Genome Mining Methods Research Articles

Mining the Biosynthetic Landscape of Lactic Acid Bacteria Unearths a New Family of RiPPs Assembled by a Novel Type of ThiF-like Adenylyltransferases.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are chemically diverse natural products of ribosomal origin. These peptides, which frequently act as signals or antimicrobials, are biosynthesized by conserved enzymatic machinery, making genome mining a powerful strategy for unearthing previously uncharacterized members of their class. Herein, we investigate the untapped biosynthetic potential of Lactobacillales (i.e., lactic acid bacteria), an order of Gram-positive bacteria closely associated with human life, including pathogenic species and industrially relevant fermenters of dairy products. Through genome mining methods, we systematically explored the distribution and diversity of ThiF-like adenylyltransferase-utilizing RiPP systems in lactic acid bacteria and identified a number of unprecedented biosynthetic gene clusters. In one of these clusters, we found a previously undescribed group of macrocyclic imide biosynthetic pathways containing multiple transporters that may be involved in a potential quorum sensing (QS) system. Through in vitro assays, we determined that one such adenylyltransferase specifically catalyzes the intracyclization of its precursor peptide through macrocyclic imide formation. Incubating the enzyme with various primary amines revealed that it could effectively amidate the C-terminus of the precursor peptide. This new transformation adds to the growing list of Nature's peptide macrocyclization strategies and expands the impressive catalytic repertoire of the adenylyltransferase family. The diverse RiPP systems identified herein represent a vast, unexploited landscape for the discovery of a novel class of natural products and QS systems.

NRPS-like ATRR in Plant-Parasitic Nematodes Involved in Glycine Betaine Metabolism to Promote Parasitism.

Plant-parasitic nematodes (PPNs) are among the most serious phytopathogens and cause widespread and serious damage in major crops. In this study, using a genome mining method, we identified nonribosomal peptide synthetase (NRPS)-like enzymes in genomes of plant-parasitic nematodes, which are conserved with two consecutive reducing domains at the N-terminus (A-T-R1-R2) and homologous to fungal NRPS-like ATRR. We experimentally investigated the roles of the NRPS-like enzyme (MiATRR) in nematode (Meloidogyne incognita) parasitism. Heterologous expression of Miatrr in Saccharomyces cerevisiae can overcome the growth inhibition caused by high concentrations of glycine betaine. RT-qPCR detection shows that Miatrr is significantly upregulated at the early parasitic life stage (J2s in plants) of M. incognita. Host-derived Miatrr RNA interference (RNAi) in Arabidopsis thaliana can significantly decrease the number of galls and egg masses of M. incognita, as well as retard development and reduce the body size of the nematode. Although exogenous glycine betaine and choline have no obvious impact on the survival of free-living M. incognita J2s (pre-parasitic J2s), they impact the performance of the nematode in planta, especially in Miatrr-RNAi plants. Following application of exogenous glycine betaine and choline in the rhizosphere soil of A. thaliana, the numbers of galls and egg masses were obviously reduced by glycine betaine but increased by choline. Based on the knowledge about the function of fungal NRPS-like ATRR and the roles of glycine betaine in host plants and nematodes, we suggest that MiATRR is involved in nematode-plant interaction by acting as a glycine betaine reductase, converting glycine betaine to choline. This may be a universal strategy in plant-parasitic nematodes utilizing NRPS-like ATRR to promote their parasitism on host plants.

Genome Mining for New Enzyme Chemistry.

A revolution in the field of biocatalysis has enabled scalable access to compounds of high societal values using enzymes. The construction of biocatalytic routes relies on the reservoir of available enzymatic transformations. A review of uncharacterized proteins predicted from genomic sequencing projects shows that a treasure trove of enzyme chemistry awaits to be uncovered. This Review highlights enzymatic transformations discovered through various genome mining methods and showcases their potential future applications in biocatalysis.

Structure Prediction and Genome Mining-Aided Discovery of the Bacterial C-Terminal Tryptophan Prenyltransferase PalQ.

