Microbial Cell Factories最新文献

{"title":"The impact of evolutionary dynamics and expression plasmid stability on long-term heterologous protein production by yeast.","authors":"Mingzhi Xu, Andrei Parker, Klaus Winzer, Simon V Avery, Christopher Finnis","doi":"10.1186/s12934-026-03071-1","DOIUrl":"10.1186/s12934-026-03071-1","url":null,"abstract":"<p><strong>Background: </strong>Baker's yeast, Saccharomyces cerevisiae, is a widely used industrial host organism for heterologous protein production. Ensuring stable, high-level production over extended production campaigns is important for low-cost manufacturing. However, maintaining consistent yields over multiple generations is challenging as strain productivity may decline over time. Understanding potential evolutionary mechanisms underlying such decline is therefore important for optimising yeast-based expression systems in continuous manufacturing.</p><p><strong>Results: </strong>In this study, diverse yeast libraries carrying high-copy-number whole-2-micron episomal expression plasmids producing a secreted SARS-CoV-2 spike protein fragment fused to mCherry were used to investigate fluorescence-based production stability during prolonged cultivation in yeast. We observed that while some strains maintained relatively stable mCherry fluorescence, most strains with high initial total fluorescence showed marked declines over 140 generations. Growth assays indicated that cultures with reduced mCherry fluorescence had a fitness advantage over high-fluorescence cultures. The decline was partly explained by phenotypic heterogeneity among genetically uniform cells; early-stage (10th generation) cultures contained distinct, non-fluorescent, low-fluorescence and high-fluorescence subpopulations (these subpopulation phenotypes were non-heritable), whereas evolved low-fluorescence cultures after 140 generations displayed loss of the high- and/or low-fluorescence subpopulations and reduced cell-to-cell heterogeneity. Despite this, the selected evolved low-fluorescence isolates had fluorescence restored to near-initial levels after being cured of their expression plasmids and retransformed with plasmids from early-stage, high-fluorescence cultures, indicating that, in the selected evolved low-fluorescence isolates analysed, plasmid-associated changes were sufficient to explain the stable reduction in mCherry fluorescence. Whole-plasmid sequencing of the episomal 2-micron expression plasmids isolated from low-fluorescence cultures at the 140th generation revealed mutations in key functional regions, including the methionine-regulated MET17 promoter driving SARS-CoV-2 protein expression, FRT recombination sites and the SARS-CoV-2 spike protein fragment coding sequence.</p><p><strong>Conclusion: </strong>The results support a model in which prolonged expression of a burdensome secreted recombinant protein can be associated with early non-genetic heterogeneity in intracellular mCherry fluorescence, growth advantages of low-fluorescence cells and evolution of plasmid variants that reduce expression. The extent of fluorescence decline varied among genetically diverse strains, with some remaining comparatively stable, highlighting the potential of exploiting yeast strain diversity to obtain production strains with improved long-term stability.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"25 1","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13682124/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149224474","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":"Optimization of biomass production, morphology, architecture, and nutrient removal by Rhizopus oryzae M10A1 cultivated on potato starch processing effluents as a fungal cell factory.","authors":"Miguel Anchundia","doi":"10.1186/s12934-026-03074-y","DOIUrl":"10.1186/s12934-026-03074-y","url":null,"abstract":"<p><strong>Background: </strong>The valorization of agro-industrial effluents through filamentous fungi represents a strategic pillar of the circular bioeconomy. Liquid residues generated during potato starch processing contain substantial amounts of carbon and nitrogen, making them suitable substrates for fungal biomass production. Despite this potential, few studies have integrated statistical optimization, morphological characterization, and nutrient assimilation efficiency in a single bioprocess using defined Rhizopus oryzae strains. This study evaluated and optimized biomass production, growth morphology, and nutrient removal efficiency of Rhizopus oryzae M10A1 cultivated in potato starch processing effluents under submerged fermentation.