Coronafacic acid

Total Syntheses of Proposed Structures of 4,10-Dihydroxy-8,12-guaianolides.[Pubmed:35446586]

Org Lett. 2022 May 6;24(17):3297-3301.

The first total syntheses of two 4,10-dihydroxy-8,12-guaianolides that were reported to be natural products were achieved. Toward the syntheses of a collection of related guaianolides, the typical 5,7-fused system of 8,12-guaianolides was constructed by a ring expansion reaction of a hydroxylated Coronafacic acid analogue that can be practically synthesized and optically resolved. The total syntheses of these compounds revealed that the previously reported structures of both natural products were incorrect.

Differential modulation of the lipoxygenase cascade during typical and latent Pectobacterium atrosepticum infections.[Pubmed:34417794]

Ann Bot. 2022 Feb 11;129(3):271-286.

BACKGROUND AND AIMS: Plant diseases caused by Pectobacterium atrosepticum are often accompanied by extensive rot symptoms. In addition, these bacteria are able to interact with host plants without causing disease for long periods, even throughout several host plant generations. There is, to date, no information on the comparative physiology/biochemistry of symptomatic and asymptomatic plant-P. atrosepticum interactions. Typical (symptomatic) P. atrosepticum infections are associated with the induction of plant responses mediated by jasmonates, which are one of the products of the lipoxygenase cascade that gives origin to many other oxylipins with physiological activities. In this study, we compared the functioning of the lipoxygenase cascade following typical and latent (asymptomatic) infections to gain better insight into the physiological basis of the asymptomatic and antagonistic coexistence of plants and pectobacteria. METHODS: Tobacco plants were mock-inoculated (control) or infected with the wild type P. atrosepticum (typical infection) or its Coronafacic acid-deficient mutant (latent infection). The expression levels of the target lipoxygenase cascade-related genes were assessed by Illumina RNA sequencing. Oxylipin profiles were analysed by GC-MS. With the aim of revising the incorrect annotation of one of the target genes, its open reading frame was cloned to obtain the recombinant protein, which was further purified and characterized using biochemical approaches. KEY RESULTS: The obtained data demonstrate that when compared to the typical infection, latent asymptomatic P. atrosepticum infection is associated with (and possibly maintained due to) decreased levels of 9-lipoxygenase branch products and jasmonic acid and increased level of cis-12-oxo-10,15-phytodienoic acid. The formation of 9-oxononanoic acid and epoxyalcohols in tobacco plants was based on the identification of the first tobacco hydroperoxide lyase (HPL) with additional epoxyalcohol synthase (EAS) activity. CONCLUSIONS: Our results contribute to the hypothesis of the oxylipin signature, indicating that different types of plant interactions with a particular pathogen are characterized by the different oxylipin profiles of the host plant. In addition, the tobacco LOC107825278 gene was demonstrated to encode an NtHPL (CYP74C43) enzyme yielding volatile aldehydes and aldoacids (HPL products) as well as oxiranyl carbinols (EAS products).

Discovery, characterization and engineering of ligases for amide synthesis.[Pubmed:34012085]

Nature. 2021 May;593(7859):391-398.

Coronatine and related bacterial phytotoxins are mimics of the hormone jasmonyl-L-isoleucine (JA-Ile), which mediates physiologically important plant signalling pathways(1-4). Coronatine-like phytotoxins disrupt these essential pathways and have potential in the development of safer, more selective herbicides. Although the biosynthesis of coronatine has been investigated previously, the nature of the enzyme that catalyses the crucial coupling of Coronafacic acid to amino acids remains unknown(1,2). Here we characterize a family of enzymes, Coronafacic acid ligases (CfaLs), and resolve their structures. We found that CfaL can also produce JA-Ile, despite low similarity with the Jar1 enzyme that is responsible for ligation of JA and L-Ile in plants(5). This suggests that Jar1 and CfaL evolved independently to catalyse similar reactions-Jar1 producing a compound essential for plant development(4,5), and the bacterial ligases producing analogues toxic to plants. We further demonstrate how CfaL enzymes can be used to synthesize a diverse array of amides, obviating the need for protecting groups. Highly selective kinetic resolutions of racemic donor or acceptor substrates were achieved, affording homochiral products. We also used structure-guided mutagenesis to engineer improved CfaL variants. Together, these results show that CfaLs can deliver a wide range of amides for agrochemical, pharmaceutical and other applications.