Post-translational prenylations, found in eukaryotic primary metabolites and bacterial secondary metabolites, play crucial roles in biomolecular interactions. Employing genome mining methods combined with AlphaFold2-based predictions of protein interactions, PalQ , a prenyltransferase responsible for the tryptophan prenylation of RiPPs produced by Paenibacillus alvei, is identified. PalQ differs from cyanobactin prenyltransferases because of its evolutionary relationship to isoprene synthases, which enables PalQ to transfer extended prenyl chains to the indole C3position. This prenylation introduces structural diversity to the tryptophan side chain and also leads to conformational dynamics in the peptide backbone, attributed to the cis/trans isomerization that arises from the formation of a pyrrolidine ring. Additionally, PalQ exhibited pronounced positional selectivity for the C-terminal tryptophan. Such enzymatic characteristics offer a toolkit for peptide therapeutic lipidation.

FunARTS, the Fungal bioActive compound Resistant TargetSeeker, an exploration engine for target-directed genome mining in fungi.

There is an urgent need to diversify the pipeline for discovering novel natural products due to the increase in multi-drug resistant infections. Like bacteria, fungi also produce secondary metabolites that have potent bioactivity and rich chemical diversity. To avoid self-toxicity, fungi encode resistance genes which are often present within the biosynthetic gene clusters (BGCs) of the corresponding bioactive compounds. Recent advances in genome mining tools have enabled the detection and prediction of BGCs responsible for the biosynthesis of secondary metabolites. The main challenge now is to prioritize the most promising BGCs that produce bioactive compounds with novel modes of action. With target-directed genome mining methods, it is possible to predict the mode of action of a compound encoded in an uncharacterized BGC based on the presence of resistant target genes. Here, we introduce the 'fungal bioactive compound resistant target seeker' (FunARTS) available at https://funarts.ziemertlab.com. This is a specific and efficient mining tool for the identification of fungal bioactive compounds with interesting and novel targets. FunARTS rapidly links housekeeping and known resistance genes to BGC proximity and duplication events, allowing for automated, target-directed mining of fungal genomes. Additionally, FunARTS generates gene cluster networking by comparing the similarity of BGCs from multi-genomes.

Discovery of new microbial natural products by genome mining

Microbial natural products are an important source of new drug creation. As more and more microbial whole genomes are sequenced, bioinformatics analysis shows that the natural prod-uct resources contained therein are greatly underestimated. Genome mining is a new strategy for natural product discovery guided by gene cluster sequences. At the same time, it can di-rectly associate the structure of natural products with synthesis pathways, which is conven-ient for biosynthesis and combinatorial biosynthesis research. This paper reviews the repre-sentative examples of successful discovery of novel natural products using genome mining strategy in recent years, which fully shows the great advantage of this strategy in exploring the potential of microbial natural metabolites. It is believed that with the continuous devel-opment and improvement of genome mining methods and technologies, it will be possible to realize the in-depth development of microbial natural product resources.This paper describes the application of combinatorial biosynthesis in new drugs, points out the existing problems and puts forward some feasible suggestions.

Genome mining of potential enzyme from Chelatococcus composti for sustainable production of D-Allulose

A novel putative D-allulose 3-epimerase (DAEase) named CcDAE with the accession number of WP_183335203.1 was discovered in this study from Chelatococcus composti genome using in silico genome mining method. The sequence, which was retrieved from the genome of Chelatococcus composti strain CGMCC 1.15283 and became the first reported D-allulose 3-epimerase from the species. The sequence length of CcDAE was 282 aa with a molecular weight prediction of 30.504 kDa. The sequence analysis disclosed a high sequence conservation at the residues building the metal binding site and substrate binding site. The phylogenetic tree also disclosed that the closest related sequence with CcDAE was from Cereibacter spaeroides. The biochemical prediction also informed that CcDAE had an isoelectric point (pI) at pH 5.74. In addition, the novel putative enzyme was predicted to withstand a high temperature up to 65 °C and was considered as a stable protein. Therefore, the research finding suggests that CcDAE was potential for further exploration.