</p><p><strong>Results: </strong>Biomass production reached 15.70 g/L after optimization using response surface methodology at 35 °C, pH 4, and 150 rpm, representing approximately a 15% increase compared to the initial conditions. The quadratic model showed strong predictive performance (adjusted R² = 89.67%), adequate precision (18.19), and a non-significant lack of fit (p = 0.089). Nutrient removal efficiencies were 85.85% for total sugars, 80.49% for total starch, 74.29% for total nitrogen, and 70.90% for reducing sugars. Morphological analysis revealed predominant pellet formation (85%) with a higher surface-to-volume ratio (1.58 ± 0.69) than clumps, suggesting improved mass transfer conditions.</p><p><strong>Conclusions: </strong>Rhizopus oryzae M10A1 efficiently converted starch-rich effluents into fungal biomass under statistically optimized conditions while substantially reducing nutrient load. The integration of statistical modeling and morphological control with physical and chemical parameters highlights its potential as a fungal cell factory within sustainable waste valorization and circular bioeconomy approaches.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"25 1","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13678927/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149220553","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":"Replication-coupled dual-deaminase mutagenesis (RCDM) enables continuous genomic evolution in Bacillus subtilis.","authors":"Xin Zhang, Pengju Wang, Ju Li, Xingmiao Zhu, Jingzhen Qian, Yajing Liu, YiWen Sun, Rong Li, Changhao Bi, Xueli Zhang","doi":"10.1186/s12934-026-03060-4","DOIUrl":"10.1186/s12934-026-03060-4","url":null,"abstract":"<p><p>Bacillus subtilis is an important Generally Recognized as Safe (GRAS) chassis widely used for industrial enzyme production, yet systematic improvement of complex host phenotypes-such as protein secretion capacity-remains difficult using rational engineering alone. Continuous evolution strategies offer a powerful alternative but remain underdeveloped in B. subtilis, largely due to the organism's efficient DNA repair systems that suppress mutagenic intermediates. Here, we report a replication-coupled dual-deaminase mutagenesis platform (RCDM) that enables efficient and continuous genome-wide diversification in B. subtilis. In this system, complementary cytidine and adenine deaminases are tethered to the helicase-loading factor DnaB to access replication-associated single-stranded DNA. Systematic architectural optimization revealed a striking topology dependence of mutagenic activity, where cytidine deamination requires C-terminal fusion whereas adenine deamination is most efficient when fused to the N-terminus of DnaB. Importantly, we show that host DNA repair strongly shapes the mutational output: uracil excision by Ung effectively masks cytidine-derived mutations in dual-deaminase constructs. Co-expression of the uracil-DNA glycosylase inhibitor Ugi alleviates this repair constraint and restores balanced C: G→T: A and A: T→G: C transitions, producing mutation frequencies of (2.80 ± 0.10) × 10⁻⁴ across the genome. To demonstrate the utility of this platform, we coupled α-amylase secretion to growth on soluble starch and performed growth-coupled continuous evolution. Within 20 iterative enrichment cycles, the evolved population exhibited a ~ 30-fold increase in extracellular α-amylase activity relative to the parental strain. Together, these results establish a replication-targeted and repair-aware continuous evolution platform for B. subtilis, providing a versatile strategy for rapid optimization of complex host phenotypes and expanding the continuous evolution toolbox for industrial Gram-positive microbes.