Polyketide Biosynthesis Research Articles

Artificial microRNA mediated silencing of cyclase and aldo-keto reductase genes reveal their involvement in the plumbagin biosynthetic pathway.

Plumbagin and other naphthoquinone derivatives from the Plumbago zeylanica L. (Plumbaginaceae) are known for their anticancer and other medicinal properties. Previous reports suggest that 3-methyl-1,8-naphthalene-diol is an intermediate of the plumbagin biosynthetic pathway and is synthesized from hexaketide backbone; a reaction catalyzed by type III polyketide synthase (PKS) along with certain accessory enzymes. Our earlier transcriptomic and metabolomic studies suggest that along with PKS, putative cyclase and aldo-keto reductase might be involved in the formation of 3-methyl-1,8-naphthalene-diol. The present study probed young leaf transcriptome and identified cyclase and aldo-keto reductase like transcripts that might be involved in the intramolecular aldol condensation of hexaketide intermediate and decarboxylation, carbonyl reduction and hydroxyl elimination of keto or enol forms of hexaketide intermediates respectively. Moreover, sequence alignment of identified cyclase1 possesses signature β-α-β-β-α-α-β topology, which belongs to the dimeric α + β barrel (DABB) protein family and is involved in the C2-C11 and C4-C9 intramolecular aldol condensation of hexaketide intermediates. Along with cyclase1, we further identified and characterized P. zeylanica specific aldo-keto reductase1 (AKR1) which is a novel member of the aldo-keto reductase (AKR) multi-gene family that possesses the conserved Asp60, Tyr65, Lys91, and His132 residues and is proposed to be involved in the C1 decarboxylation, C3 carbonyl reduction and C7 hydroxyl elimination of keto or enol form of hexaketide intermediate to form 3-methyl-1,8-naphthalene-diol. Further, the functional characterization using the artificial microRNA mediated transient silencing approach confirmed the involvement of cyclase1 and AKR1 in the plumbagin biosynthetic pathway. This is the first study reporting the identification and functional characterization of cyclase1 and AKR1 genes involved in the plumbagin biosynthetic pathway and general plant polyketide biosynthesis.

Targeted enzyme modifications enable regioselective biosynthesis of fluorinated polyketides

In attempts to enhance natural products as therapeutic agents, fluorination has emerged as a new tool for synthetic biologists and chemists. In recent articles published in Nature Chem. and Nature Chem. Bio., Grininger, Chang, and co-workers leveraged their expertise in engineering polyketide biosynthesis to incorporate fluorine into polyketide scaffolds.

Genome sequencing of Colletotrichum gloeosporioides ES026 reveals plausible pathway of HupA.

Colletotrichum gloeosporioides ES026, isolated as an endophytic fungal strain, was found to produce the important medicinal compound HuperzineA (HupA). In a genetic context, ES026 showed potential in elucidating the biosynthetic pathway of HupA. The ES026 strain was sequenced using de-novo Illumina sequencing methods in this study. Assembling the cleaned data resulted in 58,594,804bp, consisting of 404 scaffolds. The G + C mol % content of this genome was 52.53%. The genome progressive-alignment with other 4 Colletotrichum strains revealed that ES026 showed closer relation with 030206, SMCG1#C and Nara gc5. More than 60 putative biosynthetic clusters were predicted with the fungal version antiSMASH4.0 program. More than 33 types I polyketide-related biosynthetic gene clusters were distributed, containing PKS and PKS-NRPS (polyketide-nonribosomal peptides) hybrid gene clusters. Another 8 NRPS biosynthetic gene clusters were distributed among the genome of ES026. The prenyltransferases, probably involved in aromatic prenyl-compounds and terpenoid biosynthesis, were analyzed using bioinformatics tools like MEGA. We predicted a new possible biosynthetic pathway for the HupA from the pipecolic acid, based on the published HupA biosynthesis proposed pathway, the biosynthesis and pipecolic acid-derived compounds. We hypothesize that a hybrid PKS-NRPS mega-enzyme was probably involved in the biosynthesis of HupA with the pipecolic acid, the building block of rapamycin, as a HupA precursor. The rapamycin is produced from a polyketide biosynthesis pathway, and the domain incorporating the pipecolic acid is studied.

RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus

The thermotolerant yeast Kluyveromyces marxianus has gained significant attention in recent years as a promising microbial candidate for industrial biomanufacturing. Despite several contributions to the expanding molecular toolbox for gene expression and metabolic engineering of K. marxianus, there remains a need for a more efficient and versatile genome editing platform. To address this, we developed a CRISPR-based editing system that enables high efficiency marker-less gene disruptions and integrations using only 40 bp homology arms in NHEJ functional and non-functional K. marxianus strains. The use of a strong RNA polymerase II promoter allows efficient expression of gRNAs flanked by the self-cleaving RNA structures, tRNA and HDV ribozyme, from a single plasmid co-expressing a codon optimized Cas9. Implementing this system resulted in nearly 100% efficiency of gene disruptions in both NHEJ-functional and NHEJ-deficient K. marxianus strains, with donor integration efficiencies reaching 50% and 100% in the two strains, respectively. The high gRNA targeting performance also proved instrumental for selection of engineered strains with lower growth rate but improved polyketide biosynthesis by avoiding an extended outgrowth period, a common method used to enrich for edited cells but that fails to recover advantageous mutants with even slightly impaired fitness. Finally, we provide the first demonstration of simultaneous, markerless integrations at multiple loci in K. marxianus using a 2.6 kb and a 7.6 kb donor, achieving a dual integration efficiency of 25.5% in a NHEJ-deficient strain. These results highlight both the ease of use and general robustness of this system for rapid and flexible metabolic engineering in this non-conventional yeast.

Characterization of Bacillus velezensis RDA1 as a Biological Control Agent against White Root Rot Disease Caused by Rosellinia necatrix.

White root rot disease, caused by Rosellinia necatrix, poses a threat to several tree crops; hence, effective and sustainable strategies to control this disease remain warranted. This study identified an effective R. necatrix biocontrol agent by isolating 32 strains from soil samples collected from white root rot-infested organic pear orchards, among which RDA1 exhibited the most potent growth-inhibitory effects. Microbiological and 16S rRNA gene sequencing analyses revealed that the bacterial isolate belonged to the Bacillus genus and exhibited 100% nucleotide sequence similarity with Bacillus velezensis species in the GenBank. This strain showed strong antifungal activity against four Rosellinia necatrix strains and harbored genes essential for lipopeptide, polyketide, and tripeptide bacilysin biosynthesis. RDA1 produced volatile compounds that suppressed the development of phytopathogens and possessed plant growth-promoting traits, such as phosphate solubilization, and indole-3-acetic acid and siderophore production. B. velezensis RDA1 has a significant potential application in sustainable agriculture and can be used to suppress white root rot disease infections and to improve plant growth.

Genome-Based Analysis of Verticillium Polyketide Synthase Gene Clusters.