The microbial dark matter and “wanted list” in worldwide wastewater treatment plants

BackgroundWastewater treatment plants (WWTPs) are one of the largest biotechnology applications in the world and are of critical importance to modern urban societies. An accurate evaluation of the microbial dark matter (MDM, microorganisms whose genomes remain uncharacterized) proportions in WWTPs is of great value, while there is no such research yet. This study conducted a global meta-analysis of MDM in WWTPs with 317,542 prokaryotic genomes from the Genome Taxonomy Database and proposed a “wanted list” for priority targets in further investigations of activated sludge.ResultsCompared with the Earth Microbiome Project data, WWTPs had relatively lower genome-sequenced proportions of prokaryotes than other ecosystems, such as the animal related environments. Analysis showed that the median proportions of the genome-sequenced cells and taxa (100% identity and 100% coverage in 16S rRNA gene region) in WWTPs reached 56.3% and 34.5% for activated sludge, 48.6% and 28.5% for aerobic biofilm, and 48.3% and 28.5% for anaerobic digestion sludge, respectively. This result meant MDM had high proportions in WWTPs. Besides, all of the samples were occupied by a few predominant taxa, and the majority of the sequenced genomes were from pure cultures. The global-scale “wanted list” for activated sludge contained four phyla that have few representatives and 71 operational taxonomic units with the majority of them having no genome or isolate yet. Finally, several genome mining methods were verified to successfully recover genomes from activated sludge such as hybrid assembly of the second- and third-generation sequencing.ConclusionsThis work elucidated the proportion of MDM in WWTPs, defined the “wanted list” of activated sludge for future investigations, and certified potential genome recovery methods. The proposed methodology of this study can be applied to other ecosystems and improve understanding of ecosystem structure across diverse habitats.Ed3xU4yrM6Da7r9rFgJoViVideo

Bioprospecting of actinomycetes for anti-bacterial potential: A Review

Actinomycetes are gram-positive bacteria that have a high GC content compared to their counterparts. Actinomycetes were capable of producing a broad range of secondary metabolites and many of these were anti-bacterial agents. Antibiotics have been known to play a vital role in therapeutics and the clinical management of various diseases. Actinomycetes have contributed more than 70% of known antibiotics in the market today. Apart from antibiotics, actinomycetes also produce several enzymes, enzyme inhibitors, anticancer and anti-diabetic secondary metabolites. Currently, actinomycetes are being studied for medicinal, pharmaceutical, industrial, environmental and bioremediation applications. The current study focuses on a variety of antibacterial agents derived from actinomycetes and new antibiotics under clinical trial along with their mode of action. It also describes the genome mining methods for screening actinomycetes isolates for biosynthetic gene clusters and specific antibacterial secondary metabolites.

Proteomining-Based Elucidation of Natural Product Biosynthetic Pathways in Streptomyces

The genus Streptomyces is known to harbor numerous biosynthetic gene clusters (BGCs) of potential utility in synthetic biology applications. However, it is often difficult to link uncharacterized BGCs with the secondary metabolites they produce. Proteomining refers to the strategy of identifying active BGCs by correlating changes in protein expression with the production of secondary metabolites of interest. In this study, we devised a shotgun proteomics-based workflow to identify active BGCs during fermentation when a variety of compounds are being produced. Mycelia harvested during the non-producing growth phase served as the background. Proteins that were differentially expressed were clustered based on the proximity of the genes in the genome to highlight active BGCs systematically from label-free quantitative proteomics data. Our software tool is easy-to-use and requires only 1 point of comparison where natural product biosynthesis was significantly different. We tested our proteomining clustering method on three Streptomyces species producing different compounds. In Streptomyces coelicolor A3(2), we detected the BGCs of calcium-dependent antibiotic, actinorhodin, undecylprodigiosin, and coelimycin P1. In Streptomyces chrestomyceticus BCC24770, 7 BGCs were identified. Among them, we independently re-discovered the type II PKS for albofungin production previously identified by genome mining and tedious heterologous expression experiments. In Streptomyces tenebrarius, 5 BGCs were detected, including the known apramycin and tobramycin BGC as well as a newly discovered caerulomycin A BGC in this species. The production of caerulomycin A was confirmed by LC-MS and the inactivation of the caerulomycin A BGC surprisingly had a significant impact on the secondary metabolite regulation of S. tenebrarius. In conclusion, we developed an unbiased, high throughput proteomics-based method to complement genome mining methods for the identification of biosynthetic pathways in Streptomyces sp.

Genome mining methods to discover bioactive natural products.

Covering: 2016 to 2021With genetic information available for hundreds of thousands of organisms in publicly accessible databases, scientists have an unprecedented opportunity to meticulously survey the diversity and inner workings of life. The natural product research community has harnessed this breadth of sequence information to mine microbes, plants, and animals for biosynthetic enzymes capable of producing bioactive compounds. Several orthogonal genome mining strategies have been developed in recent years to target specific chemical features or biological properties of bioactive molecules using biosynthetic, resistance, or transporter proteins. These “biosynthetic hooks” allow researchers to query for biosynthetic gene clusters with a high probability of encoding previously undiscovered, bioactive compounds. This review highlights recent case studies that feature orthogonal approaches that exploit genomic information to specifically discover bioactive natural products and their gene clusters.

