CHLOROPLAST UNUSUAL POSITIONING 1 is a plant-specific actin polymerization factor regulating chloroplast movement.

Plants have unique responses to fluctuating light conditions. One such response involves chloroplast photorelocation movement, which optimizes photosynthesis under weak light by the accumulation of chloroplasts along the periclinal side of the cell, which prevents photodamage under strong light by avoiding chloroplast positioning toward the anticlinal side of the cell. This light-responsive chloroplast movement relies on the reorganization of chloroplast actin (cp-actin) filaments. Previous studies have suggested that CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) is essential for chloroplast photorelocation movement as a regulator of cp-actin filaments. In this study, we conducted comprehensive analyses to understand CHUP1 function. Functional, fluorescently tagged CHUP1 colocalized with and was coordinately reorganized with cp-actin filaments on the chloroplast outer envelope during chloroplast movement in Arabidopsis thaliana. CHUP1 distribution was reversibly regulated in a blue light- and phototropin-dependent manner. X-ray crystallography revealed that the CHUP1-C-terminal domain shares structural homology with the formin homology 2 (FH2) domain, despite lacking sequence similarity. Furthermore, the CHUP1-C-terminal domain promoted actin polymerization in the presence of profilin in vitro. Taken together, our findings indicate that CHUP1 is a plant-specific actin polymerization factor that has convergently evolved to assemble cp-actin filaments and enables chloroplast photorelocation movement.

Sulfur metabolism-mediated fungal glutathione biosynthesis is essential for oxidative stress resistance and pathogenicity in the plant pathogenic fungus Fusarium graminearum.

IMPORTANCE: Fusarium graminearum is a destructive fungal pathogen that causes Fusarium head blight (FHB) on a wide range of cereal crops. To control fungal diseases, it is essential to comprehend the pathogenic mechanisms that enable fungi to overcome host defenses during infection. Pathogens require an oxidative stress response to overcome host-derived oxidative stress. Here, we identify the underlying mechanisms of the Fgbzip007-mediated oxidative stress response in F. graminearum. ChIP-seq and subsequent genetic analyses revealed that the role of glutathione in pathogenesis is not dependent on antioxidant functions in F. graminearum. Altogether, this study establishes a comprehensive framework for the Fgbzip007 regulon on pathogenicity and oxidative stress responses, offering a new perspective on the role of glutathione in pathogenicity.

Biocontrol potential of Chitinophaga flava HK235 producing antifungal-related peptide chitinocin.

Botrytis cinerea is a necrotrophic fungal pathogen with an extremely broad host range, causing significant economic losses in agricultural production. In this study, we discovered a culture filtrate of bacterial strain HK235, which was identified as Chitinophaga flava, exhibiting high levels of antifungal activity against B. cinerea. From the HK235 culture filtrate, we isolated a new antimicrobial peptide molecule designated as chitinocin based on activity-guided fractionation followed by characterization of the amino acid composition and spectroscopic analyses. The HK235 culture filtrate and chitinocin completely inhibited both conidial germination and mycelial growth of B. cinerea at a concentration of 20% and 200 µg/mL, respectively. In addition to antibiosis against B. cinerea, the active compound chitinocin had a broad antifungal and antibacterial activity in vitro. When tomato plants were treated with the culture filtrate and chitinocin, the treatment strongly reduced the development of gray mold disease in a concentration-dependent manner compared to the untreated control. Here, considering the potent antifungal property in vitro and in vivo, we present the biocontrol potential of C. flava HK235 for the first time.

Biological Control Potential of Penicillium brasilianum against Fire Blight Disease.

Erwinia amylovora is a causative pathogen of fire blight disease, affecting apple, pear, and other rosaceous plants. Currently, management of fire blight relies on cultural and chemical practices, whereas it has been known that few biological resources exhibit disease control efficacy against the fire blight. In the current study, we found that an SFC20201208-M01 fungal isolate exhibits antibacterial activity against E. amylovora TS3128, and the isolate was identified as a Penicillium brasilianum based on the ß-tubulin (BenA) gene sequence. To identify active compounds from the P. brasilianum culture, the culture filtrate was partitioned with ethyl acetate and n-butanol sequentially. From the ethyl acetate layer, we identified two new compounds (compounds 3-4) and two known compounds (compounds 1-2) based on spectroscopic analyses and comparison with literature data. Of these active compounds, penicillic acid (1) exhibited promising antibacterial activity against E. amylovora TS3128 with a minimal inhibitory concentration value of 25 µg/ml. When culture filtrate and penicillic acid (125 µg/ml) were applied onto Chinese pearleaf crab apple seedlings prior to inoculation of E. amylovora TS3128, the development of fire blight disease was effectively suppressed in the treated plants. Our results provide new insight into the biocontrol potential of P. brasilianum SFC20201208-M01 with an active ingredient to control fire blight.

