Microbial Cell Factories最新文献

{"title":"A toolkit for facilitating markerless integration of expression cassettes in Komagataella phaffii via CRISPR/Cas9.","authors":"Laura García-Calvo, Charlotte Kummen, Solvor Rustad, Sissel Beate Rønning, Annette Fagerlund","doi":"10.1186/s12934-025-02716-x","DOIUrl":"https://doi.org/10.1186/s12934-025-02716-x","url":null,"abstract":"<p><strong>Background: </strong>The yeast Komagataella phaffii (formerly known as Pichia pastoris) has been widely used for functional expression of recombinant proteins, including plant and animal food proteins. CRISPR/Cas9 genome editing systems can be used for insertion of heterologous genes without the use of selection markers. The study aimed to create a convenient markerless knock-in method for integrating expression cassettes into the chromosome of K. phaffii using CRISPR/Cas9 technology. The approach was based on the hierarchical, modular, Golden Gate assembly employing the GoldenPiCS toolkit. Furthermore, the aim was to evaluate the system's efficiency and suitability for producing secreted recombinant food proteins.</p><p><strong>Results: </strong>Three Cas9/sgRNA plasmids were constructed, along with corresponding donor helper plasmids containing homology regions for chromosomal integration via homology-directed repair. The integration efficiency of an enhanced green fluorescent protein (eGFP) expression cassette was assessed at three genomic loci (04576, PFK1, and ROX1). The 04576 locus showed the highest integration efficiency, while ROX1 had the highest transformation efficiency. Whole genome sequencing revealed variable copy numbers of eGFP expression cassettes among clones, corresponding with increasing levels of fluorescence. Furthermore, the system's applicability for producing recombinant food proteins was validated by successfully expressing and secreting chicken ovalbumin. This constitutes the first report of CRISPR/Cas9 applied to produce recombinant chicken ovalbumin.</p><p><strong>Conclusions: </strong>The adapted GoldenPiCS toolkit combined with CRISPR/Cas9 technology enabled efficient and precise genome integration in K. phaffii. This approach holds promise for expanding the production of high-value recombinant proteins. Future research should focus on optimizing integration sites and improving cloning procedures to enhance the system's efficiency and versatility.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"97"},"PeriodicalIF":4.3,"publicationDate":"2025-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12049782/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144018466","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Probiotic spore-based antigen delivery: a novel oral vaccine strategy against Vibrio infections in aquaculture.","authors":"Marina De Stefano, Giovanni Di Gregorio Barletta, Simona Morgera, Ylenia De Luca, Chiara Belaeff, Karen Power, Loredana Baccigalupi, Gionata De Vico, Ivan Conte, Ezio Ricca, Anella Saggese","doi":"10.1186/s12934-025-02725-w","DOIUrl":"https://doi.org/10.1186/s12934-025-02725-w","url":null,"abstract":"<p><strong>Background: </strong>Vibriosis is a deadly illness caused by various species of the Vibrio genus. Due to its high incidence in aquaculture plants, vibriosis is responsible for significant economic losses. Currently, anti-vibriosis treatments rely on antibiotics. However, the global rise in antibiotic resistance necessitates the development of alternative approaches. Novel vaccines and effective probiotics have been proposed as potential alternative to antibiotics in fighting bacterial infections. Here we propose a combined vaccine/probiotic strategy based on the use of probiotic bacterial spores for the oral delivery of Vibrio antigens. Spores of various species of the Bacillus genus are widely used as probiotics and have been shown to efficiently display antigens in a non-recombinant way.</p><p><strong>Results: </strong>Spores of various probiotic strains were analyzed to assess their effectiveness in displaying a heterologous model protein, and B. megaterium MV30 was identified as the most efficient strain. MV30 spores were then used to display two antigens of Vibrio harveyi, the entire Hsp33 protein of 33 kDa and a 239 amino acids fragment of OmpK (OmpK^21/260), identified as the most immunogenic part of the protein. While Hsp33 is a stable protein, OmpK^21/260 is unstable at conditions mimicking those encountered in an aquaculture plant and the interaction with MV30 spores reduced such instability. The protective ability of the combined probiotic/vaccination strategy was assayed on Medaka fish (Oryzias latipes), as a model. In a challenge experiment with a virulent strain of Vibrio harveyi, a protective effect was observed with MV30 spores alone and such effect was significantly increased when the same spores displayed either one of the two antigens.</p><p><strong>Conclusion: </strong>Our results support the use of probiotics and oral vaccines as a valid alternative to antibiotics and point to the application of probiotic spore-based antigen delivery as a novel strategy to fight pathogenic infections.