Plant carbonic anhydrase-like enzymes in neuroactive alkaloid biosynthesis.

Plants synthesize numerous alkaloids that mimic animal neurotransmitters1. The diversity of alkaloid structures is achieved through the generation and tailoring of unique carbon scaffolds2,3, yet many neuroactive alkaloids belong to a scaffold class for which no biosynthetic route or enzyme catalyst is known. By studying highly coordinated, tissue-specific gene expression in plants that produce neuroactive Lycopodium alkaloids4, we identified an unexpected enzyme class for alkaloid biosynthesis: neofunctionalized α-carbonic anhydrases (CAHs). We show that three CAH-like (CAL) proteins are required in the biosynthetic route to a key precursor of the Lycopodium alkaloids by catalysing a stereospecific Mannich-like condensation and subsequent bicyclic scaffold generation. Also, we describe a series of scaffold tailoring steps that generate the optimized acetylcholinesterase inhibition activity of huperzine A5. Our findings suggest a broader involvement of CAH-like enzymes in specialized metabolism and demonstrate how successive scaffold tailoring can drive potency against a neurological protein target.

Production of the plant hormone gibberellin by rhizobia increases host legume nodule size.

Plant-associated microbes have evolved the ability to independently produce gibberellin (GA) phytohormones as a mechanism to influence their host. Indeed, GA was first discovered as a metabolite from the fungal rice pathogen Gibberella fujikuroi, which uses it as a virulence factor. Though some bacterial plant pathogens similarly use GA to promote infection, symbiotic nitrogen-fixing bacteria (rhizobia), which inhabit the root nodules of legumes, also can produce GA, suggesting a role in symbiosis. The bacterial GA biosynthetic operon has been identified, but in rhizobia this typically no longer encodes the final metabolic gene (cyp115), so that these symbionts can only produce the penultimate intermediate GA9. Here, we demonstrate that soybean (Glycine max) expresses functional GA 3-oxidases (GA3ox) within its nodules, which have the capability to convert GA9 produced by the enclosed rhizobial symbiont Bradyrhizobium diazoefficiens to bioactive GA4. This rhizobia-derived GA is demonstrated to cause an increase in nodule size and decrease in the number of nodules. The increase in individual nodule size correlates to greater numbers of bacterial progeny within a nodule, thereby providing a selective advantage to rhizobia that produce GA during the rhizobia-legume symbiosis. The expression of GA3ox in nodules and resultant nodulation effects of the GA product suggests that soybean has co-opted control of bioactive GA production, and thus nodule size, for its own benefit. Thus, our results suggest rhizobial GA biosynthesis has coevolved with host plant metabolism for cooperative production of a phytohormone that influences nodulation in a mutually beneficial manner.

Total Biosynthesis of the Tubulin-Binding Alkaloid Colchicine.

Colchicine (1) is a bioactive plant alkaloid from Colchicum and Gloriosa species that is used as a pharmaceutical treatment for inflammatory diseases, including gouty arthritis and familial Mediterranean fever. The activity of this alkaloid is attributed to its ability to bind tubulin dimers and inhibit microtubule assembly, which not only promotes anti-inflammatory effects, but also makes colchicine a potent mitotic poison. The biochemical origins of colchicine biosynthesis have been investigated for over 50 years, but only recently has the underlying enzymatic machinery become clear. Here, we report the discovery of multiple pathway enzymes from Gloriosa superba that allows for the reconstitution of a complete metabolic route to 1. This includes three enzymes that process a previously established tropolone-containing intermediate into 1 via tailoring of the nitrogen atom. We further demonstrate the total biosynthesis of enantiopure (-)-1 from primary metabolites via heterologous production in a model plant, thus enabling future efforts for the metabolic engineering of this medicinal alkaloid. Additionally, our results provide insight into the timing and tissue specificity for the late stage modifications required in colchicine biosynthesis, which are likely connected to the biological functions for this class of medicinal alkaloids in native producing plants.

A metabolic regulon reveals early and late acting enzymes in neuroactive Lycopodium alkaloid biosynthesis.