Simple SummaryFungi can produce many types of secondary metabolites, including mycotoxins. Poisonous mushrooms and mycotoxins that cause food spoilage have been known for a very long time. For example, Aspergillus flavus, which can grow on grains and nuts, produces highly toxic substances called Aflatoxins. Despite their menace to other living organisms, mycotoxins can be used for medicinal purposes, i.e., as antibiotics, growth-promoting compounds, and other kinds of drugs. These and other secondary metabolites produced by plant-pathogenic fungi may cause host plants to display disease symptoms and may play a substantial role in disease progression. Therefore, the identification and characterization of the genes involved in their biosynthesis are essential for understanding the molecular mechanism involved in their biosynthetic pathways and further promoting sustainable knowledge-based crop production. Polyketides are structurally diverse and physiologically active secondary metabolites produced by many organisms, including fungi. The biosynthesis of polyketides from acyl-CoA thioesters is catalyzed by polyketide synthases, PKSs. Polyketides play roles including in cell protection against oxidative stress, non-constitutive (toxic) roles in cell membranes, and promoting the survival of the host organisms. The genus Verticillium comprises many species that affect a wide range of organisms including plants, insects, and other fungi. Many are known as causal agents of Verticillium wilt diseases in plants. In this study, a comparative genomics approach involving several Verticillium species led us to evaluate the potential of Verticillium species for producing polyketides and to identify putative polyketide biosynthesis gene clusters. The next step was to characterize them and predict the types of polyketide compounds they might produce. We used publicly available sequences from ten species of Verticillium including V. dahliae, V. longisporum, V. nonalfalfae, V. alfalfae, V. nubilum, V. zaregamsianum, V. klebahnii, V. tricorpus, V. isaacii, and V. albo-atrum to identify and characterize PKS gene clusters by utilizing a range of bioinformatic and phylogenetic approaches. We found 32 putative PKS genes and possible clusters in the genomes of Verticillium species. All the clusters appear to be complete and functional. In addition, at least five clusters including putative DHN-melanin-, cytochalasin-, fusarielien-, fujikurin-, and lijiquinone-like compounds may belong to the active PKS repertoire of Verticillium. These results will pave the way for further functional studies to understand the role of these clusters.

Structure of a Promiscuous Thioesterase Domain Responsible for Branching Acylation in Polyketide Biosynthesis.

Thioesterases (TEs) are fundamentally important enzymes present in all bacteria and eukaryotes, where they have conserved functions in fatty acid biosynthesis and secondary metabolism. This work provides the first structural insights into a functionally distinct group of TEs that perform diverse acylations in polyketide and peptide biosynthesis (TEB s). Structural analysis of the oocydin (OocS) TEB domain facilitated identification and engineering of the active site to modulate acyl-group acceptance. In this way, we achieved higher reactivity using a structure-based approach, building a foundation for biocatalytic development of TEB -mediated O-acylation, a modification known to improve the bioactivity of oocydin-type polyketides. Lastly, the promiscuity of the OocS TEB motivated us to investigate, and ultimately provide evidence for, the production of longer chain branched oocydins in the native host Serratia plymuthica 4Rx13. This work frames the OocS TEB and homologs as invaluable synthetic biology tools for polyketide drug development.

Structure of a Promiscuous Thioesterase Domain Responsible for Branching Acylation in Polyketide Biosynthesis

Thioesterases (TEs) are fundamentally important enzymes present in all bacteria and eukaryotes, where they have conserved functions in fatty acid biosynthesis and secondary metabolism. This work provides the first structural insights into a functionally distinct group of TEs that perform diverse acylations in polyketide and peptide biosynthesis (TEBs). Structural analysis of the oocydin (OocS) TEB domain facilitated identification and engineering of the active site to modulate acyl-group acceptance. In this way, we achieved higher reactivity using a structure-based approach, building a foundation for biocatalytic development of TEB-mediated O-acylation, a modification known to improve the bioactivity of oocydin-type polyketides. Lastly, the promiscuity of the OocS TEB motivated us to investigate, and ultimately provide evidence for, the production of longer chain branched oocydins in the native host Serratia plymuthica 4Rx13. This work frames the OocS TEB and homologs as invaluable synthetic biology tools for polyketide drug development.

Enzyme-Catalyzed Spiroacetal Formation in Polyketide Antibiotic Biosynthesis.