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"96"},"PeriodicalIF":4.3,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12046727/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143992968","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Exchange of the L-cysteine exporter after in-vivo metabolic control analysis improved the L-cysteine production process with engineered Escherichia coli.","authors":"Daniel Alejandro Caballero Cerbon, Dirk Weuster-Botz","doi":"10.1186/s12934-025-02715-y","DOIUrl":"https://doi.org/10.1186/s12934-025-02715-y","url":null,"abstract":"<p><strong>Background: </strong>L-Cysteine is a proteinogenic amino acid of high pharmaceutical and industrial interest. However, the fermentation process for L-cysteine production is faced with multiple obstacles, like the toxicity of L-cysteine for the cells, the low carbon yield of the product, and the low selectivity of the L-cysteine exporter. In previous work, in-vivo metabolic control analysis (MCA) applied to an L-cysteine fed-batch production process with E. coli, followed by the targeted metabolic engineering to reduce an intracellular O-acetylserine (OAS) deficiency, resulted in a significant improvement of the L-cysteine production process with the new producer strain.</p><p><strong>Results: </strong>In this work, in-vivo MCA was applied to the L-cysteine fed-batch production process with the new producer strain (E. coli W3110 pCysK). The MCA indicated that a simultaneous increase in the exporter's expression and selectivity is required to increase the L-cysteine production further. The exchange of the L-cysteine exporter YdeD present in the plasmid pCysK for the potentially more selective exporter YfiK led to an increase of the maximal L-cysteine concentration by the end of the fed-batch process of 37% to a final concentration of 33.8 g L^-1. The L-cysteine production could also be extended for at least 20 h due to conserved cellular activity as a result of the reduction of carbon loss as OAS.</p><p><strong>Conclusions: </strong>It could be shown that the in-vivo MCA methodology can be utilised iteratively with cells from the production process to pinpoint targets for further strain optimisation towards a significant increase in the L-cysteine production with E. coli. The use of this technology in combination with process engineering to adapt the fed-batch process to the modified strain may achieve a further improvement of the process performance.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"95"},"PeriodicalIF":4.3,"publicationDate":"2025-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12038998/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144003984","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Improving sustainable isopropanol production in engineered Escherichia coli W via oxygen limitation.","authors":"Regina Kutscha, Dominic Uhlir, Stefan Pflügl","doi":"10.1186/s12934-025-02720-1","DOIUrl":"https://doi.org/10.1186/s12934-025-02720-1","url":null,"abstract":"<p><strong>Background: </strong>Due to ecological concerns, alternative supply lines for fuel and bulk chemicals such as isopropanol are increasingly pursued. By implementing the formation pathways from natural producers like Clostridium beijerinckii and Clostridium aurantibutyricum, isopropanol can be produced in Escherichia coli. However, developing an industrially and economically feasible microbial production process requires a robust and efficient process strategy. Therefore, this study explores microaerobic conditions in combination with lactose and sour whey as sustainable carbon source as a basis for large-scale microbial isopropanol production.</p><p><strong>Results: </strong>Different gas-liquid mass transfer regimes (affected by variations of the stirrer speed and ingas oxygen concentration) allowed the implementation of different microaerobic conditions characterized by their specific oxygen uptake rate (q_O2) in cultivations with an isopropanol producing E. coli W strain on lactose. Under microaerobic conditions, the specific isopropanol production rate (q_{p, ipa}) exhibited a direct correlation with q_O2. Moreover, isopropanol production showed a pseudo growth-coupled behavior. Monitoring of the formation rates of various by-products such as acetone, lactate, acetate, pyruvate, formate and succinate allowed to identify a q_O2 of 9.6 mmol g^- 1 h^- 1 in only slightly microaerobic cultivations as the best conditions for microbial isopropanol production. Additionally, the data suggests that a carbon bottleneck exists at the pyruvate node and the availability of the redox factor NADPH is crucial to shift the product balance from acetone to isopropanol. Finally, confirmation runs prove the effectiveness of the microaerobic production approach by yielding 8.2 g L^- 1 (135.8 ± 13.3 mmol L^- 1) and 20.6 g L^- 1 (342.9 ± 0.4 mmol L^- 1) isopropanol on lactose and whey, respectively, reaching a volumetric isopropanol formation rate of up to 2.44 g L^- 1 h^- 1 (40.6 mmol L^- 1 h^- 1).