Stereoselective Syntheses of all the Possible Stereoisomers of Coronafacic Acid.[Pubmed:33072470]

ChemistryOpen. 2020 Oct 9;9(10):1008-1017.

An efficient and stereoselective syntheses of all the possible stereoisomers of Coronafacic acid (CFA) has been developed. The stereochemistries of C3a and C7a were controlled in a diastereoselective Diels-Alder type cycloaddition using a chiral auxiliary. CFA and 6-epi-CFA were synthesized by hydrogenation of a common intermediate. During the synthesis of 6-epi-CFA, we established that its cis-fused configuration is important for the introduction of C4-C5 double bond by dehydration. This report is the first practical synthesis of both 6-epi-CFA, and its enantiomer.

A scalable synthesis of (+)-coronafacic acid.[Pubmed:31999008]

Chirality. 2020 Apr;32(4):423-430.

A facile, efficient, and scalable synthesis of optically pure Coronafacic acid by resolution of racemic Coronafacic acid obtained using an improved version of Watson's method has been developed. By optimizing the boron-mediated aldol reaction of Watson, we were able to prepare 2.1 g of racemic Coronafacic acid. This was coupled with (S)-4-isopropyl-2-oxazolidinone to give a mixture of diastereomeric coronafacyl oxazolidinones, which were readily separable by silica-gel column chromatography to give 630 mg of optically pure (+)-Coronafacic acid.

Jasmonic Acid Inhibits Auxin-Induced Lateral Rooting Independently of the CORONATINE INSENSITIVE1 Receptor.[Pubmed:29934297]

Plant Physiol. 2018 Aug;177(4):1704-1716.

Plant root systems are indispensable for water uptake, nutrient acquisition, and anchoring plants in the soil. Previous studies using auxin inhibitors definitively established that auxin plays a central role regulating root growth and development. Most auxin inhibitors affect all auxin signaling at the same time, which obscures an understanding of individual events. Here, we report that jasmonic acid (JA) functions as a lateral root (LR)-preferential auxin inhibitor in Arabidopsis (Arabidopsis thaliana) in a manner that is independent of the JA receptor, CORONATINE INSENSITIVE1 (COI1). Treatment of wild-type Arabidopsis with either (-)-JA or (+)-JA reduced primary root length and LR number; the reduction of LR number was also observed in coi1 mutants. Treatment of seedlings with (-)-JA or (+)-JA suppressed auxin-inducible genes related to LR formation, diminished accumulation of the auxin reporter DR5::GUS, and inhibited auxin-dependent DII-VENUS degradation. A structural mimic of (-)-JA and (+)-Coronafacic acid also inhibited LR formation and stabilized DII-VENUS protein. COI1-independent activity was retained in the double mutant of transport inhibitor response1 and auxin signaling f-box protein2 (tir1 afb2) but reduced in the afb5 single mutant. These results reveal JAs and (+)-Coronafacic acid to be selective counter-auxins, a finding that could lead to new approaches for studying the mechanisms of LR formation.

Scalable total synthesis and comprehensive structure-activity relationship studies of the phytotoxin coronatine.[Pubmed:29549326]

Nat Commun. 2018 Mar 16;9(1):1105.

Natural phytotoxins are valuable starting points for agrochemical design. Acting as a jasmonate agonist, coronatine represents an attractive herbicidal lead with novel mode of action, and has been an important synthetic target for agrochemical development. However, both restricted access to quantities of coronatine and a lack of a suitably scalable and flexible synthetic approach to its constituent natural product components, coronafacic and coronamic acids, has frustrated development of this target. Here, we report gram-scale production of Coronafacic acid that allows a comprehensive structure-activity relationship study of this target. Biological assessment of a >120 member library combined with computational studies have revealed the key determinants of potency, rationalising hypotheses held for decades, and allowing future rational design of new herbicidal leads based on this template.

Synthetic biology approaches to establish a heterologous production system for coronatines.[Pubmed:28951265]

Metab Eng. 2017 Nov;44:213-222.

