Benefits and concerns of probiotics: an overview of the potential genotoxicity of the colibactin-producing Escherichia coli Nissle 1917 strain.

Recently, the mounting integration of probiotics into human health strategies has gathered considerable attention. Although the benefits of probiotics have been widely recognized in patients with gastrointestinal disorders, immune system modulation, and chronic-degenerative diseases, there is a growing need to evaluate their potential risks. In this context, new concerns have arisen regarding the safety of probiotics as some strains may have adverse effects in humans. Among these strains, Escherichia coli Nissle 1917 (EcN) exhibited traits of concern due to a pathogenic locus in its genome that produces potentially genotoxic metabolites. As the use of probiotics for therapeutic purposes is increasing, the effects of potentially harmful probiotics must be carefully evaluated. To this end, in this narrative review article, we reported the findings of the most relevant in vitro and in vivo studies investigating the expanding applications of probiotics and their impact on human well-being addressing concerns arising from the presence of antibiotic resistance and pathogenic elements, with a focus on the polyketide synthase (pks) pathogenic island of EcN. In this context, the literature data here discussed encourages a thorough profiling of probiotics to identify potential harmful elements as done for EcN where potential genotoxic effects of colibactin, a secondary metabolite, were observed. Specifically, while some studies suggest EcN is safe for gastrointestinal health, conflicting findings highlight the need for further research to clarify its safety and optimize its use in therapy. Overall, the data here presented suggest that a comprehensive assessment of the evolving landscape of probiotics is essential to make evidence-based decisions and ensure their correct use in humans.

Exploration of diverse secondary metabolites from Penicillium brasilianum by co-culturing with Armillaria mellea.

Bioinformatic analysis revealed that the genomes of ubiquitous Penicillium spp. might carry dozens of biosynthetic gene clusters (BGCs), yet many clusters have remained uncharacterized. In this study, a detailed investigation of co-culture fermentation including the basidiomycete Armillaria mellea CPCC 400891 and the P. brasilianum CGMCC 3.4402 enabled the isolation of five new compounds including two bisabolene-type sesquiterpenes (arpenibisabolanes A and B), two carotane-type sesquiterpenes (arpenicarotanes A and B), and one polyketide (arpenichorismite A) along with seven known compounds. The assignments of their structures were deduced by the extensive analyses of detailed spectroscopic data, electronic circular dichroism spectra, together with delimitation of the biogenesis. Most new compounds were not detected in monocultures under the same fermentation conditions. Arpenibisabolane A represents the first example of a 6/5-fused bicyclic bisabolene. The bioassay of these five new compounds exhibited no cytotoxic activities in vitro against three human cancer cell lines (A549, MCF-7, and HepG2). Moreover, sequence alignments and bioinformatic analysis to other metabolic pathways, two BGCs including Pb-bis and Pb-car, responsible for generating sesquiterpenoids from co-culture were identified, respectively. Furthermore, based on the chemical structures and deduced gene functions of the two clusters, a hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed. These results demonstrated that the co-culture approach would facilitate bioprospecting for new metabolites even from the well-studied microbes. Our findings would provide opportunities for further understanding of the biosynthesis of intriguing sesquiterpenoids via metabolic engineering strategies. KEY POINTS: • Penicillium and Armillaria co-culture facilitates the production of diverse secondary metabolites • Arpenibisabolane A represents the first example of 6/5-fused bicyclic bisabolenes • A hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed.

Assessing and harnessing updated polyketide synthase modules through combinatorial engineering.

The modular nature of polyketide assembly lines and the significance of their products make them prime targets for combinatorial engineering. The recently updated module boundary has been successful for engineering short synthases, yet larger synthases constructed using the updated boundary have not been investigated. Here we describe our design and implementation of a BioBricks-like platform to rapidly construct 5 triketide, 25 tetraketide, and 125 pentaketide synthases to test every module combination of the pikromycin synthase. Anticipated products are detected from 60% of the triketide synthases, 32% of the tetraketide synthases, and 6.4% of the pentaketide synthases. We determine ketosynthase gatekeeping and module-skipping are the principal impediments to obtaining functional synthases. The platform is also employed to construct active hybrid synthases by incorporating modules from the erythromycin, spinosyn, and rapamycin assembly lines. The relaxed gatekeeping of a ketosynthase in the rapamycin synthase is especially encouraging in the quest to produce designer polyketides.