</p><p><strong>Conclusions: </strong>This study identifies slightly microaerobic conditions (q_O2 ~ 10 mmol g^- 1 h^- 1) as the currently best conditions for microbial isopropanol production on lactose and whey in E. coli W. In the future, optimizing the carbon flux around the pyruvate node, ensuring sufficient NADPH supply, and establishing a feedback control loop to control process variables affecting oxygen transfer to the culture, could make microbial isopropanol production feasible at an industrial scale.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"94"},"PeriodicalIF":4.3,"publicationDate":"2025-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12032697/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144027963","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structure-guided engineering of α-ketoisocaproate dioxygenase increases isobutene production in Synechocystis sp. PCC 6803.","authors":"Conrad Schumann, Amit Kugler, Bhavik Ashwin Shah, Gustav Berggren, Henrik Land, Cecilia Blikstad, Karin Stensjö","doi":"10.1186/s12934-025-02708-x","DOIUrl":"10.1186/s12934-025-02708-x","url":null,"abstract":"<p><p>Isobutene is a promising precursor for jet fuel due to its high energy density and favorable combustion properties. Light-driven bioproduction of isobutene has recently been investigated as an alternative strategy to crude oil refinement or fermentation-based manufacturing processes by harnessing the unicellular cyanobacterium Synechocystis sp. PCC 6803 and the α-ketoisocaproate dioxygenase (RnKICD) from Rattus norvegicus. However, the obtained production level was not sufficient, partially due to the promiscuous activity of RnKICD. The enzyme catalyzes both the reaction with ρ-hydroxyphenylpyruvate (HPP) for homogentisate formation, as well as the reaction with α-ketoisocaproate (KIC), the precursor for isobutene synthesis. Here, to overcome this bottleneck step in the isobutene biosynthesis, protein engineering was employed to improve RnKICD activity and in vivo isobutene production. Purified RnKICD variants were characterized by measuring in vitro KIC and HPP consumption rates, as well as isobutene formation rate. The active site mutations F336V, N363A altered the KIC and HPP consumption rates, while the KIC-to-isobutene conversion ratio was only marginally affected. Besides, the RnKICD variants F336V, N363A and F336V/N363A exhibited a substantially enhanced substrate selectivity for KIC over HPP. Among the examined engineered Synechocystis strains, Syn-F336V showed a 4-fold improvement in isobutene production, compared to the base strain (Syn-RnKICD). Our findings reveal that residues F336 and N363 play a crucial role in substrate interactions, as targeted mutations at these sites shifted the substrate selectivity towards KIC while F336V elevated the in vivo isobutene production levels significantly. We conclude that engineering the active site of RnKICD is a potent tool for improving isobutene bioproduction in Synechocystis.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"93"},"PeriodicalIF":4.3,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12020224/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143992971","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Technological application of autochthonous Meyerozyma guilliermndii cultures in Chardonnay.","authors":"Lan Mi, Wei Zang, Xuefang Zhao, Shuai Peng, Min Li, Jing Wang","doi":"10.1186/s12934-025-02669-1","DOIUrl":"https://doi.org/10.1186/s12934-025-02669-1","url":null,"abstract":"<p><p>Using Chardonnay grape, the fermentation characteristics of sequential inoculation with M. guilliermondii and S. cerevisiae in the pilot fermentation process of dry white wine were examined. In this study, the physical and chemical indexes, color indexes, volatile aroma compound composition, and sensory indexes of 2 tons of samples at the end of alcoholic fermentation (AF) and malolactic fermentation (MLF) were analyzed. The results showed that the M. guilliermondii biomass in the treatment group (inoculated M. guilliermondii and S. cerevisiae sequentially) was always higher than 10⁶ CFU/mL during AF, and the basic physicochemical indexes of samples met the requirements of the national standard GB/T15038-2006 (Wine). Also M. guilliermondii NM218 can significantly increase the color saturation of Chardonnay white wine. Regarding aromatic characteristics, the total alcohol, ester, and terpene contents of wine samples after mixed fermentation were higher than those of control group (only inoculated S. cerevisiae). Compared with control wine samples (only with S. cerevisiae), the treatment group had significantly increased ethyl caprylate, ethyl nonanoate, phenethyl acetate, and ethyl laurate contents, including n-heptanol, which can provide Chardonnay dry white wine a richer fruity fragrance. meanwhile, the sensory scores of wine samples were higher in the treatment group. In conclusion, mixed fermentation could boost the aroma quality and sensory pleasure of dry white wine, with the potential for industrial application.