Phenyl Ethers from the Marine-Derived Fungus Aspergillus tabacinus and Their Antimicrobial Activity Against Plant Pathogenic Fungi and Bacteria.

Marine fungi produce various secondary metabolites with unique chemical structures and diverse biological activities. In the continuing search for new antifungal agents from fungi isolated from marine environments, the culture filtrate of a fungus Aspergillus tabacinus SFC20160407-M11 exhibited the potential to control plant diseases caused by fungi. From the culture filtrate of A. tabacinus SFC20160407-M11, a total of seven compounds were isolated and identified by activity-guided column chromatography and spectroscopic analysis: violaceol I (1), violaceol II (2), diorcinol (3), versinol (4), orcinol (5), orsellinic acid (6), and sydowiol C (7). Based on in vitro bioassays against 17 plant pathogenic fungi and bacteria, violaceols and diorcinol (1-3) showed a broad spectrum of antimicrobial activity with minimum inhibitory concentration values in the range of 6.3-200 µg mL-1. These compounds also effectively reduced the development of rice blast, tomato late blight, and pepper anthracnose caused by plant pathogenic fungi in a dose-dependent manner. Our results suggest that A. tabacinus SFC20160407-M11 and its phenyl ether compounds could be used for developing new antimicrobial agents to protect crops from plant pathogens.

Curvicollide D, a new modified γ-lactone from the culture broth of Albifimbria verrucaria and its antifungal activity against plant pathogenic fungi.

In our screening program for new antifungal active compounds, a new modified γ-lactone curvicollide D (1) together with five known trichothecenes (2-6) were isolated from the culture filtrate of fungus Albifimbria verrucaria based on the in vitro antifungal assay. The chemical structure of new compound 1 was elucidated by NMR and HR-MS spectroscopic analyses, and the relative configurations of 1 were deduced from NOE experiments and coupling constant analysis. Compound 1 exhibited moderate antifungal activities against plant pathogenic fungi Botrytis cinerea, Colletotrichum coccodes, and Magnaporthe oryzae with MIC value in a range of 100-200 µg ml-1. Moreover, trichothecene compounds (2-6) displayed a broad spectrum of antifungal activities with MIC values in a range of 6.3-100 µg ml-1.

Characterization of Alternaria porri causing onion purple blotch and its antifungal compound magnolol identified from Caryodaphnopsis baviensis.

Alternaria porri (Ellis) Clf. causes purple blotch disease on Allium plants which results in the reduction of crop yields and quality. In this study, to efficiently find natural antifungal compounds against A. porri, we optimized the culture condition for the spore production of A. porri and the disease development condition for an in vivo antifungal assay. From tested plant materials, the methanol extracts derived from ten plant species belonging to the families Cupressaceae, Fabaceae, Dipterocarpaceae, Apocynaceae, Lauraceae, and Melastomataceae were selected as potent antifungal agents against A. porri. In particular, the methanol extract of Caryodaphnopsis baviensis (Lec.) A.-Shaw completely inhibited the growth of A. porri at a concentration of 111 µg/ml. Based on chromatographic and spectroscopic analyses, a neolignan compound magnolol was identified as the antifungal compound of the C. baviensis methanol extract. Magnolol showed a significant inhibitory activity against the spore germination and mycelial growth of A. porri with IC50 values of 4.5 and 5.4 µg/ml, respectively. Furthermore, when magnolol was sprayed onto onion plants at a concentration of 500 µg/ml, it showed more than an 80% disease control efficacy for the purple blotch diseases. In terms of the antifungal mechanism of magnolol, we explored the in vitro inhibitory activity on individual oxidative phosphorylation complexes I-V, and the results showed that magnolol acts as multiple inhibitors of complexes I-V. Taken together, our results provide new insight into the potential of magnolol as an active ingredient with antifungal inhibitory action to control purple blotch on onions.