Dysfunctional mucus structure in cystic fibrosis increases vulnerability to colibactin-mediated DNA adducts in the colon mucosa.

Colibactin is a recently characterized pro-carcinogenic genotoxin produced by pks+ Escherichia coli. We hypothesized that cystic fibrosis (CF)-associated dysfunctional mucus structure increases the vulnerability of host mucosa to colibactin-induced DNA damage. In this pilot study, we tested healthy-appearing mucosal biopsy samples obtained during screening and surveillance colonoscopies of adult CF and non-CF patients for the presence of pks+ E. coli, and we investigated the possibility of detecting a novel colibactin-specific DNA adduct that has not been yet been demonstrated in humans. While CF patients had a lower incidence of pks+ E. coli carriage (~8% vs 29%, p = 0.0015), colibactin-induced DNA adduct formation was detected, but only in CF patients and only in those who were not taking CFTR modulator medications. Moreover, the only patient found to have colon cancer during this study had CF, harbored pks+ E. coli, and had colibactin-induced DNA adducts in the mucosal samples. Larger studies with longitudinal follow-up should be done to extend these initial results and further support the development of colibactin-derived DNA adducts to stratify patients and their risk.

Induction of Three New Secondary Metabolites by the Co-Culture of Endophytic Fungi Phomopsis asparagi DHS-48 and Phomopsis sp. DHS-11 Isolated from the Chinese Mangrove Plant Rhizophora mangle.

Co-cultivation is a powerful emerging tool for awakening biosynthetic gene clusters (BGCs) that remain transcriptionally silent under artificial culture conditions. It has recently been used increasingly extensively to study natural interactions and discover new bioactive metabolites. As a part of our project aiming at the discovery of structurally novel and biologically active natural products from mangrove endophytic fungi, an established co-culture of a strain of Phomopsis asparagi DHS-48 with another Phomopsis genus fungus DHS-11, both endophytes in mangrove Rhizophora mangle, proved to be very efficient to induce the production of new metabolites as well as to increase the yields of respective target metabolites. A detailed chemical investigation of the minor metabolites produced by the co-culture of these two titled fungal strains led to the isolation of six alkaloids (1-6), two sterols (7, 8), and six polyketides (9-14). In addition, all the compounds except 8 and 10, as well as three new metabolites phomopyrazine (1), phomosterol C (7), and phomopyrone E (9), were not present in discrete fungal cultures and only detected in the co-cultures. The structures were elucidated on the basis of spectroscopic analysis, and the absolute configurations were assumed by electronic circular dichroism (ECD) calculations. Subsequently, the cytotoxic, immunosuppressive, and acetylcholinesterase inhibitory properties of all the isolated metabolites were determined in vitro. Compound 8 exhibited moderate inhibitory activity against ConA-induced T and LPS-induced B murine splenic lymphocytes, with IC50 values of 35.75 ± 1.09 and 47.65 ± 1.21 µM, respectively.

Gatekeeping Activity of Collinear Ketosynthase Domains Limits Product Diversity for Engineered Type I Polyketide Synthases.

Engineered type I polyketide synthases (type I PKSs) can enable access to diverse polyketide pharmacophores and generate non-natural natural products. However, the promise of type I PKS engineering remains modestly realized at best. Here, we report that ketosynthase (KS) domains, the key carbon-carbon bond-forming catalysts, control which intermediates are allowed to progress along the PKS assembly lines and which intermediates are excluded. Using bimodular PKSs, we demonstrate that KSs can be exquisitely selective for the upstream polyketide substrate while retaining promiscuity for the extender unit that they incorporate. It is then the downstream KS that acts as a gatekeeper to ensure the fidelity of the extender unit incorporation by the upstream KS. We also demonstrate that these findings are not universally applicable; substrate-tolerant KSs do allow engineered polyketide intermediates to be extended. Our results demonstrate the utility for evaluating the KS-induced bottlenecks to gauge the feasibility of engineering PKS assembly lines.

Giant polyketide synthase enzymes in the biosynthesis of giant marine polyether toxins.

