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.

The evolutionary origin of naturally occurring intermolecular Diels-Alderases from Morus alba.

Biosynthetic enzymes evolutionarily gain novel functions, thereby expanding the structural diversity of natural products to the benefit of host organisms. Diels-Alderases (DAs), functionally unique enzymes catalysing [4 + 2] cycloaddition reactions, have received considerable research interest. However, their evolutionary mechanisms remain obscure. Here, we investigate the evolutionary origins of the intermolecular DAs in the biosynthesis of Moraceae plant-derived Diels-Alder-type secondary metabolites. Our findings suggest that these DAs have evolved from an ancestor functioning as a flavin adenine dinucleotide (FAD)-dependent oxidocyclase (OC), which catalyses the oxidative cyclisation reactions of isoprenoid-substituted phenolic compounds. Through crystal structure determination, computational calculations, and site-directed mutagenesis experiments, we identified several critical substitutions, including S348L, A357L, D389E and H418R that alter the substrate-binding mode and enable the OCs to gain intermolecular DA activity during evolution. This work provides mechanistic insights into the evolutionary rationale of DAs and paves the way for mining and engineering new DAs from other protein families.

In Vitro Evaluation of the Potential for Drug Interactions by Salidroside.

Several studies utilizing Rhodiola rosea, which contains a complex mixture of phytochemicals, reported some positive drug-drug interaction (DDI) findings based on in vitro CYP450's enzyme inhibition, MAO-A and MAO-B inhibition, and preclinical pharmacokinetic studies in either rats or rabbits. However, variation in and multiplicity of constituents present in Rhodiola products is a cause for concern for accurately evaluating drug-drug interaction (DDI) risk. In this report, we examined the effects of bioengineered, nature-identical salidroside on the inhibition potential of salidroside on CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 utilizing human liver microsomes, the induction potential of salidroside on CYP1A2, CYP2B6 and CYP3A4 in cryopreserved human hepatocytes, the inhibitory potential of salidroside against recombinant human MAO-A and MAO-B, and the OATP human uptake transport inhibitory potential of salidroside using transfected HEK293-OATP1B1 and OATP1B3 cells. The results demonstrate that the bioengineered salidroside at a concentration exceeding the predicted plasma concentrations of <2 µM (based on 60 mg PO) shows no risk for drug-drug interaction due to CYP450, MAO enzymes, or OATP drug transport proteins. Our current studies further support the safe use of salidroside in combination with other drugs cleared by CYP or MAO metabolism or OATP-mediated disposition.

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.

Genomics and biochemical analyses reveal a metabolon key to ß-L-ODAP biosynthesis in Lathyrus sativus.

Grass pea (Lathyrus sativus L.) is a rich source of protein cultivated as an insurance crop in Ethiopia, Eritrea, India, Bangladesh, and Nepal. Its resilience to both drought and flooding makes it a promising crop for ensuring food security in a changing climate. The lack of genetic resources and the crop's association with the disease neurolathyrism have limited the cultivation of grass pea. Here, we present an annotated, long read-based assembly of the 6.5 Gbp L. sativus genome. Using this genome sequence, we have elucidated the biosynthetic pathway leading to the formation of the neurotoxin, ß-L-oxalyl-2,3-diaminopropionic acid (ß-L-ODAP). The final reaction of the pathway depends on an interaction between L. sativus acyl-activating enzyme 3 (LsAAE3) and a BAHD-acyltransferase (LsBOS) that form a metabolon activated by CoA to produce ß-L-ODAP. This provides valuable insight into the best approaches for developing varieties which produce substantially less toxin.

Emergence of a proton exchange-based isomerization and lactonization mechanism in the plant coumarin synthase COSY.

Plants contain rapidly evolving specialized enzymes that support the biosynthesis of functionally diverse natural products. In coumarin biosynthesis, a BAHD acyltransferase-family enzyme COSY was recently discovered to accelerate coumarin formation as the only known BAHD enzyme to catalyze an intramolecular acyl transfer reaction. Here we investigate the structural and mechanistic basis for COSY's coumarin synthase activity. Our structural analyses reveal an unconventional active-site configuration adapted to COSY's specialized activity. Through mutagenesis studies and deuterium exchange experiments, we identify a unique proton exchange mechanism at the α-carbon of the o-hydroxylated trans-hydroxycinnamoyl-CoA substrates during the catalytic cycle of COSY. Quantum mechanical cluster modeling and molecular dynamics further support this key mechanism for lowering the activation energy of the rate-limiting trans-to-cis isomerization step in coumarin production. This study unveils an unconventional catalytic mechanism mediated by a BAHD-family enzyme, and sheds light on COSY's evolutionary origin and its recruitment to coumarin biosynthesis in eudicots.

Safety of a Sustainably Produced, Bioengineered, Nature-Identical Salidroside Compound.