Clerodane Diterpenoids Identified from Polyalthia longifolia Showing Antifungal Activity against Plant Pathogens.

In the search for new natural resources showing plant disease control effects, we found that the methanol extract of Polyalthia longifolia suppressed fungal disease development in plants. To identify the bioactive substances, the methanol extract of P. longifolia was extracted by organic solvents, and consequently, four new 2-oxo-clerodane diterpenes (1-4), a new 4(3 → 2)-abeo-clerodane diterpene (5), together with ten known compounds (6-16) were isolated and identified from the extracts. Of the new compounds, compound 2 showed a broad spectrum of antifungal activity with moderated minimum inhibitory concentration (MIC) values in a range of 50-100 µg/mL against tested fungal pathogens. Considering with the known compounds, compound 6 showed the most potent antifungal activity with an MIC value in the range of 6.3-12.5 µg/mL. When compound 6 was evaluated for an in vivo antifungal activity against rice blast, tomato late blight, and pepper anthracnose, compound 6 reduced the plant disease by at least 60% compared to the untreated control at concentrations of 250 and 500 µg/mL. Together, our results suggested that the methanol extract of twigs and leaves of P. longifolia and its major compound 6 could be used as a source for the development of eco-friendly plant protection agents.

Plant Disease Control Efficacy of Platycladus orientalis and Its Antifungal Compounds.

Plants contain a number of bioactive compounds that exhibit antimicrobial activity, which can be recognized as an important source of agrochemicals for plant disease control. In searching for natural alternatives to synthetic fungicides, we found that a methanol extract of the plant species Platycladus orientalis suppressed the disease development of rice blast caused by Magnaporthe oryzae. Through a series of chromatography procedures in combination with activity-guided fractionation, we isolated and identified a total of eleven compounds including four labdane-type diterpenes (1-4), six isopimarane-type diterpenes (5-10), and one sesquiterpene (11). Of the identified compounds, the MIC values of compounds 1, 2, 5 & 6 mixture, 9, and 11 ranged from 100 to 200 µg/mL against M. oryzae, whereas the other compounds were over 200 µg/mL. When rice plants were treated with the antifungal compounds, compounds 1, 2, and 9 effectively suppressed the development of rice blast at all concentrations tested by over 75% compared to the non-treatment control. In addition, a mixture of compounds 5 & 6 that constituted 66% of the P. orientalis ethyl acetate fraction also exhibited a moderate disease control efficacy. Together, our data suggest that the methanol extract of P. orientalis including terpenoid compounds has potential as a crop protection agent.

Genome Sequence of Brevibacillus brevis HK544, an Antimicrobial Bacterium Isolated from Soil in Daejeon, South Korea.

The Brevibacillus brevis HK544 strain, which was isolated from soil, exhibited antimicrobial activity against plant pathogens such as Botrytis cinerea, Phytophthora infestans, and Erwinia amylovora. Here, we report the draft genome sequence of the B. brevis HK544 strain, which consists of one circular chromosome of 6,486,246 bp with a GC content of 47.3%.

In Vitro and In Vivo Antifungal Activity of Sorbicillinoids Produced by Trichoderma longibrachiatum.

In the search for antifungal agents from marine resources, we recently found that the culture filtrate of Trichoderma longibrachiatum SFC100166 effectively suppressed the development of tomato gray mold, rice blast, and tomato late blight. The culture filtrate was then successively extracted with ethyl acetate and n-butanol to identify the fungicidal metabolites. Consequently, a new compound, spirosorbicillinol D (1), and a new natural compound, 2',3'-dihydro-epoxysorbicillinol (2), together with 11 known compounds (3-13), were obtained from the solvent extracts. The chemical structures were determined by spectroscopic analyses and comparison with literature values. The results of the in vitro antifungal assay showed that of the tested fungal pathogens, Phytophthora infestans was the fungus most sensitive to the isolated compounds, with MIC values ranging from 6.3 to 400 µg/mL, except for trichotetronine (9) and trichodimerol (10). When tomato plants were treated with the representative compounds (4, 6, 7, and 11), bisvertinolone (6) strongly reduced the development of tomato late blight disease compared to the untreated control. Taken together, our results revealed that the culture filtrate of T. longibrachiatum SFC100166 and its metabolites could be useful sources for the development of new natural agents to control late blight caused by P. infestans.