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"25 1","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13673809/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149207188","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":"Comparative analysis of precursor supply and pathway reinforcement strategies for shikimic acid biosynthesis in Pseudomonas putida.","authors":"İrem İlter, Tanya Beril Korkmaz, Hatice Arslan, Ahmet Kalaycı, Emrah Kırdök, Tomás Aparicio, Özlem Akkaya","doi":"10.1186/s12934-026-03042-6","DOIUrl":"10.1186/s12934-026-03042-6","url":null,"abstract":"<p><p>The shikimate pathway is a central metabolic route for the biosynthesis of high-value aromatic compounds. Traditional metabolic engineering strategies have largely focused on overexpressing pathway-specific genes, enhancing precursor supply, or introducing feedback-resistant variants of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase to increase pathway flux. However, how these distinct strategies differentially affect flux distribution and the accumulation of key intermediates, specifically 3-dehydroshikimate (DHS) and shikimic acid (SA), remains largely unexplored in Pseudomonas putida (P. putida). Here, we systematically assess the effects of commonly applied engineering strategies on DHS and SA production using distinct SA pathway modules. The results show that overexpression of transketolase I (tktA), phosphoenolpyruvate synthase (ppsA), and SA pathway genes (aroE, aroB, aroQ-II), together with heterologous expression of feedback-resistant aroG, in a genome-edited P. putida EM42 strain harboring pykA/pykF deletions and a hexR knockout, yielded approximately 71 mg L⁻¹ shikimic acid, corresponding to a > 284-fold increase relative to the wild-type EM42. Notably, the findings reveal that following the relief of precursor constraints, the achievable maximum titer is unaffected by the particular DAHP synthase isoform utilized. Furthermore, the standalone overexpression of ppsA and tktA yielded SA and DHS levels comparable to those achieved with the additional expression of the aforementioned pathway genes. However, despite this phenotypic convergence in titers, comparative transcriptomic analysis revealed that the genetic route taken to reach these levels is associated with markedly distinct transcriptional configurations of the host metabolic network. In the same genetic background, amplification of ppsA-tktA alone is associated with an increased transcriptional investment in the biosynthesis of essential cofactors and metabolic precursors, whereas overexpression of the SA pathway is accompanied by enrichment of protocatechuate catabolic genes, consistent with the intrinsic aromatic funneling capacity of P. putida under conditions of elevated shikimate pathway flux. These results demonstrate that evaluating engineering success solely based on final product yields obscures substantial differences in underlying metabolic states. Accordingly, we propose that tailored engineering strategies guided by key pathway intermediates are critical for the rational optimization of P. putida as an efficient cell factory.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"25 1","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13678936/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149220592","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":"Transcriptional reprogramming of the stress response and translational-secretory machinery enhances recombinant secreted protein production in Yarrowia lipolytica.","authors":"Maria Gorczyca, Piotr Kubiak, Ewelina Celińska","doi":"10.1186/s12934-026-03062-2","DOIUrl":"10.1186/s12934-026-03062-2","url":null,"abstract":"<p><p>Protein synthesis and secretion processes are non-pathway traits. The genetic complexity makes them very challenging to engineer and genetically optimize. Therefore, we exploit transcriptional reprogramming to create a favorable cellular environment for a trait of interest. In this study, we investigate the effects of transcription factor (TF) engineering on secretory recombinant protein (rProt) production in Y. lipolytica. High-throughput genome-wide pre-screening guided the TFs selection. Here, we run high-throughput functional studies, followed by transcriptome analysis for selected TF-modified strains from semi-steady state. We contrasted the overexpression (OE) vs. deletion (KO) genotypes for their capacity to produce two reporter proteins (fluorescent model and glucoamylase). Despite a wide set of TF-modified strains analyzed here, only several displayed consistent, unbiased phenotypes. Collected data indicated that the known general stress response TFs, Msn4 and Hsf1, elicit their highly beneficial rProt-promoting function specifically towards a \"challenging\" rProt, while the levels of \"simple\" rProt remain unaffected. Deletion of the previously identified strong repressor of intracellular rProt synthesis, Azf1, significantly enhanced production of secretory rProt. At the transcriptomic level, deletion