A key step in the biosynthesis of numerous polyketides is the stereospecific formation of a spiroacetal (spiroketal). We report here that spiroacetal formation in the biosynthesis of the macrocyclic polyketides ossamycin and oligomycin involves catalysis by a novel spiroacetal cyclase. OssO from the ossamycin biosynthetic gene cluster (BGC) is homologous to OlmO, the product of an unannotated gene from the oligomycin BGC. The deletion of olmO abolished oligomycin production and led to the isolation of oligomycin-like metabolites lacking the spiroacetal structure. Purified OlmO catalyzed complete conversion of the major metabolite into oligomycin C. Crystal structures of OssO and OlmO reveal an unusual 10-strand β-barrel. Three conserved polar residues are clustered together in the β-barrel cavity, and site-specific mutation of any of these residues either abolished or substantially diminished OlmO activity, supporting a role for general acid/general base catalysis in spiroacetal formation.

Pathogenic bacteria remodel central metabolic enzyme to build a cyclopropanol warhead

Bacteria of the Burkholderia pseudomallei (BP) group pose a global health threat, causing the infectious diseases melioidosis, a common cause of pneumonia and sepsis, and glanders, a contagious zoonosis. A trait of BP bacteria is a conserved gene cluster coding for the biosynthesis of polyketides (malleicyprols) with a reactive cyclopropanol unit that is critical for virulence. Enzymes building this warhead represent ideal targets for antivirulence strategies but the biochemical basis of cyclopropanol formation is unknown. Here we describe the formation of the malleicyprol warhead. We show that BurG, an unusual NAD+-dependent member of the ketol-acid reductoisomerase family, constructs the strained cyclopropanol ring. Biochemical assays and a suite of eight crystal structures of native and mutated BurG with bound analogues and inhibitors provide snapshots of each step of the complex reaction mechanism, involving a concealed oxidoreduction and a C–S bond cleavage. Our findings illustrate a remarkable case of neofunctionalisation, where a biocatalyst from central metabolism has been evolutionarily repurposed for warhead production in pathogens.

Polyketide reductases in defense-related parasorboside biosynthesis in Gerbera hybrida share processing strategies with microbial polyketide synthase systems.

Summary Plant polyketides are well‐known for their crucial functions in plants and their importance in the context of human health. They are synthesized by type III polyketide synthases (PKSs) and their final functional diversity is determined by post‐PKS tailoring enzymes. Gerbera hybrida is rich in two defense‐related polyketides: gerberin and parasorboside. Their synthesis is known to be initiated by GERBERA 2‐PYRONE SYNTHASE 1 (G2PS1), but the polyketide reductases (PKRs) that determine their final structure have not yet been identified.We identified two PKR candidates in the pathway, GERBERA REDUCTASE 1 (GRED1) and GRED2. Gene expression and metabolite analysis of different gerbera tissues, cultivars, and transgenic gerbera plants, and in vitro enzyme assays, were performed for functional characterization of the enzymes.GRED1 and GRED2 catalyze the second reduction step in parasorboside biosynthesis. They reduce the proximal keto domain of the linear CoA bound intermediate before lactonization.We identified a crucial tailoring step in an important gerbera PKS pathway and show that plant polyketide biosynthesis shares processing strategies with fungi and bacteria. The two tailoring enzymes are recruited from the ancient sporopollenin biosynthetic pathway to a defense‐related PKS pathway in gerbera. Our data provide an example of how plants recruit conserved genes to new functions in secondary metabolism that are important for environmental adaptation.

A Nonfunctional Halogenase Masquerades as an Aromatizing Dehydratase in Biosynthesis of Pyrrolic Polyketides by Type I Polyketide Synthases.

The bacterial modular type I polyketide synthases (PKSs) typically furnish nonaromatic lactone and lactam natural products. Here, by the complete in vitro enzymatic production of the polyketide antibiotic pyoluteorin, we describe the biosynthetic mechanism for the construction of an aromatic resorcylic ring by a type I PKS. We find that the pyoluteorin type I PKS does not produce an aromatic product, rather furnishing an alicyclic dihydrophloroglucinol that is later enzymatically dehydrated and aromatized. The aromatizing dehydratase is encoded in the pyoluteorin biosynthetic gene cluster (BGC), and its presence is conserved in other BGCs encoding production of pyrrolic polyketides. Sequence similarity and mutational analysis demonstrates that the overall structure and position of the active site for the aromatizing dehydratase is shared with flavin-dependent halogenases albeit with a loss in ability to perform redox catalysis. We demonstrate that the post-PKS dehydrative aromatization is critical for the antibiotic activity of pyoluteorin.