Coronatine (COR) represents a phytotoxin produced by several pathovars of Pseudomonas syringae. It mediates multiple virulence activities by mimicking the plant stress hormone jasmonoyl-l-isoleucine. Structurally, COR consists of a bicyclic polyketide moiety, Coronafacic acid (CFA), which is linked via an amide bond to an unusual ethylcyclopropyl amino acid moiety, coronamic acid (CMA). In our studies, we aimed at establishing and engineering of heterologous COR and CFA production platforms using P. putida KT2440 as host. Based on genetic information of the native producer P. syringae pv. tomato DC3000 a COR biosynthetic gene cluster was designed and reconstituted from synthetic DNA fragments. The applied constructional design facilitated versatile pathway modifications and the generation of various expression constructs, which were evaluated for the production of CFA, COR and its derivatives. By modifications of the gene cluster composition production profiles were directed towards target compounds and valuable information about the function and impact of selected pathway proteins on COR biosynthesis were obtained. Additional engineering of expression vector features, including the use of the constitutive PrpsH promoter and a p15Aori-based transposon backbone, led to the development of an expression strain with promising CFA production yields of > 90mg/l.

Coronafacoyl Phytotoxin Biosynthesis and Evolution in the Common Scab Pathogen Streptomyces scabiei.[Pubmed:28754703]

Appl Environ Microbiol. 2017 Sep 15;83(19):e01169-17.

Coronafacoyl phytotoxins are an important family of plant toxins that are produced by several different phytopathogenic bacteria, including the gammaproteobacterium Pseudomonas syringae and the actinobacterium Streptomyces scabiei (formerly Streptomyces scabies). The phytotoxins consist of Coronafacic acid (CFA) linked via an amide bond to different amino acids or amino acid derivatives. Previous work suggested that S. scabiei and P. syringae use distinct biosynthetic pathways for producing CFA, which is subsequently linked to its amino acid partner to form the complete phytotoxin. Here, we provide further evidence that the S. scabiei CFA biosynthetic pathway is novel by characterizing the role of CYP107AK1, a predicted cytochrome P450 that has no homologue in P. syringae Deletion of the CYP107AK1 gene abolished production of coronafacoyl-isoleucine (CFA-Ile), the primary coronafacoyl phytotoxin produced by S. scabiei Structural elucidation of accumulated biosynthetic intermediates in the DeltaCYP107AK1 mutant indicated that CYP107AK1 is required for introducing the oxygen atom that ultimately forms the carbonyl group in the CFA backbone. The CYP107AK1 gene along with two additional genes involved in CFA-Ile biosynthesis in S. scabiei were found to be associated with putative CFA biosynthetic genes in other actinobacteria but not in other organisms. Analysis of the overall genetic content and organization of known and putative CFA biosynthetic gene clusters, together with phylogenetic analysis of the core biosynthetic genes, indicates that horizontal gene transfer has played an important role in the dissemination of the gene cluster and that rearrangement, insertion, and/or deletion events have likely contributed to the divergent biosynthetic evolution of coronafacoyl phytotoxins in bacteria.IMPORTANCE The ability of plants to defend themselves against invading pathogens relies on complex signaling pathways that are controlled by key phytohormones such as jasmonic acid (JA). Some phytopathogenic bacteria have evolved the ability to manipulate JA signaling in order to overcome host defenses by producing coronatine (COR), which functions as a potent JA mimic. COR and COR-like molecules, collectively referred to as coronafacoyl phytotoxins, are produced by several different plant-pathogenic bacteria, and this study provides supporting evidence that different biosynthetic pathways are utilized by different bacteria for production of these phytotoxins. In addition, our study provides a greater understanding of how coronafacoyl phytotoxin biosynthesis may have evolved in phylogenetically distinct bacteria, and we demonstrate that production of these compounds may be more widespread than previously recognized and that their role for the producing organism may not be limited to host-pathogen interactions.

Pseudomonas syringae pv. tomato DC3000 polymutants deploying coronatine and two type III effectors produce quantifiable chlorotic spots from individual bacterial colonies in Nicotiana benthamiana leaves.[Pubmed:28677296]

Mol Plant Pathol. 2018 Apr;19(4):935-947.