Prymnesium parvum are harmful haptophyte algae that cause massive environmental fish kills. Their polyketide polyether toxins, the prymnesins, are among the largest nonpolymeric compounds in nature and have biosynthetic origins that have remained enigmatic for more than 40 years. In this work, we report the "PKZILLAs," massive P. parvum polyketide synthase (PKS) genes that have evaded previous detection. PKZILLA-1 and -2 encode giant protein products of 4.7 and 3.2 megadaltons that have 140 and 99 enzyme domains. Their predicted polyene product matches the proposed pre-prymnesin precursor of the 90-carbon-backbone A-type prymnesins. We further characterize the variant PKZILLA-B1, which is responsible for the shorter B-type analog prymnesin-B1, from P. parvum RCC3426 and thus establish a general model of haptophyte polyether biosynthetic logic. This work expands expectations of genetic and enzymatic size limits in biology.

From the forest floor to the lab: Insights into the diversity and complexity of mushroom polyketide synthases.

Mushroom-forming fungi exhibit a distinctive ecology, which is unsurprisingly also reflected in unique and divergent biosynthetic pathways. We review this phenomenon through the lens of the polyketide metabolism, where mushrooms often deviate from established principles and challenge conventional paradigms. This is evident not only by non-canonical enzyme architectures and functions but also by their propensity for multi-product synthases rather than single-product pathways. Nevertheless, mushrooms also feature many polyketides familiar from plants, bacteria, and fungi of their sister division Ascomycota, which, however, are the result of an independent evolution. In this regard, the captivating biosynthetic pathways of mushrooms might even help us understand the biological pressures that led to the simultaneous production of the same natural products (via convergent evolution, co-evolution, and/or metaevolution) and thus address the question of their raison d'être.

Analysis of the partial sequencing of clbA, clbB and clbQ in Escherichia coli isolates that produce colibactin and multilocus sequence typing.

Colibactin, is a cyclomodulin expressed from polyketide synthase (pk) genomic islands. These bacterial toxins interfere with the eukaryotic cell cycle and induce DNA damage. The aim of the present study was to investigate the prevalence of colibactin production among E. coli strains recovered from different infections, determine the similarity of clb nucleotide sequences, and identify genotype of isolates using multilocus sequence typing(MLST). This was a prospective, cross-sectional study conducted from January 2022 to February 2023. A total of 117 clinical isolates were obtained from various sample types collected from outpatients and inpatients recruited to the Department of Bacteriology Labs in different hospitals in Baghdad, Iraq. clbA/clbR, clbB and clbP/clbQ were detected via conventional PCR, and partial sequencing of amplicons was performed via Sanger sequencing. For select isolates, MLST genotyping was performed. The most common phylogenetic group was B2 (61/106; 57.54%). Among the E. coli strains, 27/106 (25.47%) were clb + ve, and the most common type was clbB (13/27; 48.14%). Analysis of the partial sequencing of clb among the strains revealed high molecular similarity. Genotyping of 37 selected E. coli strains via MLST revealed 28 different genotypes. There was a high prevalence of colibactin production in phylogroup B2, and it seems that the clb + ve strains had conserved molecular structures. There was high genetic diversity among the strains tested.

De Novo Genome Assembly of Toniniopsis dissimilis (Ramalinaceae, Lecanoromycetes) from Long Reads Shows a Comparatively High Composition of Biosynthetic Genes Putatively Involved in Melanin Synthesis.

Lichens have developed numerous adaptations to optimize their survival in various environmental conditions, largely by producing secondary compounds by the fungal partner. They often have antibiotic properties and are involved in protection against intensive UV radiation, pathogens, and herbivores. To contribute to the knowledge of the arsenal of secondary compounds in a crustose lichen species, we sequenced and assembled the genome of Toniniopsis dissimilis, an indicator of old-growth forests, using Oxford Nanopore Technologies (ONT, Oxford, UK) long reads. Our analyses focused on biosynthetic gene clusters (BGCs) and specifically on Type I Polyketide (T1PKS) genes involved in the biosynthesis of polyketides. We used the comparative genomic approach to compare the genome of T. dissimilis with six other members of the family Ramalinaceae and twenty additional lichen genomes from the database. With only six T1PKS genes, a comparatively low number of biosynthetic genes are present in the T. dissimilis genome; from those, two-thirds are putatively involved in melanin biosynthesis. The comparative analyses showed at least three potential pathways of melanin biosynthesis in T. dissimilis, namely via the formation of 1,3,6,8-tetrahydroxynaphthalene, naphthopyrone, or YWA1 putative precursors, which highlights its importance in T. dissimilis. In addition, we report the occurrence of genes encoding ribosomally synthesized and posttranslationally modified peptides (RiPPs) in lichens, with their highest number in T. dissimilis compared to other Ramalinaceae genomes. So far, no function has been assigned to RiPP-like proteins in lichens, which leaves potential for future research on this topic.