Biocontrol Potential of Aspergillus Species Producing Antimicrobial Metabolites.

Microbial metabolites have been recognized as an important source for the discovery of new antifungal agents because of their diverse chemical structures with novel modes of action. In the course of our screening for new antifungal agents from microbes, we found that culture filtrates of two fungal species Aspergillus candidus SFC20200425-M11 and Aspergillus montenegroi SFC20200425-M27 have the potentials to reduce the development of fungal plant diseases such as tomato late blight and wheat leaf rust. From these two Aspergillus spp., we isolated a total of seven active compounds, including two new compounds (4 and 6), and identified their chemical structures based on the NMR spectral analyses: sphaeropsidin A (1), (R)-formosusin A (2), (R)-variotin (3), candidusin (4), asperlin (5), montenegrol (6), and protulactone A (7). Based on the results of the in vitro bioassays of 11 plant pathogenic fungi and bacteria, sphaeropsidin A (1), (R)-formosusin A (2), (R)-variotin (3), and asperlin (5) exhibited a wide range of antimicrobial activity. Furthermore, when plants were treated with sphaeropsidin A (1) and (R)-formosusin A (2) at a concentration of 500 µg/ml, sphaeropsidin A (1) exhibited an efficacy disease control value of 96 and 90% compared to non-treated control against tomato late blight and wheat leaf rust, and (R)-formosusin A (2) strongly reduced the development of tomato gray mold by 82%. Asperlin (5) at a concentration of 500 µg/ml effectively controlled the development of tomato late blight and wheat leaf rust with a disease control value of 95%. Given that culture filtrates and active compounds derived from two Aspergillus spp. exhibited disease control efficacies, our results suggest that the Aspergillus-produced antifungal compounds could be useful for the development of new natural fungicides.

The Antifungal Test: An Efficient Screening Tool for the Discovery of Microbial Metabolites with Respiratory Inhibitory Activity.

Valuable natural compounds produced by a variety of microorganisms can be used as lead molecules for development of new agrochemicals. Furthermore, high-throughput in vitro screening systems with specific modes of action can increase the probability of discovery of new fungicides. In the current study, a rapid assay tested with various microbes was developed to determine the degree of respiratory inhibition of Saccharomyces cerevisiae in two different liquid media, YG (containing a fermentable carbon source) and NFYG (containing a non-fermentable carbon source). Based on this system, we screened 100 fungal isolates that were classified into basidiomycetes, to find microbial secondary metabolites that act as respiratory inhibitors. Consequently, of the 100 fungal species tested, the culture broth of an IUM04881 isolate inhibited growth of S. cerevisiae in NFYG medium, but not in YG medium. The result is comparable to that from treatment with kresoxim-methyl used as a control, suggesting that the culture broth of IUM04881 isolate might contain active compounds showing the inhibition activity for respiratory chain. Based on the assay developed in this study and spectroscopic analysis, we isolated and identified an antifungal compound (-)-oudemansin A from culture broth of IUM04881 that is identified as Oudemansiella venosolamellata. This is the first report that (-)-oudemansin A is identified from O. venosolamellata in Korea. Taken together, the development of this assay will accelerate efforts to find and identify natural respiratory inhibitors from various microbes.

Transcription Factor PdeR Is Involved in Fungal Development, Metabolic Change, and Pathogenesis of Gray Mold Botrytis cinerea.

The necrotrophic fungus Botrytis cinerea releases extracellular enzymes that facilitate its penetration into a host. This study functionally characterized the gene pdeR of B. cinerea, which is predicted to encode a Zn(II)2Cys6 zinc finger transcription factor. To investigate the role of pdeR, deleted and complemented strains of pdeR in B. cinerea were generated, which were designated as ΔpdeR and PdeRc, respectively. The ΔpdeR strain exhibited impaired germination and growth compared to the wild-type and PdeRc strains, particularly when provided with maltose as the sole carbon source. When all of the strains were grown on a minimal medium containing polysaccharide as the sole carbon source, the ΔpdeR exclusively showed defects in polysaccharide hydrolysis with reduced gene expression encoding for amylase and cellulase. As far as the involvement of pdeR in carbon metabolism is concerned, metabolic changes were investigated in the ΔpdeR mutant. Comparisons of relative, normalized concentrations of each metabolite showed that the amounts of six metabolites including glucose and trehalose were significantly changed in the ΔpdeR strain. Based on pleiotropic changes derived from the deletion of pdeR, we hypothesized that pdeR has an important role in pathogenesis. When the ΔpdeR strain was inoculated onto pepper plant, the ΔpdeR strain did not cause expansion of the disease lesions from the infection sites, which grew on the surface without any penetration. Taken together, these results show that the deletion of pdeR affected the extracellular enzymatic activity, leading to changes in fungal development, metabolism, and virulence.