Acetyl‐CoA Electron Density: Acyl‐CoA Reactivity and Crystallographic Data

Acetyl‐CoA and other acyl‐CoAs are the building blocks used in carbon‐carbon bond forming reactions underlying fatty acid and polyketide biosynthesis and many other biological activities. Since many enzymes use acetyl‐CoA, understanding how the substrate binds in the active site is key to determining interactions leading to enzymatic catalysis and substrate selectivity. We examined all of the PDB deposited crystal structures that report acetyl‐CoA bound and report stats for the reactive acetyl‐thioester moiety. We quantified this data by generating the real‐space R‐factor (RSR) and real‐space correlation coefficient (RSCC) by running a few rounds of refinement with Refmac to normalize the data. We examined the modeled thioester ends in comparison to the rest of CoA by creating a Linux script that splits acetyl‐CoA into the nucleic acid, pantetheine, and acetyl‐thioester moieties. We also analyzed the 3’phosphate of acetyl‐CoA to examine how splitting the molecule affects the RSR/RSCC values of small portions of the molecules along with the ribonucleotide containing substrates NAD+, NADH, NADP, and NADPH. Preliminary analysis of the output data reveals the nucleic acid and pantetheine RSR/RSCC values indicate good fits of models to electron density, but there is a portion of structures where RSR/RSCC values indicate the acetyl‐thioester moiety is poorly fit. We also find that the RSR method of calculation shows a significant difference between the acetyl‐CoA thioester and the 3’phosphate where the 3’phosphate is better than the average of the entire structure while the thioester is poorly fit. This is also consistent with the RSR values of NADP and NADPH 3’ phosphates. Conversely, the RSCC values of the acetyl‐CoA thioester end and the 3’phosphate were both equally lower than the entire structure. It appears there is an overfitting of the acetyl‐thioester fitting in crystal structures, likely due to a pressure to claim mechanistic inference from the structures.

Identification of a polyketide biosynthesis gene cluster by transcriptional regulator activation in Aspergillus terreus

In filamentous fungi, the secondary metabolism is environmentally sensitive. Most of the biosynthetic gene clusters of secondary metabolites are silent under laboratory conditions. In this study, a highly conserved naphthopyrone PKS ATEG_06206 was identified in the genome sequence of A. terreus MEFC01. This gene is silent under laboratory conditions. To study the function of this PKS, we activated the silent biosynthetic pathway by replacing the promoter of cluster-specific transcriptional factor ATEG_06205 in A. terreus MEFC01. With this strategy, we confirmed that the products of this cryptic PKS are naphthoquinones. These naphthoquinones are soluble pigments, which could be secreted into the agar medium and culture broth. For this reason, the colour of mycelium and conidia of the activated mutant was significantly darker than that of the parental strain. The gene cluster and biosynthetic pathway were further elucidated through the reverse genetics approach and enzymatic assay in vitro.

Crystal structure of Acetyl-CoA carboxylase (AccB) from Streptomyces antibioticus and insights into the substrate-binding through in silico mutagenesis and biophysical investigations

