Microbial Cell Factories最新文献

{"title":"Enhanced myco-synthesis of selenium and zinc oxide nanoparticles and evaluating their anticancer activities and role against antibiotic resistance genes in certain bacterial strains.","authors":"Amira Mohamed, El-Sayed R El-Sayed, Zainab Zakaria, Mona H Mohamed, Heba K A Elhakim","doi":"10.1186/s12934-025-02795-w","DOIUrl":"10.1186/s12934-025-02795-w","url":null,"abstract":"<p><strong>Background: </strong>In an array to check microbial resistance against generally used antibiotics, it is essential to create innovative and efficient antimicrobial agents. Therefore, nanoparticles (NPs) with their antimicrobial activities describe an effective solution. In this study, we synthesized Selenium nanoparticles (Se-NPs) and zinc oxide nanoparticles (ZnO-NPs) using Alternaria alternata fungus, then their characterization were evaluated using several techniques.</p><p><strong>Results: </strong>We explored the potential of antimicrobial impact of Se-NPs and ZnO-NPs against negative and positive grams antibiotic resistance bacterial strains in combination with penicillin, Ceftriaxone and Cefipime. Moreover, antibiotic resistance gene expression was assessed after those treatments. The results demonstrated that Se-NPs and ZnO-NPs displayed antibacterial properties, while the expression of antibiotic resistance genes decreased when exposed to a combination of NPs and antibiotics. This suggests the presence of both synergistic and additive effects in these treatments. Furthermore, the cytotoxic effects of Se-NPs and ZnO-NPs were assessed, revealing their potent anticancer properties against MCF-7, A549, and HepG2 cancer cells and lower cytotoxic values for HFB-4 standard cell line. Ultimately, the production efficiency of both NPs was enhanced through gamma irradiation.</p><p><strong>Conclusions: </strong>According to the results, it seems that the green synthesis of Se-NPs and ZnO-NPs promotes environmental sustainability and cost-effective approach. This study provides insights into the development of new antibacterial and anticancer agents . The eco-friendly production of nanoparticles suggests also a sustainable approach to combating bacteria resistant to antibiotics.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"214"},"PeriodicalIF":4.9,"publicationDate":"2025-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12495769/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145228627","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":"Scaling up the production of fungal perylenequinones and investigating their biosynthesis through stable isotope labeling.","authors":"Reema A Al-Qiam, Sachin Dumbare, Tyler N Graf, Warren S Vidar, Huzefa A Raja, Cedric J Pearce, Shabnam Hematian, Nicholas H Oberlies","doi":"10.1186/s12934-025-02842-6","DOIUrl":"10.1186/s12934-025-02842-6","url":null,"abstract":"<p><strong>Background: </strong>Perylenequinones, such as hypocrellins and hypomycins, are fungal secondary metabolites with potential for pharmaceutical and industrial applications due to both their physical and biological properties. This study focused on their sustainable production. Additionally, stable isotope labeling was used to probe the biosynthesis of these compounds, demonstrating how sugars are likely incorporated into the perylenequinone scaffold.</p><p><strong>Methods: </strong>Shiraia sp. (strain MSX60519; Shiraiaceae, Pleosporales) was cultivated under varying nutrient conditions to evaluate the production of perylenequinones, with sugars serving as primary carbon sources. Five metabolites were isolated (from oatmeal cultures) using environmentally friendly solvent-based techniques, and the process was further optimized to maximize yields. High-performance liquid chromatography (HPLC) and liquid chromatography-high-resolution mass spectrometry (LC-HRMS) were employed to detect, characterize, and quantify the major compounds. Furthermore, feeding experiments were performed using ¹³C-labeled glucose, with droplet probe mass spectrometry used to monitor stable isotope incorporation in situ.</p><p><strong>Results: </strong>This study yielded three key findings. First, the production of perylenequinones was significantly enhanced by supplementing fermentation media with sugars, and disaccharides significantly enhanced the production of perylenequinones compared to monosaccharides. Optimizing sugar concentrations during the fermentation further influenced the profile of secondary metabolites. Second, stable isotope labeling experiments confirmed that sugars are the primary building blocks of perylenequinones, as noted by tracing ¹³C-labeling into ent-shiraiachrome A (1). Finally, a green, scalable, and sustainable strategy for producing these compounds on the gram scale was developed by optimizing fermentation conditions, refining purification methods, and improving extraction efficiency.