Plants synthesize many diverse small molecules that affect function of the mammalian central nervous system, making them crucial sources of therapeutics for neurological disorders. A notable portion of neuroactive phytochemicals are lysine-derived alkaloids, but the mechanisms by which plants produce these compounds have remained largely unexplored. To better understand how plants synthesize these metabolites, we focused on biosynthesis of the Lycopodium alkaloids that are produced by club mosses, a clade of plants used traditionally as herbal medicines. Hundreds of Lycopodium alkaloids have been described, including huperzine A (HupA), an acetylcholine esterase inhibitor that has generated interest as a treatment for the symptoms of Alzheimer's disease. Through combined metabolomic profiling and transcriptomics, we have identified a developmentally controlled set of biosynthetic genes, or potential regulon, for the Lycopodium alkaloids. The discovery of this putative regulon facilitated the biosynthetic reconstitution and functional characterization of six enzymes that act in the initiation and conclusion of HupA biosynthesis. This includes a type III polyketide synthase that catalyzes a crucial imine-polyketide condensation, as well as three Fe(II)/2-oxoglutarate-dependent dioxygenase (2OGD) enzymes that catalyze transformations (pyridone ring-forming desaturation, piperidine ring cleavage, and redox-neutral isomerization) within downstream HupA biosynthesis. Our results expand the diversity of known chemical transformations catalyzed by 2OGDs and provide mechanistic insight into the function of noncanonical type III PKS enzymes that generate plant alkaloid scaffolds. These data offer insight into the chemical logic of Lys-derived alkaloid biosynthesis and demonstrate the tightly coordinated coexpression of secondary metabolic genes for the biosynthesis of medicinal alkaloids.

Discovery and engineering of colchicine alkaloid biosynthesis.

Few complete pathways have been established for the biosynthesis of medicinal compounds from plants. Accordingly, many plant-derived therapeutics are isolated directly from medicinal plants or plant cell culture1. A lead example is colchicine, a US Food and Drug Administration (FDA)-approved treatment for inflammatory disorders that is sourced from Colchicum and Gloriosa species2-5. Here we use a combination of transcriptomics, metabolic logic and pathway reconstitution to elucidate a near-complete biosynthetic pathway to colchicine without prior knowledge of biosynthetic genes, a sequenced genome or genetic tools in the native host. We uncovered eight genes from Gloriosa superba for the biosynthesis of N-formyldemecolcine, a colchicine precursor that contains the characteristic tropolone ring and pharmacophore of colchicine6. Notably, we identified a non-canonical cytochrome P450 that catalyses the remarkable ring expansion reaction that is required to produce the distinct carbon scaffold of colchicine. We further used the newly identified genes to engineer a biosynthetic pathway (comprising 16 enzymes in total) to N-formyldemecolcine in Nicotiana benthamiana starting from the amino acids phenylalanine and tyrosine. This study establishes a metabolic route to tropolone-containing colchicine alkaloids and provides insights into the unique chemistry that plants use to generate complex, bioactive metabolites from simple amino acids.

Publisher Correction: Discovery and engineering of colchicine alkaloid biosynthesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Unraveling a Tangled Skein: Evolutionary Analysis of the Bacterial Gibberellin Biosynthetic Operon.