Acetyl-CoA carboxylase (ACC) is crucial for polyketides biosynthesis and acts as an essential metabolic checkpoint. It is also an attractive drug target against obesity, cancer, microbial infections, and diabetes. However, the lack of knowledge, particularly sequence-structure function relationship to narrate ligand-enzyme binding, has hindered the progress of ACC-specific therapeutics and unnatural “natural” polyketides. Structural characterization of such enzymes will boost the opportunity to understand the substrate binding, designing new inhibitors and information regarding the molecular rules which control the substrate specificity of ACCs. To understand the substrate specificity, we determined the crystal structure of AccB (Carboxyl-transferase, CT) from Streptomyces antibioticus with a resolution of 2.3 Å and molecular modeling approaches were employed to unveil the molecular mechanism of acetyl-CoA recognition and processing. The CT domain of S. antibioticus shares a similar structural organization with the previous structures and the two steps reaction was confirmed by enzymatic assay. Furthermore, to reveal the key hotspots required for the substrate recognition and processing, in silico mutagenesis validated only three key residues (V223, Q346, and Q514) that help in the fixation of the substrate. Moreover, we also presented atomic level knowledge on the mechanism of the substrate binding, which unveiled the terminal loop (500–514) function as an opening and closing switch and pushes the substrate inside the cavity for stable binding. A significant decline in the hydrogen bonding half-life was observed upon the alanine substitution. Consequently, the presented structural data highlighted the potential key interacting residues for substrate recognition and will also help to re-design ACCs active site for proficient substrate specificity to produce diverse polyketides.

A Pair of Atypical KAS III Homologues with Initiation and Elongation Functions Program the Polyketide Biosynthesis in Asukamycin.

β-Ketoacyl-ACP synthase III (KAS III) is a class of important C-C bond-forming enzymes that mostly catalyze the initiation of polyketide and fatty acid biosynthesis. In this study, we elucidated an unusual polyketide synthase (PKS) system that involves two unique KAS IIIs (AsuC3 and C4) in the biosynthesis of the upper triene chain of asukamycin. Significantly, AsuC3 and C4 have both initiation and iterative elongation activity, while being functionally biased toward the elongation and initiation steps, respectively. Mutational analysis revealed that their catalytic activities rely on the catalytic triad Cys-His-Asn. Unlike other KAS IIIs, AsuC3 and C4 are very promiscuous and can accept various lengths of acyl-CoAs with either cyclic, branched or linear acyl moieties. By cooperation with the permissive ketoreductase (AsuC7) and dehydratase (AsuC8/C9), a large variety of polyenes can be efficiently synthesized. This study significantly broadens the understanding of KAS IIIs and polyketide biosynthesis.

A Pair of Atypical KAS III Homologues with Initiation and Elongation Functions Program the Polyketide Biosynthesis in Asukamycin

Abstractβ‐Ketoacyl‐ACP synthase III (KAS III) is a class of important C−C bond‐forming enzymes that mostly catalyze the initiation of polyketide and fatty acid biosynthesis. In this study, we elucidated an unusual polyketide synthase (PKS) system that involves two unique KAS IIIs (AsuC3 and C4) in the biosynthesis of the upper triene chain of asukamycin. Significantly, AsuC3 and C4 have both initiation and iterative elongation activity, while being functionally biased toward the elongation and initiation steps, respectively. Mutational analysis revealed that their catalytic activities rely on the catalytic triad Cys‐His‐Asn. Unlike other KAS IIIs, AsuC3 and C4 are very promiscuous and can accept various lengths of acyl‐CoAs with either cyclic, branched or linear acyl moieties. By cooperation with the permissive ketoreductase (AsuC7) and dehydratase (AsuC8/C9), a large variety of polyenes can be efficiently synthesized. This study significantly broadens the understanding of KAS IIIs and polyketide biosynthesis.

Redirection of acyl donor metabolic flux for lipopeptide A40926B0 biosynthesis.

