme-substrate reactions heavily influenced by diffusion. In these cases, binding non-native substrates 'off-center' provides an effective rationale to develop substrate permissiveness while maintaining the native functions of promiscuous enzymes.

Natural product modulators of human sensations and mood: molecular mechanisms and therapeutic potential.

Humans perceive physical information about the surrounding environment through their senses. This physical information is registered by a collection of highly evolved and finely tuned molecular sensory receptors. A multitude of bioactive, structurally diverse ligands have evolved in nature that bind these molecular receptors. The complex, dynamic interactions between the ligands and the receptors lead to changes in our sensory perception or mood. Here, we review our current knowledge of natural products and their derived analogues that interact specifically with human G protein-coupled receptors, ion channels, and nuclear hormone receptors to modulate the sensations of taste, smell, temperature, pain, and itch, as well as mood and its associated behaviour. We discuss the molecular and structural mechanisms underlying such interactions and highlight cases where subtle differences in natural product chemistry produce drastic changes in functional outcome. We also discuss cases where a single compound triggers complex sensory or behavioural changes in humans through multiple mechanistic targets. Finally, we comment on the therapeutic potential of the reviewed area of research and draw attention to recent technological developments in genomics, metabolomics, and metabolic engineering that allow us to tap the medicinal properties of natural product chemistry without taxing nature.

Crystalline-Sponge-Based Structural Analysis of Crude Natural Product Extracts.

Characterization of complex natural product mixtures to the absolute structural level of their components often requires significant amounts of starting materials and lengthy purification process, followed by arduous structure elucidation efforts. The crystalline sponge (CS) method has demonstrated utility in the absolute structure elucidation of isolated organic compounds at miniscule quantities compared to conventional methods. In this work, we developed a new CS-based workflow that greatly expedites the in-depth structural analysis of crude natural product extracts. Using a crude extract of the red alga Laurencia pacifica, we showed that CS affinity screening prior to compound isolation enables prioritization of analytes present in the extract, and we subsequently resolved the molecular structures of six sesquiterpenes with stereochemical clarity from around 10 mg crude extract. This study demonstrates a new chemotyping workflow that can greatly accelerate natural product discovery from complex samples.