</p><p><strong>Conclusion: </strong>These findings provide critical insights into optimizing fermentation conditions for the scaled and sustainable production of perylenequinones. This approach offers a cost-effective and environmentally friendly pipeline for harnessing these valuable compounds, paving the way for broader pharmaceutical and industrial applications.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"213"},"PeriodicalIF":4.9,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12492911/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145225618","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":"Biosynthesis of tauro-ursodeoxycholic acid (TUDCA) in Saccharomyces cerevisiae.","authors":"Seonyun Moon, Michael J Smanski","doi":"10.1186/s12934-025-02829-3","DOIUrl":"10.1186/s12934-025-02829-3","url":null,"abstract":"<p><p>Tauroursodeoxycholic acid (TUDCA) is a microbial bile acid known for its diverse yet still largely unexplored biological activities. Traditionally used in Asian medicine, TUDCA and the taurine-free form, ursodeoxycholic acid (UDCA), share similar therapeutic properties. Recent studies suggest that TUDCA is a promising lead compound for developing neuroprotective agents to prevent neurodegenerative diseases such as Parkinson's disease and Huntington's disease. However, sustainable and ethical sources for large-scale TUDCA production remain unavailable. To address this, we are engineering a yeast-based microbial platform capable of producing TUDCA via fermentation. Here we report strains capable of converting the more widely available primary bile acid, chenodeoxycholic acid (CDCA) into TUDCA. This was achieved by introducing heterologous genes enabling taurine conjugation and taurine biosynthesis, providing a sustainable and scalable approach for TUDCA production.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"212"},"PeriodicalIF":4.9,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12492934/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145213169","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":"Effect of different colloidal gold nanomaterials on Ganoderma lingzhi fermentation for production of ganoderma polysaccharide and triterpenoid through macroscopic and microscopic investigation.","authors":"Mengqiu Luo, Muling Shi, Yang Li, Yiqing Yang, Hanqi Wei, Shengwen Luo, Wenhuan Huang, Yida Deng, Gao-Qiang Liu","doi":"10.1186/s12934-025-02810-0","DOIUrl":"10.1186/s12934-025-02810-0","url":null,"abstract":"<p><p>Ganoderma lingzhi (G. lingzhi) is a Basidiomycete macrofungus valued for its secondary metabolites with pharmacological activity. To enhance the biosynthesis of secondary metabolites, various exogenous additives have been introduced to fungal fermentation processes. Metal nanomaterials, while known to influence cellular metabolism in mammalian systems, exhibit unclear effects when applied to macrofungal cultivation systems. The study systematically evaluates the impact of three distinct gold nanostructures-nanoparticles (AuNPs), nanorods (AuNRs), and nanoclusters (AuNCs)-on bioactive metabolite production during G. lingzhi submerged fermentation, employing integrated macroscopic process analytics and microscopic characterization. The results demonstrate that their impact on mycelial growth and bioactive metabolite production varied with the type, concentration, and addition timing of gold nanomaterials. Microscopic survey on cell surface morphology and nanoparticle distribution also reveals the different patterns of nanomaterial-mycelia cell interaction. Under the optimized addition conditions, AuNPs increased total polysaccharide content by 50.37% compared to the control group, while AuNRs increased triterpenoid content by 42.78%. The work confirms the potential of colloidal gold nanomaterials to facilitate the submerged fermentation of G. lingzhi, which is expected to encourage the development of nanomaterial additives-based approach for efficient microbial bioactive substances production.