SummaryThe metabolic flux of fatty acyl‐CoAs determines lipopeptide biosynthesis efficiency, because acyl donor competition often occurs from polyketide biosynthesis and homologous pathways. We used A40926B0 as a model to investigate this mechanism. The lipopeptide A40926B0 with a fatty acyl group is the active precursor of dalbavancin, which is considered as a new lipoglycopeptide antibiotic. The biosynthetic pathway of fatty acyl‐CoAs in the A40926B0 producer Nonomuraea gerenzanensis L70 was efficiently engineered using endogenous replicon CRISPR (erCRISPR). A polyketide pathway and straight‐chain fatty acid biosynthesis were identified as major competitors in the malonyl‐CoA pool. Therefore, we modified both pathways to concentrate acyl donors for the production of the desired compound. Combined with multiple engineering approaches, including blockage of an acetylation side reaction, overexpression of acetyl‐CoA carboxylase, duplication of the dbv gene cluster and optimization of the fermentation parameters, the final strain produced 702.4 mg l‐1 of A40926B0, a 2.66‐fold increase, and the ratio was increased from 36.2% to 81.5%. Additionally, an efficient erCRISPR‐Cas9 editing system based on an endogenous replicon was specifically developed for L70, which increased conjugation efficiency by 660% and gene‐editing efficiency was up to 90%. Our strategy of redirecting acyl donor metabolic flux can be widely adopted for the metabolic engineering of lipopeptide biosynthesis.

Engineered EryF hydroxylase improving heterologous polyketide erythronolide B production in Escherichia coli.

SummaryIn the last two decades, the production of complex polyketides such as erythromycin and its precursor 6‐deoxyerythronolide B (6‐dEB) was demonstrated feasible in Escherichia coli. Although the heterologous production of polyketide skeleton 6‐dEB has reached 210 mg l−1 in E. coli, the yield of its post‐modification products erythromycins remains to be improved. Cytochrome P450EryF catalyses the C6 hydroxylation of 6‐dEB to form erythronolide B (EB), which is the initial rate‐limiting modification in a multi‐step pathway to convert 6‐dEB into erythromycin. Here, we engineered hydroxylase EryF to improve the production of heterologous polyketide EB in E. coli. By comparative analysis of various versions of P450EryFs, we confirmed the optimal SaEryF for the biosynthesis of EB. Further mutation of SaEryF based on the crystal structure of SaEryF and homology modelling of AcEryF and AeEryF afforded the enhancement of EB production. The designed mutant of SaEryF, I379V, achieved the yield of 131 mg l−1 EB, which was fourfold to that produced by wild‐type SaEryF. Moreover, the combined mutagenesis of multiple residues led to further boost the EB concentration by another 41%, which laid the foundation for efficient heterologous biosynthesis of erythromycin or other complex polyketides.

Pseudomonas bijieensis Strain XL17 within the P. corrugata Subgroup Producing 2,4-Diacetylphloroglucinol and Lipopeptides Controls Bacterial Canker and Gray Mold Pathogens of Kiwifruit.

Kiwifruit worldwide suffers from the devastating diseases of bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) and gray mold caused by Botrytis cinerea. Here, an endophytic bacterium XL17 isolated from a rape crown gall was screened out for its potent antagonistic activities against Psa and B. cinerea. Strain XL17 and its cell-free culture filtrate (CF) inhibited the growth of Psa and B. cinerea, Psa-associated leaf necrosis, and B. cinerea-associated kiwifruit necrosis. Electron microscopy showed that XL17 CF could damage the cell structures of Psa and B. cinerea. Genome-based taxonomy revealed that strain XL17 belongs to Pseudomonas bijieensis within the P. corrugata subgroup of the P. fluorescens species complex. Among the P. corrugata subgroup containing 31 genomospecies, the presence of the phl operon responsible for the biosynthesis of the phenolic polyketide 2,4-diacetylphloroglucinol (DAPG) and the absence of the lipopeptide/quorum sensing island can serve as the genetic marker for the determination of a plant-protection life style. HPLC detected DAPG in extracts from XL17 CF. MALDI-TOF-MS analysis revealed that strain XL17 produced cyclic lipopeptides of the viscosin family and orfamide family. Together, phenotypic, genomic, and metabolic analyses identified that P. bijieensis XL17 producing DAPG and cyclic lipopeptides can be used to control bacterial canker and gray mold pathogens of kiwifruit.