Primary virulence factors of Pseudomonas syringae pv. tomato DC3000 include the phytotoxin coronatine (COR) and a repertoire of 29 effector proteins injected into plant cells by the type III secretion system (T3SS). DC3000 derivatives differentially producing COR, the T3SS machinery and subsets of key effectors were constructed and assayed in leaves of Nicotiana benthamiana. Bacteria were inoculated by the dipping of whole plants and assayed for population growth and the production of chlorotic spots on leaves. The strains fell into three classes. Class I strains are T3SS(+) but functionally effectorless, grow poorly in planta and produce faint chlorotic spots only if COR(+) . Class II strains are T3SS(-) or, if T3SS(+) , also produce effectors AvrPtoB and HopM1. Class II strains grow better than class I strains in planta and, if COR(+) , produce robust chlorotic spots. Class III strains are T3SS(+) and minimally produce AvrPtoB, HopM1 and three other effectors encoded in the P. syringae conserved effector locus. These strains differ from class II strains in growing better in planta, and produce chlorotic spots without COR if the precursor Coronafacic acid is produced. Assays for chlorotic spot formation, in conjunction with pressure infiltration of low-level inoculum and confocal microscopy of fluorescent protein-labelled bacteria, revealed that single bacteria in the apoplast are capable of producing colonies and associated leaf spots in a 1 : 1 : 1 manner. However, COR makes no significant contribution to the bacterial colonization of the apoplast, but, instead, enables a gratuitous, semi-quantitative, surface indicator of bacterial growth, which is determined by the strain's effector composition.

Endogenous Bioactive Jasmonate Is Composed of a Set of (+)-7-iso-JA-Amino Acid Conjugates.[Pubmed:27756820]

Plant Physiol. 2016 Dec;172(4):2154-2164.

Jasmonates (JAs) regulate a wide range of plant defense and development processes. The bioactive JA is perceived by its receptor COI1 to trigger the degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins and subsequently derepress the JAZ-repressed transcription factors for activation of expression of JA-responsive genes. So far, (+)-7-iso-JA-l-Ile has been the only identified endogenous bioactive JA molecule. Here, we designed Coronafacic acid (CFA) conjugates with all the amino acids (CFA-AA) to mimic the JA amino acid conjugates, and revealed that (+)-7-iso-JA-Leu, (+)-7-iso-JA-Val, (+)-7-iso-JA-Met, and (+)-7-iso-JA-Ala are new endogenous bioactive JA molecules. Furthermore, our studies uncover the general characteristics for all the bioactive JA molecules, and provide a new strategy to synthetically generate novel active JA molecules.

Pectobacterium atrosepticum and Pectobacterium carotovorum Harbor Distinct, Independently Acquired Integrative and Conjugative Elements Encoding Coronafacic Acid that Enhance Virulence on Potato Stems.[Pubmed:27065965]

Front Microbiol. 2016 Mar 31;7:397.

Integrative and conjugative elements (ICEs) play a central role in the evolution of bacterial virulence, their transmission between bacteria often leading to the acquisition of virulence factors that alter host range or aggressiveness. Much is known about the functions of the virulence determinants that ICEs harbor, but little is understood about the cryptic effects of ICEs on their host cell. In this study, the importance of horizontally acquired island 2 (HAI2), an ICE in the genome of Pectobacterium atrosepticum SCRI1043, was studied using a strain in which the entire ICE had been removed by CRISPR-Cas-mediated genome editing. HAI2 encodes Coronafacic acid, a virulence factor that enhances blackleg disease of potato stems caused by P. atrosepticum SCRI1043. As expected, deletion of HAI2 resulted in reduced blackleg symptoms in potato stems. A subsequent screen for HAI2-related ICEs in other strains of the Pectobacterium genus revealed their ubiquitous nature in P. atrosepticum. Yet, HAI2-related ICEs were only detected in the genomes of a few P. carotovorum strains. These strains were notable as blackleg causing strains belonging to two different subspecies of P. carotovorum. Sequence analysis of the ICEs in different strains of both P. atrosepticum and P. carotovorum confirmed that they were diverse and were present in different locations on the genomes of their bacterial host, suggesting that the cfa cluster was probably acquired independently on a number of occasions via chromosomal insertion of related ICEs. Excision assays also demonstrated that the ICEs in both P. atrosepticum and P. carotovorum are mobilized from the host chromosome. Thus, the future spread of these ICEs via lateral gene transfer might contribute to an increase in the prevalence of blackleg-causing strains of P. carotovorum.