MFS Transporter as the Molecular Switch Unlocking the Production of Cage-Like Acresorbicillinol C.

Sorbicillinoids are a class of fungal polyketides with diverse structures and distinguished bioactivities. Although remarkable progress has been achieved in their chemistry and biosynthesis, the efflux of sorbicillinoids is poorly understood. Here, we found MFS transporter AcsorT was responsible for the biosynthesis of sorbicillinoids in Acremonium chrysogenum. Combinatorial knockout and subcellular location demonstrated that the plasma membrane-associated AcsorT was responsible for the transportation of sorbicillinol and subsequent formation of oxosorbicillinol and acresorbicillinol C via the berberine bridge enzyme-like oxidase AcsorD in the periplasm. Homology modeling and site-directed mutation revealed that Tyr303 and Arg436 were the key residues of AcsorT, which was further explained by molecular dynamics simulation. Based on our study, it was suggested that AcsorT modulates sorbicillinoid production by coordinating its biosynthesis and export, and a transport model of sorbicillinoids was proposed in A. chrysogenum.

Biosynthesis of Atypical Angucyclines Unveils New Ring Rearrangement Reactions Catalyzed by Flavoprotein Monooxygenases.

Six new angucycline structures, including spirocyclione A (1), which contains an unusual oxaspiro[5.5]undecane architecture, and its ring-A-cleaved product spirocyclione B (2), were discovered by heterologous expression of a type II polyketide biosynthetic gene cluster captured from a marine actinomycete strain Streptomyces sp. HDN155000. Three flavoprotein monooxygenases are confirmed to be responsible for the oxidative carbon skeleton rearrangements in the biosynthesis of compounds 1 and 2. The obtained compounds showed promising cytotoxicity against different types of cancer cells.

Recent progresses in the cyclization and oxidation of polyketide biosynthesis.

Polyketides represent an important class of natural products, renowned for their intricate structures and diverse biological activities. In contrast to common fatty acids, polyketides possess relatively more rigid carbon skeletons, more complex ring systems, and chiral centers. These structural features are primarily achieved through distinctive enzymatic cyclizations and oxidations as tailoring steps. In this opinion, we discuss the recent progress in deciphering the mechanisms of cyclization and oxidation within polyketide biosynthesis. By shedding light on these enzymatic processes, this article seeks to motivate the community to unravel the remaining mysteries surrounding cyclase and oxidase functionalities and to explore novel polyketide natural products through genome mining.

Bdelloid rotifers deploy horizontally acquired biosynthetic genes against a fungal pathogen.

Coevolutionary antagonism generates relentless selection that can favour genetic exchange, including transfer of antibiotic synthesis and resistance genes among bacteria, and sexual recombination of disease resistance alleles in eukaryotes. We report an unusual link between biological conflict and DNA transfer in bdelloid rotifers, microscopic animals whose genomes show elevated levels of horizontal gene transfer from non-metazoan taxa. When rotifers were challenged with a fungal pathogen, horizontally acquired genes were over twice as likely to be upregulated as other genes - a stronger enrichment than observed for abiotic stressors. Among hundreds of upregulated genes, the most markedly overrepresented were clusters resembling bacterial polyketide and nonribosomal peptide synthetases that produce antibiotics. Upregulation of these clusters in a pathogen-resistant rotifer species was nearly ten times stronger than in a susceptible species. By acquiring, domesticating, and expressing non-metazoan biosynthetic pathways, bdelloids may have evolved to resist natural enemies using antimicrobial mechanisms absent from other animals.

Time-Resolved Ion Mobility-Mass Spectrometry Reveals Structural Transitions in the Disassembly of Modular Polyketide Syntheses.