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"91"},"PeriodicalIF":4.3,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12012932/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144008297","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Vacuole and mitochondria patch protein Mcp1 of Saccharomyces boulardii impairs the oxidative stress response of Candida albicans by regulating 2-phenylethanol.","authors":"Yunyun Wei, Xiaohui Zhao, Chuanqi Li, Jianhao Fu, Wanli Gao, Xiaolong Mao","doi":"10.1186/s12934-025-02721-0","DOIUrl":"https://doi.org/10.1186/s12934-025-02721-0","url":null,"abstract":"<p><strong>Background: </strong>Vacuole and mitochondria patch (vCLAMP) protein Mcp1 is crucial in eukaryotic cells response to environmental stress, but the mechanism of Mcp1 in Saccharomyces boulardii (S. boulardii) against pathogenic fungi is unclear.</p><p><strong>Results: </strong>This work first explored the role of Mcp1 in S. boulardii against Candida albicans (C. albicans). The results showed that Mcp1 located on the vacuolar and mitochondrial membrane of S. boulardii. Overexpression of Mcp1 inhibited the adhesion and hyphal formation of C. albicans in vitro. The mice model of intestinal infection revealed that WT-pGK1-MCP1 mutant enhanced the ability of S. boulardii antagonize C. albicans infecting gut. High performance liquid chromatography-mass spectrometry analysis demonstrated that overexpressing Mcp1 promoted the production of 2-phenylethanol. The latter is a secondary metabolite of S. boulardii, and can inhibit the adhesion and biofilm formation of C. albicans. The reverse transcription polymerase chain reaction and western blotting results confirmed Mcp1 promoted the production of 2-phenylethanol by regulating the expression level of Aro10. Notably, RNA-sequencing and Gene Ontology enrichment analyses showed that 2-phenylethanol impaired the oxidative stress response of C. albicans.</p><p><strong>Conclusion: </strong>This work reveals the critical role of Mcp1 in S. boulardii against C. albicans by regulating 2-phenylethanol metabolism, which provide a theoretical basis for S. boulardii as antifungal biologic therapy to prevent and treat of Candida infection.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"92"},"PeriodicalIF":4.3,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12013155/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144005375","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering a PhrC-RapC-SinR quorum sensing molecular switch for dynamic fine-tuning of menaquinone-7 synthesis in Bacillus subtilis.","authors":"Xuli Gao, Yani Luo, Elvis Kwame Adinkra, Yu Chen, Wei Tao, Yongyuan Liu, Mingyu Guo, Jing Wu, Chuanchao Wu, Yan Liu","doi":"10.1186/s12934-025-02714-z","DOIUrl":"https://doi.org/10.1186/s12934-025-02714-z","url":null,"abstract":"<p><strong>Background: </strong>Menaquinone-7 (MK-7) is a valuable vitamin K₂ produced by Bacillus subtilis. Although many strategies have been adopted to increase the yield of MK-7 in B. subtilis, the effectiveness of these common approaches is not high because long metabolic synthesis pathways and numerous bypass pathways competing for precursors with MK-7 synthesis. Regarding the modification of bypass pathways, studies of common static metabolic engineering method such as knocking out genes involved in side pathway have been reported previously. Since byproductsphenylalanine(Phe), tyrosine (Tyr), tryptophan (Trp), folic acid, dihydroxybenzoate, hydroxybutanone in the MK-7 synthesis pathway are indispensable for cell growth, the complete knockout of the bypass pathway restricts cell growth, resulting in limited increase in MK-7 synthesis. Dynamic regulation via quorum sensing (QS) provides a cost-effective strategy to harmonize cell growth and product synthesis, eliminating the need for pricey inducers. SinR, a transcriptional repressor, is crucial in suppressing biofilm formation, a process closely intertwined with MK-7 biosynthesis. Given this link, we targeted SinR to construct a dynamic regulatory system, aiming to modulate MK-7 production by leveraging SinR's regulatory influence.</p><p><strong>Results: </strong>A modular PhrC-RapC-SinR QS system is developed to dynamic regulate side pathway of MK-7. In this study, first, we analyzed the SinR-based gene expression regulation system in B. subtilis 168 (BS168). We constructed a promoter library of different abilities, selected suitable promoters from the library, and performed mutation screening on the selected promoters. Furthermore, we constructed a PhrC-RapC-SinR QS system to dynamically control the synthesis of Phe, Tyr, Trp, folic acid, dihydroxybenzoate, hydroxybutanone in MK-7 synthesis in BS168. Cell growth and efficient synthesis of the MK-7 production can be dynamically balanced by this QS system. Using this system to balance cell growth and product fermentation, the MK-7 yield was ultimately increased by 6.27-fold, from 13.95 mg/L to 87.52 mg/L.</p><p><strong>Conclusion: </strong>In summary, the PhrC-RapC-SinR QS system has been successfully integrated with biocatalytic functions to achieve dynamic metabolic pathway control in BS168, which has potential applicability to a large number of microorganisms to fine-tune gene expression and enhance the production of metabolites.