Production of the Streptomyces scabies coronafacoyl phytotoxins involves a novel biosynthetic pathway with an F420 -dependent oxidoreductase and a short-chain dehydrogenase/reductase.[Pubmed:26991928]

Mol Microbiol. 2016 Jul;101(1):122-35.

Coronafacoyl phytotoxins are secondary metabolites that are produced by various phytopathogenic bacteria, including several pathovars of the Gram-negative bacterium Pseudomonas syringae as well as the Gram-positive potato scab pathogen Streptomyces scabies. The phytotoxins are composed of the polyketide Coronafacic acid (CFA) linked via an amide bond to amino acids or amino acid derivatives, and their biosynthesis involves the cfa and cfa-like gene clusters that are found in P. syringae and S. scabies, respectively. The S. scabies cfa-like gene cluster was previously reported to contain several genes that are absent from the P. syringae cfa gene cluster, including one (oxr) encoding a putative F420 -dependent oxidoreductase, and another (sdr) encoding a predicted short-chain dehydrogenase/reductase. Using gene deletion analysis, we demonstrated that both oxr and sdr are required for normal production of the S. scabies coronafacoyl phytotoxins, and structural analysis of metabolites that accumulated in the Deltasdr mutant cultures revealed that Sdr is directly involved in the biosynthesis of the CFA moiety. Our results suggest that S. scabies and P. syringae use distinct biosynthetic pathways for producing coronafacoyl phytotoxins, which are important mediators of host-pathogen interactions in various plant pathosystems.

Mobilization of horizontally acquired island 2 is induced in planta in the phytopathogen Pectobacterium atrosepticum SCRI1043 and involves the putative relaxase ECA0613 and quorum sensing.[Pubmed:26271942]

Environ Microbiol. 2015 Nov;17(11):4730-44.

Integrative and conjugative elements (ICEs) contribute to the rapid evolution of bacterial pathogens via horizontal gene transfer of virulence determinants. ICEs have common mechanisms for transmission, yet the cues triggering this process under natural environmental or physiological conditions are largely unknown. In this study, mobilization of the putative ICE horizontally acquired island 2 (HAI2), present in the chromosome of the phytopathogen Pectobacterium atrosepticum SCRI1043, was examined during infection of the host plant potato. Under these conditions, mobilization of HAI2 increased markedly compared with in vitro cultures. In planta-induced mobilization of HAI2 was regulated by quorum sensing and involved the putative ICE-encoded relaxase ECA0613. Disruption of ECA0613 also reduced transcription of genes involved in production of Coronafacic acid (Cfa), the major virulence factor harboured on HAI2, whereas their expression was unaffected in the quorum-sensing (expI) mutant. Thus, suppression of cfa gene expression was not regulated by the mobilization of the ICE per se, but was due directly to inactivation of the relaxase. The identification of genetic factors associated solely with in planta mobilization of an ICE demonstrates that this process is highly adapted to the natural environment of the bacterial host and can influence the expression of virulence determinants.

Characterization of the Coronatine-Like Phytotoxins Produced by the Common Scab Pathogen Streptomyces scabies.[Pubmed:25423263]

Mol Plant Microbe Interact. 2015 Apr;28(4):443-54.

Streptomyces scabies is an important causative agent of common scab disease of potato tubers and other root crops. The primary virulence factor produced by this pathogen is a phytotoxic secondary metabolite called thaxtomin A, which is essential for disease development. In addition, the genome of S. scabies harbors a virulence-associated biosynthetic gene cluster called the Coronafacic acid (CFA)-like gene cluster, which was previously predicted to produce metabolites that resemble the Pseudomonas syringae coronatine (COR) phytotoxin. COR consists of CFA linked to an ethylcyclopropyl amino acid called coronamic acid, which is derived from L-allo-isoleucine. Using a combination of genetic and chemical analyses, we show that the S. scabies CFA-like gene cluster is responsible for producing CFA-L-isoleucine as the major product as well as other minor COR-like metabolites. Production of the metabolites was shown to require the cfl gene, which is located within the CFA-like gene cluster and encodes an enzyme involved in ligating CFA to its amino acid partner. CFA-L-isoleucine purified from S. scabies cultures was shown to exhibit bioactivity similar to that of COR, though it was found to be less toxic than COR. This is the first report demonstrating the production of coronafacoyl phytotoxins by S. scabies, which is the most prevalent scab-causing pathogen in North America.