of Azf1 was associated with massive upregulation of ribosome biogenesis and cytoplasmic translation, which underwent alternative splicing (global differential exon usage analysis). The results of contrasting the Azf1- and Dep1-driven regulomes (the latter identified as a strong repressor of intracellular rProt synthesis, with no positive effect on the secretory rProt production) suggest that for efficient rProt secretion, upregulation in the vesicle-mediated transportation must co-occur, as in the case of Azf1-KO. In our experimental setting, OE of the general stress response TFs was beneficial for the production of \"problematic\", UPR-awakening rProt, and deletion of Azf1 for tuning a molecular background beneficial for secretory rProt.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13670810/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148536312","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":"A versatile toolset for Staphylococcus aureus gene inactivation enables testing of anti-phage defense systems in native genetic backgrounds.","authors":"Łukasz Kałuski, Stephen Amankwah, Laura Wicke, Ján Bíňovský, Emil Stefańczyk, Ivana Mašlaňová, Lucie Kuntová, Roman Pantůček, Małgorzata Łobocka","doi":"10.1186/s12934-026-03063-1","DOIUrl":"10.1186/s12934-026-03063-1","url":null,"abstract":"<p><strong>Background: </strong>Functional gene analysis of Staphylococcus aureus across different strains requires the development of genetic engineering tools. In studies on phage therapy, such analysis is especially important for understanding the genomic differences between strains, underlying the differences in sensitivity to phages. Meanwhile, difficulties in introducing DNA constructs for genetic manipulations into non-laboratory S. aureus strains hinder the elucidation of staphylococcal anti-phage defense strategies.</p><p><strong>Results: </strong>We used a transducing S. aureus bacteriophage and an E. coli -S. aureus shuttle plasmid with a DNA fragment of this phage to develop a toolset for recombinational gene replacement in difficult-to-transform S. aureus isolates. The plasmid carried an erythromycin-resistance cassette flanked by unique restriction sites for cloning the flanking regions of target DNA to be replaced. Plasmid loss from recombinants with the desired gene replacement was achieved by removing the plasmid par gene, which is essential for plasmid inheritance. Recombinants that acquired the cassette but lost the plasmid were resistant to erythromycin and susceptible to chloramphenicol selective for plasmid backbone. We applied our system to inactivate the predicted anti-phage defense gene avs2 in a clinical S. aureus isolate and inactivated the identical gene in a laboratory strain. While the mutation sensitized the laboratory strain to three phages, it did not affect the clinical strain's sensitivity to phages, demonstrating the complexity of phage-bacterial interactions and limitations of bioinformatic phage susceptibility predictions without experimental validation. The large terminase subunits of Avs2-sensitive phages were predicted to form complexes with Avs2, suggesting their direct involvement in triggering Avs2.</p><p><strong>Conclusions: </strong>Our new, simple toolset for introducing plasmids carrying donor DNA for genetic manipulation into S. aureus strains that are difficult to transform proved suitable for generating chromosomal gene knockouts in clinical S. aureus isolates. Its use to knock out an exemplary anti-phage defense gene, avs2, in a clinical S. aureus strain and comparative phenotypic analysis of the obtained mutant with that of a laboratory strain revealed differences resulting presumably from different genomic backgrounds of target genes in each strain. This supports the validity of our approach and shows the importance of empirical phage sensitivity testing of strains in native backgrounds.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13660305/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148471696","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":"Biocatalytic synthesis of alkoxylated phenazine derivatives using methyltransferases.","authors":"Chaozhi Wang, Shuo Zhang, Sijia Xu, Chuanzeng Wang, Zhe Zhang, Mohd Sadeeq, Yupeng Wan, Chen Gao, Wei Huang, Peng Xiong, Feifei Hou","doi":"10.1186/s12934-026-03069-9","DOIUrl":"10.1186/s12934-026-03069-9","url":null,"abstract":"<p><strong>Background: </strong>Phenazines are important nitrogen-containing heterocycles with diverse applications in the chemical and pharmaceutical industry. Alkoxylated phenazines, in particular, exhibit promising acaricidal and fungicidal properties. Currently, chemical synthesis is the main approach for alkoxylated phenazines and derivatives production. However, these processes are associated with harsh reaction conditions, accumulation of chemical waste (e.g., organic solvents, noble metal catalysts), and environmental concerns.