Discovery of New Triterpenoid Saponins Isolated from Maesa japonica with Antifungal Activity against Rice Blast Fungus Magnaporthe oryzae.

While searching for new antifungal compounds, we revealed that a methanol extract of plant species Maesa japonica has a potent antifungal activity in vivo against rice blast fungus Magnaporthe oryzae. To identify the antifungal substances, the methanol extract of M. japonica was extracted by organic solvents, and consequently, six active compounds were isolated from the n-butanol layer. The isolated compounds were five new acylated triterpenoid saponins including maejaposide I (1), maejaposides C-1, C-2, and C-3 (2-4), and maejaposide A-1 (5), along with a known one, maejaposide A (6). These chemical structures were determined by NMR and a comparison of their NMR and MS data with those reported in the literature. Based on the in vitro antifungal bioassay, the five compounds (2-6) exhibited strong antifungal activity against M. oryzae with MIC values ranging from 4 to 32 µg/mL, except for maejaposide I (1) (MIC > 250 µg/mL). When the compounds were evaluated at concentrations of 125, 250, and 500 µg/mL for an in vivo antifungal activity against rice blast, compounds 2-6 strongly reduced the development of blast by at least 85% to 98% compared to the untreated control. However, compound 1 did not show any in vivo antifungal activity up to a concentration of 500 µg/mL. Taken together, our results suggest that the methanol extract of M. japonica and the new acylated triterpenoid saponins can be used as a source for the development of natural fungicides.

Antimicrobial aromatic polyketides: a review of their antimicrobial properties and potential use in plant disease control.

Aromatic polyketides are secondary metabolites widely found in bacteria, fungi, and plants, which are well-known for their diverse chemical structures and biological functions. The structural diversity of aromatic polyketides arises from a series of enzymatic modifications of the linear poly-ß-ketone intermediates during biosynthesis. Their versatile bioactivities are exemplified by reports of their use as antibacterials, antifungals, antivirals, and antiparasitics. Despite many reports on the antimicrobial nature of aromatic polyketides, their potential use as plant disease control agents has still not been systematically explored and discussed. This review highlights examples of the use of aromatic polyketides as plant disease control agents and discusses their function and merits as agrochemicals.

Crinipellins A and I, Two Diterpenoids from the Basidiomycete Fungus Crinipellis rhizomaticola, as Potential Natural Fungicides.

In the course of screening for microbes with antifungal activity, we found that the culture filtrate of the IUM00035 isolate exhibited strong antifungal activity against Magnaporthe oryzae and Colletotrichum coccodes in planta. Based on the phylogenetic analysis with the ITS region, the IUM00035 isolate was identified as Crinipellis rhizomaticola. To identify antifungal compounds from the C. rhizomaticola IUM00035 isolate, the culture filtrate of the isolate was partitioned with ethyl acetate and n-butanol and, consequently, two active compounds were isolated from the ethyl acetate extract. The chemical structures of the isolated compounds were determined as crinipellin A (1) and a new crinipellin derivative, crinipellin I (2), by NMR spectral analyses and a comparison of their NMR and MS data with those reported in the literature. Crinipellin A (1) exhibited a wide range of antifungal activity in vitro against C. coccodes, M. oryzae, Botrytis cinerea, and Phytophthora infestans (MICs = 1, 8, 31, and 31 µg/mL, respectively). Furthermore, when plants were treated with crinipellin A (1) (500 µg/mL) prior to inoculation with fungal pathogens, crinipellin A (1) exhibited disease control values of 88%, 65%, and 60% compared with non-treatment control against tomato late blight, pepper anthracnose, and wheat leaf rust, respectively. In contrast to crinipellin A (1), crinipellin I (2) showed weak or no activity (MICs > 250 µg/mL). Taken together, our results show that the C. rhizomaticola IUM00035 isolate suppresses the development of plant fungal diseases, in part through the production of crinipellin A (1).