Gibberellin (GA) phytohormones are ubiquitous regulators of growth and developmental processes in vascular plants. The convergent evolution of GA production by plant-associated bacteria, including both symbiotic nitrogen-fixing rhizobia and phytopathogens, suggests that manipulation of GA signaling is a powerful mechanism for microbes to gain an advantage in these interactions. Although orthologous operons encode GA biosynthetic enzymes in both rhizobia and phytopathogens, notable genetic heterogeneity and scattered operon distribution in these lineages, including loss of the gene for the final biosynthetic step in most rhizobia, suggest varied functions for GA in these distinct plant-microbe interactions. Therefore, deciphering GA operon evolutionary history should provide crucial evidence toward understanding the distinct biological roles for bacterial GA production. To further establish the genetic composition of the GA operon, two operon-associated genes that exhibit limited distribution among rhizobia were biochemically characterized, verifying their roles in GA biosynthesis. This enabled employment of a maximum parsimony ancestral gene block reconstruction algorithm to characterize loss, gain, and horizontal gene transfer (HGT) of GA operon genes within alphaproteobacterial rhizobia, which exhibit the most heterogeneity among the bacteria containing this biosynthetic gene cluster. Collectively, this evolutionary analysis reveals a complex history for HGT of the entire GA operon, as well as the individual genes therein, and ultimately provides a basis for linking genetic content to bacterial GA functions in diverse plant-microbe interactions, including insight into the subtleties of the coevolving molecular interactions between rhizobia and their leguminous host plants.IMPORTANCE While production of phytohormones by plant-associated microbes has long been appreciated, identification of the gibberellin (GA) biosynthetic operon in plant-associated bacteria has revealed surprising genetic heterogeneity. Notably, this heterogeneity seems to be associated with the lifestyle of the microbe; while the GA operon in phytopathogenic bacteria does not seem to vary to any significant degree, thus enabling production of bioactive GA, symbiotic rhizobia exhibit a number of GA operon gene loss and gain events. This suggests that a unique set of selective pressures are exerted on this biosynthetic gene cluster in rhizobia. Through analysis of the evolutionary history of the GA operon in alphaproteobacterial rhizobia, which display substantial diversity in their GA operon structure and gene content, we provide insight into the effect of lifestyle and host interactions on the production of this phytohormone by plant-associated bacteria.

A Third Class: Functional Gibberellin Biosynthetic Operon in Beta-Proteobacteria.

The ability of plant-associated microbes to produce gibberellin A (GA) phytohormones was first described for the fungal rice pathogen Gibberella fujikuroi in the 1930s. Recently the capacity to produce GAs was shown for several bacteria, including symbiotic alpha-proteobacteria (α-rhizobia) and gamma-proteobacteria phytopathogens. All necessary enzymes for GA production are encoded by a conserved operon, which appears to have undergone horizontal transfer between and within these two phylogenetic classes of bacteria. Here the operon was shown to be present and functional in a third class, the beta-proteobacteria, where it is found in several symbionts (ß-rhizobia). Conservation of function was examined by biochemical characterization of the enzymes encoded by the operon from Paraburkholderia mimosarum LMG 23256T. Despite the in-frame gene fusion between the short-chain alcohol dehydrogenase/reductase and ferredoxin, the encoded enzymes exhibited the expected activity. Intriguingly, together these can only produce GA9, the immediate precursor to the bioactive GA4, as the cytochrome P450 (CYP115) that catalyzes the final hydroxylation reaction is missing, similar to most α-rhizobia. However, phylogenetic analysis indicates that the operon from ß-rhizobia is more closely related to examples from gamma-proteobacteria, which almost invariably have CYP115 and, hence, can produce bioactive GA4. This indicates not only that ß-rhizobia acquired the operon by horizontal gene transfer from gamma-proteobacteria, rather than α-rhizobia, but also that they independently lost CYP115 in parallel to the α-rhizobia, further hinting at the possibility of detrimental effects for the production of bioactive GA4 by these symbionts.

D2O Labeling to Measure Active Biosynthesis of Natural Products in Medicinal Plants.

Plant natural products have served as a prominent source of medicines throughout human history, and are still used today as clinically-approved pharmaceuticals. However, many medicinal plants that produce useful compounds are slow-growing or recalcitrant to cultivation, making it difficult to investigate the underlying genetic/enzymatic machinery responsible for biosynthesis. To better understand the metabolism of bioactive natural products in slow-growing medicinal plants, we used D2O labeling and LC-MS-based metabolomics to explore the biosynthesis of medically-relevant alkaloids in three plant species. Our results provide evidence for sites of active biosynthesis for these alkaloids, and demonstrate that D2O labeling can be used as a general method to determine sites of active secondary metabolism over relatively short time scales. We anticipate that these results will facilitate discovery of complete metabolic pathways for plant natural products of medicinal importance, especially for approaches that rely upon transcriptomics and knowledge of active metabolism to identify biosynthetic enzymes.