Bioactive phytochemicals such as salidroside have been studied to understand the beneficial effects of Rhodiola rosea, an herbaceous plant used in traditional medicine to increase energy and treat a variety of health issues. However, Rhodiola plants are often slow-growing, and many are endangered in their native habitats. Thus, there is a need for safe, alternative supplies of key phytochemicals from Rhodiola. The salidroside subject of this safety study is a synthetic biology product from fermentation of a bioengineered E. coli that produces salidroside. Here, we present comprehensive test results that support the safety of salidroside manufactured via a patented sustainable bioengineering manufacturing process. In vitro bacterial reverse mutation assays with the bioengineered salidroside show no mutagenicity in any of the concentrations tested. In vivo toxicity studies in rats show no adverse effects from the salidroside product. Based on the results of these studies, we conclude that the bioengineered salidroside discussed here is not genotoxic and demonstrates a no-observed-adverse-effect level (NOAEL) at least 2000 mg/kg bw/day in male and female Sprague-Dawley rats. This study supports that the salidroside compound produced using bioengineered E. coli is a viable alternative to salidroside produced from harvested Rhodiola plants for use as a dietary supplement, food ingredient, or potentially as a pharmaceutical product.

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.

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.

Sporopollenin-inspired design and synthesis of robust polymeric materials.

Sporopollenin is a mechanically robust and chemically inert biopolymer that constitutes the outer protective exine layer of plant spores and pollen grains. Recent investigation of the molecular structure of pine sporopollenin revealed unique monomeric units and inter-unit linkages distinct from other previously known biopolymers, which could be harnessed for new material design. Herein, we report the bioinspired synthesis of a series of sporopollenin analogues. This exercise confirms large portions of our previously proposed pine sporopollenin structural model, while the measured chemical, thermal, and mechanical properties of the synthetic sporopollenins constitute favorable attributes of a new kind of robust material. This study explores a new design framework of robust materials inspired by natural sporopollenins, and provides insights and reagents for future elucidation and engineering of sporopollenin biosynthesis in plants.

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.

Imine chemistry in plant metabolism.

Imine chemistry represents an important category of chemical reactions involved in the biosynthesis of plant natural products, ranging from the newly discovered mobile defense hormone N-hydroxy-pipecolic acid to the red-to-yellow tyrosine-derived betalain pigments. Spontaneous imine formation reactions have also served as the basis for the evolution of numerous plant metabolic enzymes, such as specialized Pictet-Spenglerases that produce the backbone structures of benzylisoquinoline and monoterpene indole alkaloids and pyridoxal 5'-phosphate-dependent enzymes of diverse functions. Here, we review occurrences of imine chemistry in plant metabolism and their chemical and biochemical mechanisms. A better understanding of plant imine chemistry will ultimately facilitate synthetic biology approaches to further expand the scope of imine natural product biosynthesis for broad biotechnological applications.

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.

Evidence for de novo Biosynthesis of the Luminous Substrate Coelenterazine in Ctenophores.

Coelenterazine is a key substrate involved in marine bioluminescence which is used for light-production by at least nine phyla. Some luminous animals, such as the hydromedusa Aequorea, lack the ability to produce coelenterazine endogenously and instead depend on dietary sources. Little is known about the source organisms or the metabolic process of coelenterazine biosynthesis. Here, we present evidence that ctenophores are both producers and suppliers of coelenterazine in marine ecosystems. Using biochemical assays and mass spectrometry analyses, we detected coelenterazine from cultured ctenophores fed with a non-luminous coelenterazine-free diet. We propose that ctenophores are an emerging model organism to study coelenterazine biosynthesis and the origins of bioluminescence.

Precision Delivery of Multiscale Payloads to Tissue-Specific Targets in Plants.

The precise deployment of functional payloads to plant tissues is a new approach to help advance the fundamental understanding of plant biology and accelerate plant engineering. Here, the design of a silk-based biomaterial is reported to fabricate a microneedle-like device, dubbed "phytoinjector," capable of delivering a variety of payloads ranging from small molecules to large proteins into specific loci of various plant tissues. It is shown that phytoinjector can be used to deliver payloads into plant vasculature to study material transport in xylem and phloem and to perform complex biochemical reactions in situ. In another application, it is demonstrated Agrobacterium-mediated gene transfer to shoot apical meristem (SAM) and leaves at various stages of growth. Tuning of the material composition enables the fabrication of another device, dubbed "phytosampler," which is used to precisely sample plant sap. The design of plant-specific biomaterials to fabricate devices for drug delivery in planta opens new avenues to enhance plant resistance to biotic and abiotic stresses, provides new tools for diagnostics, and enables new opportunities in plant engineering.

Partitioning of cancer therapeutics in nuclear condensates.

The nucleus contains diverse phase-separated condensates that compartmentalize and concentrate biomolecules with distinct physicochemical properties. Here, we investigated whether condensates concentrate small-molecule cancer therapeutics such that their pharmacodynamic properties are altered. We found that antineoplastic drugs become concentrated in specific protein condensates in vitro and that this occurs through physicochemical properties independent of the drug target. This behavior was also observed in tumor cells, where drug partitioning influenced drug activity. Altering the properties of the condensate was found to affect the concentration and activity of drugs. These results suggest that selective partitioning and concentration of small molecules within condensates contributes to drug pharmacodynamics and that further understanding of this phenomenon may facilitate advances in disease therapy.