Computer-aided search for a cold-active cellobiose 2-epimerase

Cellobiose 2-epimerase (CE) is a promising industrial enzyme that can catalyze bioconversion of lactose to its high-value derivatives, namely epilactose and lactulose. A need exists in the dairy industry to catalyze lactose bioconversions at low temperatures to avoid microbial growth. We focused on the discovery of cold-active CE in this study. A genome mining method based on computational prediction was used to screen the potential genes encoding cold-active enzymes. The CE-encoding gene from Roseburia intestinalis, with a predicted high structural flexibility, was expressed heterologously in Escherichia coli. The catalytic property of the recombinant enzyme was extensively studied. The optimum temperature and pH of the enzyme were 45°C and 7.0, respectively. The specific activity of this enzyme to catalyze conversion of lactose to epilactose was measured to be 77.3 ± 1.6 U/mg. The kinetic parameters, including turnover number (kcat), Michaelis constant (Km), and catalytic efficiency (kcat/Km) using lactose as a substrate were 117.0 ± 7.7 s-1, 429.9 ± 57.3 mM, and 0.27 mM-1s-1, respectively. In situ production of epilactose was carried out at 8°C: 20.9% of 68.4 g/L lactose was converted into epilactose in 4 h using 0.02 mg/mL (1.5 U/mL, measured at 45°C) of recombinant enzyme. The enzyme discovered by this in silico method is suitable for low-temperature applications.

Stereoselective synthesis of the key intermediate of ticagrelor and its diverse analogs using a new alcohol dehydrogenase from Rhodococcus kyotonensis

Bioreduction catalyzed by alcohol dehydrogenase/reductase is one of the most valuable biotransformation processes widely used in industry. The (S)-2-Chloro-1-(3, 4-difluorophenyl) ethanol is a key chiral synthon for synthesizing the antithrombotic agent ticagrelor. Herein, a new alcohol dehydrogenase (named Rhky-ADH) identified from Rhodococcus kyotonensis by an enzyme promiscuity-based genome mining method was successfully cloned and functionally expressed in Escherichia coli. The whole cell biocatalyst harboring Rhky-ADH was biochemically characterized and was shown to be able to convert 2-Chloro-1-(3, 4-difluorophenyl) ethanone to (S)-2-Chloro-1-(3, 4-difluorophenyl) ethanol with more than 99 % enantiomeric excess (ee) and 99 % conversion. Our data showed that the optimum temperature and pH for Rhky-ADH were 25 °C and pH 8.0, respectively. The addition of NADH and an appropriate concentration of isopropanol enhanced the activity of Rhky-ADH, and 1 mM Mn2+ increased the enzyme activity by about 8 %. Substrate specificity experiments showed that Rhky-ADH had notable enzyme promiscuity and could reduce several ketones with high stereoselectivity. Our investigation on this novel enzyme adds another rare biocatalyst to the toolbox for producing chiral alcohols, which are widely used in the pharmaceutical industry.

Activation of three natural product biosynthetic gene clusters from Streptomyces lavendulae CGMCC 4.1386 by a reporter-guided strategy

Along with the fast developing of DNA sequencing technology, a great number of natural product biosynthetic gene clusters have been discovered by bioinformatic analysis, which demands novel high-throughput genome mining methods to obtain the diverse compounds dictated by those gene clusters. In this work, a method based on the reporter gene xylE was established to screen for the activation conditions of thirteen different gene clusters from Streptomyces lavendulae CGMCC 4.1386. In this reporter-guided method, the key structure gene was replaced by a xylE-kanaR cassette with the xylE gene being controlled by the transcription and translation machinery of the key structure gene. It not only facilitated the screening of activation conditions, but also provided the null mutants of specific natural product gene clusters as controls to link those clusters with their products conveniently. The potential activation conditions of eleven gene clusters from S. lavendulae CGMCC 4.1386 were obtained. In addition, activation of three of the eleven gene clusters was confirmed and their products were identified.