The type 1 polyketide synthase (PKS) assembly line uses its modular structure to produce polyketide natural products that form the basis of many pharmaceuticals. Currently, several cryoelectron microscopy (cryo-EM) structures of a multidomain PKS module have been constructed, but much remains to be learned. Here we utilize ion-mobility mass spectrometry (IM-MS) to record size and shape information and detect different conformational states of a 207 kDa didomain dimer comprised of ketosynthase (KS) and acyl transferase (AT), excised from full-length module. Furthermore, gas-phase stability differences between these different conformations are captured by collision induced unfolding (CIU) technology. Additionally, through tracking these forms as a function of time, we elucidate a detailed disassembly pathway for KS-AT dimers for the first time.

Discovering type I cis-AT polyketides through computational mass spectrometry and genome mining with Seq2PKS.

Type 1 polyketides are a major class of natural products used as antiviral, antibiotic, antifungal, antiparasitic, immunosuppressive, and antitumor drugs. Analysis of public microbial genomes leads to the discovery of over sixty thousand type 1 polyketide gene clusters. However, the molecular products of only about a hundred of these clusters are characterized, leaving most metabolites unknown. Characterizing polyketides relies on bioactivity-guided purification, which is expensive and time-consuming. To address this, we present Seq2PKS, a machine learning algorithm that predicts chemical structures derived from Type 1 polyketide synthases. Seq2PKS predicts numerous putative structures for each gene cluster to enhance accuracy. The correct structure is identified using a variable mass spectral database search. Benchmarks show that Seq2PKS outperforms existing methods. Applying Seq2PKS to Actinobacteria datasets, we discover biosynthetic gene clusters for monazomycin, oasomycin A, and 2-aminobenzamide-actiphenol.

Preclinical and clinical evidence of the association of colibactin-producing Escherichia coli with anxiety and depression in colon cancer.

BACKGROUND: The association between the intestinal microbiota and psychiatric disorders is becoming increasingly apparent. The gut microbiota contributes to colorectal carcinogenesis (CRC), as demonstrated with colibactin-producing Escherichia coli (CoPEC). AIM: To evaluate the association between CoPEC prevalence and anxiety- and depressive-like behaviors with both preclinical and clinical approaches. METHODS: Patients followed after a CRC surgery and for whom the prevalence of CoPEC has been investigated underwent a psychiatric interview. Results were compared according to the CoPEC colonization. In parallel C57BL6/J wild type mice and mice with a CRC susceptibility were chronically infected with a CoPEC strain. Their behavior was assessed using the Elevated Plus Maze test, the Forced Swimming Test and the Behavior recognition system PhenoTyper®. RESULTS: In a limited cohort, all patients with CoPEC colonization presented with psychiatric disorders several years before cancer diagnosis, whereas only one patient (17%) without CoPEC did. This result was confirmed in C57BL6/J wild-type mice and in a CRC susceptibility mouse model (adenomatous polyposis colimultiple intestinal neoplasia/+). Mice exhibited a significant increase in anxiety- and depressive-like behaviors after chronic infection with a CoPEC strain. CONCLUSION: This finding provides the first evidence that CoPEC infection can induce microbiota-gut-brain axis disturbances in addition to its procarcinogenic properties.

A systematic comparison of natural product potential, with an emphasis on RiPPs, by mining of bacteria of three large ecosystems.

The implementation of several global microbiome studies has yielded extensive insights into the biosynthetic potential of natural microbial communities. However, studies on the distribution of several classes of ribosomally synthesized and post-translationally modified peptides (RiPPs), non-ribosomal peptides (NRPs) and polyketides (PKs) in different large microbial ecosystems have been very limited. Here, we collected a large set of metagenome-assembled bacterial genomes from marine, freshwater and terrestrial ecosystems to investigate the biosynthetic potential of these bacteria. We demonstrate the utility of public dataset collections for revealing the different secondary metabolite biosynthetic potentials among these different living environments. We show that there is a higher occurrence of RiPPs in terrestrial systems, while in marine systems, we found relatively more terpene-, NRP-, and PK encoding gene clusters. Among the many new biosynthetic gene clusters (BGCs) identified, we analyzed various Nif-11-like and nitrile hydratase leader peptide (NHLP) containing gene clusters that would merit further study, including promising products, such as mersacidin-, LAP- and proteusin analogs. This research highlights the significance of public datasets in elucidating the biosynthetic potential of microbes in different living environments and underscores the wide bioengineering opportunities within the RiPP family.