Identification of novel compounds, oleanane- and ursane-type triterpene glycosides, from Trevesia palmata: their biocontrol activity against phytopathogenic fungi.

Plants contain a number of bioactive compounds that exhibit antimicrobial activity, which can be recognized as an important source of agrochemicals for plant disease control. As part of our search for new antimicrobial agents from natural sources, we found that a crude methanol extract of Trevesia palmata exhibited a promising antifungal activity against phytopathogenic fungi, such as Magnaporthe oryzae and Botrytis cinerea. Furthermore, based on activity-guided fractionation, we isolated five antifungal compounds from the methanol extract of T. palmata: two new triterpene glycosides (TPGs), TPG1 (hederagenin-3-O-ß-D-glucopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranoside) and TPG5 (3-O-α-L-rhamnopyranosyl asiatic acid), along with three known TPGs (TPG2 [macranthoside A], TPG3 [α-hederin], and TPG4 [ilekudinoside D]). The chemical structures of the TPGs were determined by spectroscopic analyses and by comparison with literature data. An in vitro antifungal bioassay revealed that except for TPG4 (ilekudinoside D; IC50 >256 µg/ml), the other TPGs exhibited strong antifungal activities against the rice blast pathogen M. oryzae with IC50 values ranging from 2-5 µg/ml. In particular, when the plants were treated with compound TPG1 (500 µg/ml), disease control values against rice blast, tomato grey mold, tomato late blight, and wheat leaf rust were 84, 82, 88, and 70%, respectively, compared to the non-treatment control. Considering the in vitro and in vivo antifungal activities of the TPGs and the T. palmata methanol extracts, our results suggest that T. palmata can be a useful source to develop new natural fungicides.

Nematicidal and Plant Growth-Promoting Activity of Enterobacter asburiae HK169: Genome Analysis Provides Insight into Its Biological Activities.

In the course of screening for microbes with nematicidal activity, we found that Enterobacter asburiae HK169 displayed promising nematicidal activity against the root-knot nematode Meloidogyne incognita, along with plant growth-promoting properties. Soil drenching of a culture of HK169 reduced gall formation by 66% while also increasing root and shoot weights by 251% and 160%, respectively, compared with an untreated control. The cell-free culture filtrate of the HK169 culture killed all juveniles of M. incognita within 48 h. In addition, the nematicidal activity of the culture filtrate was dramatically reduced by a protease inhibitor, suggesting that proteolytic enzymes contribute to the nematicidal activity of HK169. In order to obtain genomic information about the HK169 isolate related to its nematicidal and plant growth-promoting activities, we sequenced and analyzed the whole genome of the HK169 isolate, and the resulting information provided evidence that the HK169 isolate has nematicidal and plant growth-promoting activities. Taken together, these observations enable the future application of E. asburiae HK169 as a biocontrol agent for nematode control and promote our understanding of the beneficial interactions between E. asburiae HK169 and plants.

The chejuenolide biosynthetic gene cluster harboring an iterative trans-AT PKS system in Hahella chejuensis strain MB-1084.

Hahella chejuensis MB-1084 is a Gram-negative marine bacterial strain that produces unusual 17-membered carbocyclic tetraenes, chejuenolide A and B. Two fosmid clones responsible for chejuenolide production were identified from the genomic DNA library of the MB-1084 strain. Systematic inactivation of the open reading frames (ORFs) in the sequenced region defines the boundaries of the chejuenolide (che) biosynthetic gene cluster (24.9 kbp) that encodes one non-ribosomal peptide synthase (NRPS)-polyketide synthase (PKS) hybrid protein, three modular PKSs, two PKS domains, and an amine oxidase homolog. Based on the results, we found that the che PKSs have non-canonical features such as trans-AT system and insufficient number of KS domains (five KS domains) for chejuenolide production (requires eight rounds of Claisen condensation reaction). Heterologous expression of the che PKSs in the E. coli BAP1 strain provides strong evidence of the iterative characteristic of the modular PKSs. Additionally, the phylogenetic relatedness of the KS domains of che PKSs and other trans-AT PKSs was analyzed to propose a possible pathway for chejuenolide biosynthesis.