Characterization of CYP115 As a Gibberellin 3-Oxidase Indicates That Certain Rhizobia Can Produce Bioactive Gibberellin A4.

The gibberellin (GA) phytohormones are produced not only by plants but also by fungi and bacteria. Previous characterization of a cytochrome P450 (CYP)-rich GA biosynthetic operon found in many symbiotic, nitrogen-fixing rhizobia led to the elucidation of bacterial GA biosynthesis and implicated GA9 as the final product. However, GA9 does not exhibit hormonal/biological activity and presumably requires further transformation to elicit an effect in the legume host plant. Some rhizobia that contain the GA operon also possess an additional CYP (CYP115), and here we show that this acts as a GA 3-oxidase to produce bioactive GA4 from GA9. This is the first GA 3-oxidase identified for rhizobia, and provides a more complete scheme for biosynthesis of bioactive GAs in bacteria. Furthermore, phylogenetic analyses suggest that rhizobia acquired CYP115 independently of the core GA operon, adding further complexity to the horizontal gene transfer of GA biosynthetic enzymes among bacteria.

An operon for production of bioactive gibberellin A4 phytohormone with wide distribution in the bacterial rice leaf streak pathogen Xanthomonas oryzae pv. oryzicola.

Phytopathogens have developed elaborate mechanisms to attenuate the defense response of their host plants, including convergent evolution of complex pathways for production of the GA phytohormones, which were actually first isolated from the rice fungal pathogen Gibberella fujikuroi. The rice bacterial pathogen Xanthomonas oryzae pv. oryzicola (Xoc) has been demonstrated to contain a biosynthetic operon with cyclases capable of producing the universal GA precursor ent-kaurene. Genetic (knock-out) studies indicate that the derived diterpenoid serves as a virulence factor for this rice leaf streak pathogen, serving to reduce the jasmonic acid-mediated defense response. Here the functions of the remaining genes in the Xoc operon are elucidated and the distribution of the operon in X. oryzae is investigated in over 100 isolates. The Xoc operon leads to production of the bioactive GA4 , an additional step beyond production of the penultimate precursor GA9 mediated by the homologous operons recently characterized from rhizobia. Moreover, this GA biosynthetic operon was found to be widespread in Xoc (> 90%), but absent in the other major X. oryzae pathovar. These results indicate selective pressure for production of GA4 in the distinct lifestyle of Xoc, and the importance of GA to both fungal and bacterial pathogens of rice.

Elucidation of gibberellin biosynthesis in bacteria reveals convergent evolution.

Gibberellins (GAs) are crucial phytohormones involved in many aspects of plant growth and development, including plant-microbe interactions, which has led to GA production by plant-associated fungi and bacteria as well. While the GA biosynthetic pathways in plants and fungi have been elucidated and found to have arisen independently through convergent evolution, little has been uncovered about GA biosynthesis in bacteria. Some nitrogen-fixing, symbiotic, legume-associated rhizobia, including Bradyrhizobium japonicum-the symbiont of soybean-and Sinorhizobium fredii-a broad-host-nodulating species-contain a putative GA biosynthetic operon, or gene cluster. Through functional characterization of five unknown genes, we demonstrate that this operon encodes the enzymes necessary to produce GA9, thereby elucidating bacterial GA biosynthesis. The distinct nature of these enzymes indicates that bacteria have independently evolved a third biosynthetic pathway for GA production. Furthermore, our results also reveal a central biochemical logic that is followed in all three convergently evolved GA biosynthetic pathways.

Labeling Studies Clarify the Committed Step in Bacterial Gibberellin Biosynthesis.

Bacteria have evolved gibberellin phytohormone biosynthesis independently of plants and fungi. Through 13C-labeling and NMR analysis, the mechanistically unusual "B" ring contraction catalyzed by a cytochrome P450 (CYP114), which is the committed step in gibberellin biosynthesis, was shown to occur via oxidative extrusion of carbon-7 from ent-kaurenoic acid in bacteria. This is identical to the convergently evolved chemical transformation in plants and fungi, suggesting a common semipinacol rearrangement mechanism potentially guided by carbon-4α carboxylate proximity.