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"211"},"PeriodicalIF":4.9,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12487537/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145200196","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":"Unlocking efficient polyhydroxyalkanoate production by Gram-positive Priestia megaterium using waste-derived feedstocks.","authors":"Xinyi Bai, Libo Xu, Kang Li, Guangbao Zhang, Mengjun Zhang, Yi Huang","doi":"10.1186/s12934-025-02803-z","DOIUrl":"10.1186/s12934-025-02803-z","url":null,"abstract":"<p><p>Polyhydroxyalkanoates (PHA) are sustainable alternatives to conventional plastics due to biodegradability and biocompatibility. However, most PHA-producing strains are Gram-negative, which co-produce endotoxins that limit their applicability in high-quality biomedical fields. Additionally, industrial-scale PHA production is hindered by high costs, with feedstocks accounting for half the total expenses. In this study, a Gram-positive strain, GM-4, was isolated and evaluated for industrial potential. This strain achieved a dry cell weight (DCW) of 5.4 g/L and a PHA content of 63% with glucose, exhibiting the highest production rates at the genus level. GM-4 could efficiently utilize sugarcane molasses and corn steep liquor, yielding 13.60 g/L DCW and 9.84 g/L PHA, which represents one of the highest PHA production observed from a wild bacterial strain utilizing waste-derived feedstocks at the flask scale. This feedstock combination significantly enhanced biomass growth and PHA production by 2.6-fold and 3.1-fold, respectively, offering economic and environmental benefits. The produced PHA was determined as polyhydroxybutyrate with excellent material properties through comprehensive characterization. Whole-genome analysis clarified the metabolic pathways that convert diverse substrates into PHA. These findings position GM-4 as a promising candidate for sustainable and cost-effective PHA production, with potential for biomedical and other applications.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"210"},"PeriodicalIF":4.9,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12487134/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145200190","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":"Conversion and upgrading of syringate by Acinetobacter baylyi ADP1.","authors":"Heidi Tuomela, Johanna Koivisto, Elena Efimova, Suvi Santala","doi":"10.1186/s12934-025-02839-1","DOIUrl":"10.1186/s12934-025-02839-1","url":null,"abstract":"<p><strong>Background: </strong>Lignin holds great potential as an abundant and sustainable source of aromatic compounds, offering a viable alternative to fossil-based resources for producing chemicals and materials. Biological upgrading of lignin-derived aromatics can lead to more comprehensive lignocellulose utilization, thereby enhancing the overall feasibility of production. However, exploring a broader range of potential microbial hosts, pathways, and enzymes is crucial for developing efficient conversion processes. In particular, improving the conversion of S-lignin-related aromatics, such as syringate, remains a key area for future research.</p><p><strong>Results: </strong>In this study, we aimed to investigate the conversion of S-lignin-related syringate in Acinetobacter baylyi ADP1 by exploiting its native vanillate demethylase, VanAB. We discovered that the wild-type strain can efficiently O-demethylate syringate to 3-O-methylgallate (3MGA) and then to gallate, revealing a previously unknown activity of VanAB of A. baylyi ADP1. Conversion dynamics and in vitro characterization showed that VanAB prefers syringate as a substrate over 3MGA. Overexpression of vanAB resulted in simultaneous conversion of syringate and 3MGA, but negatively impacted growth, potentially due to toxic side product formaldehyde and redox imbalance caused by high NADH consumption of the O-demethylation reactions. Native vanAB expression resulted in 3MGA accumulation if syringate was available. We took advantage of this by constructing a strain with heterologous expression of galA, a gallate dioxygenase from Pseudomonas putida KT2440, and demonstrated the conversion of the intermediate 3MGA into 2-pyrone-4,6,-dicarboxylate (PDC), a precursor for high-quality polyesters.</p><p><strong>Conclusions: </strong>In this study, we discovered a previously unknown activity of syringate conversion in A. baylyi ADP1. By adjusting the expression level of vanAB, syringate can be directed either into gallate or 3MGA, which could be further converted into PDC through the heterologous expression of galA. Our results further highlight the potential and versatility of A. baylyi ADP1 for the conversion and upgrading of lignin-related aromatic compounds.