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"88"},"PeriodicalIF":4.3,"publicationDate":"2025-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12010548/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144063832","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Linking a polyketide synthase gene cluster to 6-pentyl-alpha-pyrone, a Trichoderma metabolite with diverse bioactivities.","authors":"Daniel Flatschacher, Alexander Eschlböck, Siebe Pierson, Ulrike Schreiner, Valentina Stock, Arne Schiller, David Ruso, Maria Doppler, Veronika Ruzsanyi, Mario Gründlinger, Christoph Büschl, Rainer Schuhmacher, Susanne Zeilinger","doi":"10.1186/s12934-025-02718-9","DOIUrl":"https://doi.org/10.1186/s12934-025-02718-9","url":null,"abstract":"<p><strong>Background: </strong>Members of the fungal genus Trichoderma are well-known for their mycoparasitic and plant protecting activities, rendering them important biocontrol agents. One of the most significant specialized metabolites (SMs) produced by various Trichoderma species is the unsaturated lactone 6-pentyl-alpha-pyrone (6-PP). Although first identified more than 50 years ago and having pronounced antifungal and plant growth-promoting properties, the biosynthetic pathway of 6-PP still remains unresolved.</p><p><strong>Results: </strong>Here, we demonstrate that 6-PP is biosynthesized via the polyketide biosynthesis pathway. We identified Pks1, an iterative type I polyketide synthase, as crucial for its biosynthesis in Trichoderma atroviride, a species recognized for its prominent 6-PP production abilities. Phylogenetic and comparative genomic analyses revealed that the pks1 gene is part of a biosynthetic gene cluster conserved in those Trichoderma species that are known to produce 6-PP. Deletion of pks1 caused a complete loss of 6-PP production in T. atroviride and a significant reduction in antifungal activity against Botrytis cinerea and Rhizoctonia solani. Surprisingly, the absence of pks1 led to enhanced lateral root formation in Arabidopsis thaliana during interaction with T. atroviride. Transcriptomic analysis revealed co-regulation of pks1 with adjacent genes, including candidates coding for a C3H1-type zinc finger protein and lytic polysaccharide monooxygenase, suggesting coordination between 6-PP biosynthesis and environmental response mechanisms.</p><p><strong>Conclusion: </strong>Our findings establish pks1 as an essential gene for 6-PP biosynthesis in T. atroviride, providing novel insights into the production of one of the most significant compounds of this mycoparasite. These findings may pave the way for the development of improved biocontrol agents and the application of 6-PP as potent biopesticide contributing to an eco-friendly and sustainable way of plant disease management.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"89"},"PeriodicalIF":4.3,"publicationDate":"2025-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12010586/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144021842","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Effects of Magnaporthe oryzae cell-free filtrate on the secondary metabolism of Streptomyces bikiniensis HD-087: a non-targeted metabolomics analysis.","authors":"Jiahan Gang, Qingqing Tian, Chunmei Du","doi":"10.1186/s12934-025-02711-2","DOIUrl":"https://doi.org/10.1186/s12934-025-02711-2","url":null,"abstract":"<p><p>Rice blast, a disease caused by Magnaporthe oryzae, significantly threatens global rice production. To improve the anti-M. oryzae activity of Streptomyces bikiniensis HD-087 metabolites, the effects of inducer, Magnaporthe oryzae acellular filtrate, on secondary metabolism of S. bikiniensis HD-087 were studied. The results showed that M. oryzae cell-free filtrate cultured for 96 h served as the most effective inducer, significantly enhancing the anti-M. oryzae activity of metabolites of S. bikiniensis HD-087 and increasing the diameter of the inhibitory zone by 2.96 mm. The inhibition rates of M. oryzae colony diameter and spore germination in the induced group were 12.39% and 39.6% higher than those in the non-induced group, respectively. Metabolomic profiling of strain HD-087 highlighted substantial differences between the induced and non-induced groups. At 48 h of fermentation, a total of 705 distinct metabolites were identified, while at 96 h this number decreased to 321. Moreover, induction markedly altered primary pathways such as the tricarboxylic acid cycle, amino acid biosynthesis, and fatty acid metabolism in S. bikiniensis HD-087. qPCR analysis showed that nrps genes and pks genes in the induced group were significantly up-regulated by 9.92 ± 0.51 and 2.71 ± 0.17 times, respectively, and biotin carboxylase activity was also increased 26.63%. These results provide a theoretical basis for using inducers to enhance the antimicrobial ability of Streptomyces.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"90"},"PeriodicalIF":4.3,"publicationDate":"2025-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12013144/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144007803","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}