Putrescine Supplementation Limits the Expansion of pks+ Escherichia coli and Tumor Development in the Colon.

Escherichia coli that harbor the polyketide synthase (pks) genomic island produce colibactin and are associated with sporadic colorectal cancer development. Given the considerable prevalence of pks+ bacteria in healthy individuals, we sought to identify strategies to limit the growth and expansion of pks+ E. coli. We found that culture supernatants of the probiotic strain E. coli Nissle 1917 were able to inhibit the growth of the murine pathogenic strain pks+ E. coli NC101 (EcNC101). We performed a nontargeted analysis of the metabolome in supernatants from several E. coli strains and identified putrescine as a potential postbiotic capable of suppressing EcNC101 growth in vitro. The effect of putrescine supplementation was then evaluated in the azoxymethane/dextran sulfate sodium mouse model of colorectal cancer in mice colonized with EcNC101. Putrescine supplementation inhibited the growth of pks+ E. coli, reduced the number and size of colonic tumors, and downmodulated the release of inflammatory cytokines in the colonic lumen. Additionally, putrescine supplementation led to shifts in the composition and function of gut microbiota, characterized by an increase in the Firmicutes/Bacteroidetes ratio and enhanced acetate production. The effect of putrescine was further confirmed in vitro using a pks+ E. coli strain isolated from a patient with colorectal cancer. These results suggest that probiotic-derived metabolites can be used as an alternative to live bacteria in individuals at risk of developing colorectal cancer due to the presence of pks+ bacteria in their colon. SIGNIFICANCE: Putrescine supplementation inhibits the growth of cancer-promoting bacteria in the gut, lowers inflammation, and reduces colon cancer development. The consumption of healthy foods rich in putrescine may be a potential prophylactic approach for individuals at risk of developing colorectal cancer due to the presence of pks+ bacteria in their colon.

A limited concentration range of diaphorin, a polyketide produced by a bacterial symbiont of the Asian citrus psyllid, promotes the in vitro gene expression with bacterial ribosomes.

Diaphorin is a polyketide produced by "Candidatus Profftella armatura" (Gammaproteobacteria: Burkholderiales), an obligate symbiont of a devastating agricultural pest, the Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae). Physiological concentrations of diaphorin, which D. citri contains at levels as high as 2-20 mM, are inhibitory to various eukaryotes and Bacillus subtilis (Firmicutes: Bacilli) but promote the growth and metabolic activity of Escherichia coli (Gammaproteobacteria: Enterobacterales). Our previous study demonstrated that 5-mM diaphorin, which exhibits significant inhibitory and promoting effects on cultured B. subtilis and E. coli, respectively, inhibits in vitro gene expression utilizing purified B. subtilis and E. coli ribosomes. This suggested that the adverse effects of diaphorin on B. subtilis are partly due to its influence on gene expression. However, the result appeared inconsistent with the positive impact on E. coli. Moreover, the diaphorin concentration in bacterial cells, where genes are expressed in vivo, may be lower than in culture media. Therefore, the present study analyzed the effects of 50 and 500 µM of diaphorin on bacterial gene expression using the same analytical method. The result revealed that this concentration range of diaphorin, in contrast to 5-mM diaphorin, promotes the in vitro translation with the B. subtilis and E. coli ribosomes, suggesting that the positive effects of diaphorin on E. coli are due to its direct effects on translation. This study demonstrated for the first time that a pederin-type compound promotes gene expression, establishing a basis for utilizing its potential in pest management and industrial applications.IMPORTANCEThis study revealed that a limited concentration range of diaphorin, a secondary metabolite produced by a bacterial symbiont of an agricultural pest, promotes cell-free gene expression utilizing substrates and proteins purified from bacteria. The unique property of diaphorin, which is inhibitory to various eukaryotes and Bacillus subtilis but promotes the growth and metabolic activity of Escherichia coli, may affect the microbial flora of the pest insect, potentially influencing the transmission of devastating plant pathogens. Moreover, the activity may be exploited to improve the efficacy of industrial production by E. coli, which is often used to produce various important materials, including pharmaceuticals, enzymes, amino acids, and biofuels. This study elucidated a part of the mechanism by which the unique activity of diaphorin is expressed, constructing a foundation for applying the distinct property to pest management and industrial use.