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"209"},"PeriodicalIF":4.9,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12481942/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145192026","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 the oleaginous yeast Yarrowia lipolytica for co-production of phenolic monoterpenes thymol and carvacrol.","authors":"Junkai Zhu, Yi Zhang, Haibo Jiang, Mingming Zheng, Yangmin Gong","doi":"10.1186/s12934-025-02836-4","DOIUrl":"10.1186/s12934-025-02836-4","url":null,"abstract":"<p><strong>Background: </strong>Thymol and carvacrol are natural phenolic compounds with various biological activities. They are widely used in the spice and medicine industries. Production of thymol and carvacrol using microbial cell factories is considered a viable alternative to extracting them from plants of the Thymus genus or the Lamiaceae family.</p><p><strong>Results: </strong>In this study, the complete synthetic pathways of thymol and carvacrol were constructed using the oleaginous yeast Yarrowia lipolytica Po1f as a chassis. The total titer of thymol and carvacrol was increased 18.44-fold by enhancing the mevalonate pathway during the modification process and reducing the metabolic flux from geranyl diphosphate (GPP) to farnesyl diphosphate (FPP) through the modification of ERG20. Next, by increasing the copy number of the TvCYP71D507 gene combination (TvCYP71D507 + TvTPS2 + TvSDR1) in the synthetic pathway, the total titer was increased by 1.75-fold. Finally, the engineered strain CT18, which was reintroduced the 3-isopropylmalate dehydrogenase (LEU2) gene, achieved a titer of thymol and carvacrol of 7.14 mg/L in shake flasks and 61.31 mg/L in a 5-L bioreactor.</p><p><strong>Conclusion: </strong>This study demonstrates the de novo synthesis of thymol and carvacrol in Y. lipolytica for the first time and provides a valuable reference for constructing microbial cell factories for phenolic monoterpenes biosynthesis.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"208"},"PeriodicalIF":4.9,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12482179/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145192044","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":"Bacteria-based immunosuppressive tumor microenvironment reprogramming: a promising dawn in cancer therapy.","authors":"Mostafa Akbariqomi, Peyman Kheirandish Zarandi, Azam Abedi, Mehrdad Moosazadeh Moghaddam, Abbas Ali Imani Fooladi","doi":"10.1186/s12934-025-02838-2","DOIUrl":"10.1186/s12934-025-02838-2","url":null,"abstract":"<p><p>Traditional chemotherapy, a prevalent cancer treatment modality, is associated with significant side effects and often leads to treatment failure. Non-specific drug distribution and chemoresistance are the main factors contributing to this failure. Certain distinctive characteristics of the tumor microenvironment (TME), including hypoxia, acidic pH, and increased interstitial fluid pressure, render cancer cells resistant to conventional treatments. Multiple approaches have been devised to enhance the treatment efficiency of neoplasms and overcome chemoresistance. Nowadays, bacteria-based cancer therapy has garnered significant interest in both preclinical and clinical research, owing to its distinctive mechanism and various applications in eliciting host antitumor immunity. Due to their inherent tumor tropism, elevated motility, and capacity for quick colonization in the conducive TME, bacteria are increasingly being considered for targeted tumor treatment. Bacteria, rich in pathogen-associated molecular patterns (PAMPs), can efficiently stimulate immune cells even inside the immunosuppressive TME, boosting the particular immune detection and eradication of tumor cells. Furthermore, outer membrane vesicles (OMVs), cytoplasmic membrane vesicles (CMVs), and their derived physiological components exhibit analogous functionalities to their parental cells. This review article is representative of the latest innovations in bacteria-based immunosuppressive TME reprogramming. Additionally, the article discusses future directions in this research area, drawing on current advances.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"207"},"PeriodicalIF":4.9,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12465396/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145176534","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":"Characterizing the growth of PHA-producing microorganisms on short-chain carboxylic acids.","authors":"Steven Leonhardt, Pravesh Tamang, Günter E M Tovar, Susanne Zibek","doi":"10.1186/s12934-025-02840-8","DOIUrl":"10.1186/s12934-025-02840-8","url":null,"abstract":"<p><p>Short-chain carboxylic acids are important chemical intermediates in anaerobic treatment or thermal hydrolysis of biowaste. They are a crucial precursor for the production of Polyhydroxyalkanoate (PHA) by various microorganisms. However, there is limited data on the growth behavior of PHA-producing microorganisms on carboxylic acids and their tolerance range. Therefore, this study aims to systematically determine the growth behavior of four commonly used PHA-producing microorganisms (Cupriacidus necator, Pseudomonas putida, Azohydromonas australica, and Haloferax mediterranei) on different carboxylic acids (formic, acetic, propionic, valeric, and levulinic acid). Batch experiments were conducted in a microbioreactor to determine the maximum specific growth rates (µ_max) on varying acid concentrations. Additionally, the data was analyzed using the Han-Levenspiel growth model to determine the inhibitory effect of the acids on the microorganisms. Our experiments showed that P. putida had the highest µ_max on propionic acid (0.165 h^- 1) and levulinic acid (0.16 h^- 1). Likewise, H. mediterranei also showed the highest µ_max on propionic acid (0.174 h^- 1). For C. necator, the highest µ_max was determined in acetic (0.102 h^- 1) and levulinic acid (0.109 h^- 1). Among all the tested carboxylic acids, acetic acid was found to be the least toxic acid and was also the only substrate that enabled meaningful growth of A. australica. Furthermore, formic acid seems to be the least suitable substrate for the growth of these microorganisms except H. mediterranei as only the growth of this microorganism was observed. Based on these results, we concluded that carboxylic acids are not a suitable carbon source for the growth of A. australica. While C. necator and P. putida are more versatile in utilizing various acids, except formic acid. They showed the largest optimum growth or tolerance range in acetic and levulinic acid, making these acids the most preferred substrate for growth. Lastly, H. mediterranei could grow in all carboxylic acids, however, the requirement of a highly saline medium would be challenging.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"206"},"PeriodicalIF":4.9,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12455835/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145131360","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":"Development of a highly degenerate primer-based molecular tool for detecting and classifying the four major classes of polyhydroxyalkanoate synthase (phaC) genes in bacteria.","authors":"Abdiqani Ibrahim Osman, Brendon Noble, Linda Percy, Pooja Basnett","doi":"10.1186/s12934-025-02831-9","DOIUrl":"10.1186/s12934-025-02831-9","url":null,"abstract":"<p><p>Polyhydroxyalkanoate synthase (phaC) gene encodes for PHA synthase enzyme which plays a key role in PHA polymerisation. To screen, unknown bacterial strains for their potential to produce PHAs, the presence of phaC gene is essential. Currently published primer sets targeting phaC gene are inadequate and often work only for well-studied genera (e.g., Pseudomonas, Cupriavidus). Few studies validate them in vitro, even fewer use degenerate primers to address phaC sequence diversity, and many fail to target all four phaC classes. In this study, nine novel highly degenerate primers were designed using the HYDEN (HighlY DEgeNerate) tool. The design included 65 phaC gene sequences from class I, 10 from class II, 19 from class III, 30 from class III/IV, and 6 from class IV, carefully selected as a representative sample size to capture the variations among bacterial strains and phaC sequences. The primer specificity was then assessed in silico with De-MetaST-BLAST against all known phaC sequences in the NCBI database. This was followed by in vitro screening of seven bacterial strains known to express the four major classes of phaC genes and 15 novel marine bacterial strains in which phaC presence were unknown. Seven strains-namely Halomonas alkaliphila DINO, Marinobacter sp. MB2, Halomonas profundus NQ7, Halomonas titanicae MC2, Bacillus pacificus C4, Bacillus pacificus B4 and Bacillus mycoides B12 tested positive. All 15 strains were subjected to nutrient limiting growth conditions to assess PHA production with results confirming molecular screening. This study demonstrates the successful development and validation of a highly degenerate primer-based molecular screening tool capable of detecting and differentiating the four major classes of phaC genes in well-known non-marine and novel marine PHA-storing bacteria.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"205"},"PeriodicalIF":4.9,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12455824/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145124973","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}