</p><p><strong>Results: </strong>In this study, we developed biocatalytic systems for the synthesis of alkoxylated phenazines by designing enzymatic systems and de novo biosynthetic pathways. The O-methyltransferase LaphzM from Lysobacter antibioticus OH13 was shown to catalyze the alkoxylation of phenazines using SAM analogs generated in situ by halide methyltransferases (HMTs) from Burkholderia xenovorans and Arabidopsis thaliana. Using these enzymatic systems, we successfully synthesized six alkoxylated phenazine derivatives, including three novel compounds. We further established de novo biosynthetic pathways for 1-ethoxyphenazine and 2-ethoxyphenazine in Pseudomonas chlororaphis via the direct sulfurylation pathway from S. cerevisiae. To improve production, we optimized whole-cell systems, achieving 486.3 mg/L (54% yield) of 2-ethoxyphenazine, 218.1 mg/L (22.8% yield) of 2-propoxyphenazine and 378.2 mg/L (78.4% yield) of 1-ethoxyphenazine-N'10-oxide within 7 h using microbially produced 2-hydroxyphenazine (for 2-ethoxyphenazine and 2-propoxyphenazine) or 1-hydroxyphenazine (for 1-ethoxyphenazine-N'10-oxide) as the substrate, along with ethyl iodide (EtI) or propyl iodide (PrI) accordingly.</p><p><strong>Conclusions: </strong>Overall, we successfully established biocatalytic platforms for alkoxylated phenazines through enzymatic catalysis and de novo biosynthetic pathways. Methyltransferases derived from diverse species, including LaphzM and HMTs, play key roles in the SAM-analog-mediated alkylation of phenazines. This study provides a promising alternative to conventional chemical synthesis, with significant potential for the manufacturing of alkoxylated phenazine derivatives.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13660096/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148471730","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":"Establishment of SRLC: a multiplex genome editing technology for Saccharomyces cerevisiae and its application in metabolic engineering of malonyl-CoA pathway.","authors":"Wentao Zheng, Miao Wang, Qiang Tu, Xiaoying Bian, Youming Zhang, Xue Wang","doi":"10.1186/s12934-026-03068-w","DOIUrl":"10.1186/s12934-026-03068-w","url":null,"abstract":"<p><p>The development of advanced genome engineering tools is crucial for optimizing metabolic pathways in Saccharomyces cerevisiae and achieving efficient biomanufacturing. This study proposes an enhancing multiplex genome editing strategy in S. cerevisiae by employing Escherichia coli-derived single-stranded annealing proteins (SSAPs) combined with S. cerevisiae-derived homologous recombinases (Rad51 and Rad52). The strategy utilizes an SSAP-Rad-Linearized CRISPR (SRLC) platform, which supports efficient simultaneous editing of multiple genomic loci without constructing complex multi-gRNA expression vectors. Co-overexpressing Rad51/Rad52 and E. coli SSAP proteins significantly enhances homologous recombination (HR), allowing precise multi-locus genome editing mediated by short homologous arms. Furthermore, SRLC employs a linearized CRISPR-Cas system to stimulate homologous recombination and enable counter-selection in S. cerevisiae, thereby improving precise multiplex genome editing efficiency. We applied SRLC to engineer the malonyl-CoA metabolic pathway in S. cerevisiae. Through a single round of editing and screening, we constructed a chassis strain with 9 targets simultaneously modification and achieved a 9.6-fold increase in intracellular malonyl-CoA. Using this chassis, 3-hydroxypropionic acid production increased 4.5-fold relative to wild-type S. cerevisiae. This platform offers a robust and scalable tool for S. cerevisiae manipulation and a practical pathway-engineering strategy for building for malonyl-CoA-derived factories.