Enantioselective resolution of γ-lactam utilizing a novel (+)-γ-lactamase from Bacillus thuringiensis

Abstract (-)-γ-Lactam ((-)-2-azabicyclo[2.2.1]hept-5-en-3-one) is a significant chiral synthon that can be used to synthesize a large number of carbocyclic nucleosides such as antiviral drugs abacavir and carbovir. As a new emerging group of enantioselective biocatalysts, (+)-γ-lactamase has drawn more and more attention because of its application for kinetic resolution of racemic γ-lactam to obtain optically pure (-)-γ-lactam. In this paper, a novel (+)-γ-lactamase (designated as SYJ322B5) derived from Bacillus thuringiensis was identified by genome mining method. The gene was cloned, functionally expressed in Escherichia coli system and the protein was purified to homogeneity. Subsequently, biochemical characterization of SYJ322B5 showed that the optimal temperature and pH were 50 °C and 7.0, respectively. Enzyme assay showed that the recombinant enzyme could enantioselectively catalyze the bioresolution of racemic γ-lactam with a high enantiomeric excess (ee) of 99.8% and enantiomeric ratio (E) >200. More importantly, SYJ322B5 exhibited the highest enzyme activity among all reported (+)-γ-lactamases. Bioinformatics analysis and molecular docking studies showed that the enzyme was not a conventional amidase superfamily member but belonged to the isochorismatase-like hydrolases superfamily. It possesses a conserved Asp9-Lys81-Cys114 catalytic triad like other members in which the cysteine is considered to perform nucleophilic attack in the enzymatic catalysis process. Overall, this novel (+)-γ-lactamase may become a potential tool for the production of (-)-γ-lactam.

Fluostatins M-Q Featuring a 6-5-6-6 Ring Skeleton and High Oxidized A-Rings from Marine Streptomyces sp. PKU-MA00045.

Aromatic polyketides from marine actinomycetes have received increasing attention due to their unusual structures and potent bioactivities. Compared to their terrestrial counterparts, marine aromatic polyketides have been less discovered and their structural and biological diversities are far from being fully investigated. In this study, we employed a PCR-based genome mining method to discover aromatic polyketides in our marine bacteria collection. Five new atypical angucyclinones, fluostatins M–Q (1–5) featuring a unique 6-5-6-6 ring skeleton, were discovered from one “positive” Streptomyces sp. PKU-MA00045. The structures of fluostatins M–Q (1–5) were elucidated based on comprehensive spectroscopic analyses and the crystallographic structure of fluostatin P (4), which contains the most oxidized A-ring, was solved by X-ray diffraction analysis with Cu Kα radiation. Compared to the published 16 fluostatin analogues, fluostatins M–Q (1–5) contained a different methoxy group attached at C-7 and hydroxy group attached at C-4, enriching the structural diversity of aromatic polyketides from marine actinomycetes. Genome sequencing of Streptomyces sp. PKU-MA00045 revealed the biosynthetic gene cluster of fluostatins M–Q (1–5), which contained different genes and gene organizations compared to known fluostatin gene clusters, facilitating the investigation of the biosynthesis of the unique 6-5-6-6 ring skeleton in all fluostatins.

The Antibiotic Resistant Target Seeker (ARTS), an exploration engine for antibiotic cluster prioritization and novel drug target discovery.

With the rise of multi-drug resistant pathogens and the decline in number of potential new antibiotics in development there is a fervent need to reinvigorate the natural products discovery pipeline. Most antibiotics are derived from secondary metabolites produced by microorganisms and plants. To avoid suicide, an antibiotic producer harbors resistance genes often found within the same biosynthetic gene cluster (BGC) responsible for manufacturing the antibiotic. Existing mining tools are excellent at detecting BGCs or resistant genes in general, but provide little help in prioritizing and identifying gene clusters for compounds active against specific and novel targets. Here we introduce the ‘Antibiotic Resistant Target Seeker’ (ARTS) available at https://arts.ziemertlab.com. ARTS allows for specific and efficient genome mining for antibiotics with interesting and novel targets. The aim of this web server is to automate the screening of large amounts of sequence data and to focus on the most promising strains that produce antibiotics with new modes of action. ARTS integrates target directed genome mining methods, antibiotic gene cluster predictions and ‘essential gene screening’ to provide an interactive page for rapid identification of known and putative targets in BGCs.