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13656355/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148471653","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":"Enhancing recombinant eGFP secretion in P. tricornutum by optimizing culture conditions.","authors":"Nesrine Gargouch, Aurélie Charrier, Charlotte Toustou, Gaël Bougaran, Bruno Saint-Jean, Muriel Bardor","doi":"10.1186/s12934-026-03061-3","DOIUrl":"10.1186/s12934-026-03061-3","url":null,"abstract":"<p><p>Phaeodactylum tricornutum is a promising candidate for the production of recombinant therapeutic proteins including antibodies. So far, low secretion yields remain a significant bottleneck for pharmaceutical applications. Towards the objective to increase production yield of secreted recombinant protein, this study investigates the optimization of environmental and nutritional parameters, under laboratory conditions, to maximize the production of a model secreted recombinant protein (eGFPsec) under the control of the Nitrate Reductase promoter (P_NR). We evaluated the interplay between nitrate availability, Photosynthetic Photon Flux Density (PPFD), and pH level within both natural and artificial seawater matrices. Our results demonstrate that while P_NR activity does not increase with nitrate concentrations beyond non-limiting levels, a PPFD of 150 µmol photons m^- 2 s^- 1 significantly enhances eGFPsec accumulation in the medium. Interestingly, cultures grown in Natural Sea Water under pH-regulated conditions (pH = 8) exhibited markedly higher secreted-eGFP fluorescence compared to those in artificial seawater. These findings highlight the importance of optimizing key environmental and nutritional parameters to improve secretion recombinant protein yields. By identifying key drivers of eGFP fluorescence-based productivity and accumulation in the medium, this work provides actionable insights for process optimization, reinforcing the potential of P. tricornutum as a robust and cost-effective platform for industrial microalgae-based biomanufacturing.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13656395/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148471705","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":"Functional redundancy driven by isocitrate dehydrogenase enhances metabolic robustness in Pseudomonas putida KT2440.","authors":"Maria-Tsampika Manoli, Sandra Herrera Alarcon, Álvaro Gargantilla-Becerra, M Auxiliadora Prieto, Juan Nogales","doi":"10.1186/s12934-026-03053-3","DOIUrl":"10.1186/s12934-026-03053-3","url":null,"abstract":"<p><strong>Background: </strong>Prokaryotic isocitrate dehydrogenase (PIDH) is a conserved and highly versatile enzyme regulating carbon flux between the TCA cycle and the glyoxylate shunt. While most mesophilic organisms encode a single PIDH, the industrial chassis Pseudomonas putida KT2440 harbours two isoforms: a monomeric (IDH) and a dimeric (ICD). The physiological significance of this dual-system in mesophiles remains poorly understood, yet it may provide a framework for understanding metabolic flexibility and flux regulation in biotechnological applications.</p><p><strong>Results: </strong>Here, we biochemically characterized both PIDHs and, through modelling and site-directed mutagenesis, identified key residues (Ser133, Asn136, and Arg140) critical for monomeric catalytic activity and substrate binding. Genetic analysis revealed that the monomeric IDH is essential for growth, whereas the dimeric ICD appears to facilitate high-flux oxidative metabolism. System-level analysis integrating transcriptomics and flux modelling further demonstrated that these isoforms might fuel distinct TCA functional modes. Our results suggest that IDH sustains a basal, carbon-independent mode critical for biosynthetic precursor supply. In contrast, ICD drives a high-flux oxidative mode under glycolytic conditions and is post-translationally inactivated under gluconeogenic conditions, favouring the glyoxylate cycle.</p><p><strong>Conclusions: </strong>Taken together, these findings support a model in which specialized isoenzymes with conditional redundancy, rather than simple redundancy, in P. putida. This dual-enzyme system enables the cell to balance energy production with biosynthetic demands, which might facilitate metabolic adaptation under fluctuating environmental conditions. Our results provide a potential modular framework for engineering central metabolism to optimize precursor provisioning in microbial cell factories.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13660168/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148471735","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}