Enzymatic preparation of optically pure t -butyl 6-chloro-(3 R ,5 S )-dihydroxyhexanoate by a novel alcohol dehydrogenase discovered from Klebsiella oxytoca

Abstract Alcohol dehydrogenases can catalyze the inter-conversion of aldehydes and alcohols. The t -butyl 6-chloro-(3 R ,5 S )-dihydroxyhexanoate is a key chiral intermediate in the synthesis of statin-type drugs such as Crestor (rosuvastatin calcium) and Lipitor (atorvastatin). Herein, a novel alcohol dehydrogenase (named as KleADH) discovered from Klebsiella oxytoca by a genome mining method was cloned and characterized. The KleADH was functionally overexpressed in Escherichia coli Rosetta (DE3) and the whole cell biocatalyst was able to convert t -butyl 6-chloro-(5 S )-hydroxy-3-oxohexanoate to t -butyl 6-chloro-(3 R ,5 S )-dihydroxyhexanoate with more than 99% diastereomeric excess (de) and 99% conversion in 24 h without adding any expensive cofactors. Several factors influencing the whole cell catalyst activity such as temperature, pH, the effects of metal ions and organic solvent were determined. The optimum enzyme activity was achieved at 30 °C and pH 7.0 and it was shown that 1 mM Fe 3+ can increase the enzyme activity by 1.2 times. N-hexane/water and n-heptane/water biphasic systems can also increase the activity of KleADH. Substrate specificity studies showed that KleADH also exhibited notable activity towards several aryl ketones with high stereoselectivity. Our investigation on this novel alcohol dehydrogenase KleADH reveals a promising biocatalyst for producing chiral alcohols for preparation of valuable pharmaceuticals.

Titer improvement and pilot-scale production of platensimycin from Streptomyces platensis SB12026.

Platensimycin (PTM) and platencin (PTN), isolated from several strains of Streptomyces platensis are potent antibiotics against multi-drug resistant bacteria. PTM was also shown to have antidiabetic and antisteatotic activities in mouse models. Through a novel genome-mining method, we have recently identified six PTM and PTN dual-producing strains, and generated several mutants with improved production of PTM or PTN by inactivating the pathway-specific transcriptional repressor gene ptmR1. Among them, S. platensis SB12026 gave the highest titer of 310mg/L for PTM. In this study, we now report titer improvement by medium and fermentation optimization and pilot-scale production and isolation of PTM from SB12026. The fermentation medium optimization was achieved by manipulating the carbon and nitrogen sources, as well as the inorganic salts. The highest titer of 1560mg/L PTM was obtained in 15-L fermentors, using a formulated medium mainly containing soluble starch, soybean flour, morpholinepropanesulfonic acid sodium salt and CaCO3. In addition, a polyamide chromatographic step was applied to facilitate the purification and 45.14g of PTM was successfully obtained from a 60L scale fermentation. These results would speed up the future development of PTM as human medicine.

Discovery of pentangular polyphenols hexaricins A–C from marine Streptosporangium sp. CGMCC 4.7309 by genome mining

Many novel microbial nature products were discovered from Actinobacteria by genome mining methods. However, only a few number of genome mining works were carried out in rare actinomycetes. An important reason precluding the genome mining efforts in rare actinomycetes is that most of them are recalcitrant to genetic manipulation. Herein, we chose the rare marine actinomycete Streptosporangium sp. CGMCC 4.7309 to explore its secondary metabolite diversity by genome mining. The genetic manipulation method has never been established for Streptosporangium strains. At first, we set up the genetic system of Streptosporangium sp. CGMCC 4.7309 unprecedentedly. The draft genome sequencing of Streptosporangium sp. CGMCC 4.7309 revealed that it contains more than 20 cryptic secondary metabolite biosynthetic clusters. A type II polyketide synthases-containing cluster (the hex cluster) was predicted to encode compounds with a pentangular polyphenol scaffold by in silico analysis. The products of the hex cluster were uncovered by comparing the metabolic profile of Streptosporangium sp. CGMCC 4.7309 with that of the hex30 inactivated mutant, in which a key ketoreductase gene was disrupted. Finally, three pentangular polyphenols were isolated and named as hexaricins A (1), B (2), and C (3). The inconsistency of the stereochemistry of C-15 in hexaricins A, B, and C indicates a branch point in their biosynthesis. Finally, the biosynthetic pathway of the hexaricins was proposed based on bioinformatics analysis.