Microbial Biotechnology最新文献

{"title":"Correction to ‘Metagenomic Exploration Uncovers Several Novel “Candidatus” Species Involved in Acetate Metabolism in High-Ammonia Thermophilic Biogas Processes’","authors":"","doi":"10.1111/1751-7915.70217","DOIUrl":"10.1111/1751-7915.70217","url":null,"abstract":"<p>Cheng, G. B., E. Bongcam-Rudloff, and A. Schnürer. 2025. \"Metagenomic Exploration Uncovers Several Novel ‘<i>Candidatus</i>’ Species Involved in Acetate Metabolism in High-Ammonia Thermophilic Biogas Processes.\" <i>Microbial Biotechnology</i> 18: e70133. https://doi.org/10.1111/1751-7915.70133.</p><p>In our original publication, we mistakenly acknowledged Prof. Oren for assisting with the naming of the candidate species. While we did discuss general nomenclatural principles with Prof. Oren, the specific names proposed in the original version were developed independently by the authors, and he did not advise on or approve those names. We regret this misrepresentation and in this corrigendum, we present revised naming suggestions that better reflect appropriate taxonomic practices. These revisions have benefited from input by Prof Oren, whose guidance on naming conventions and principles we gratefully acknowledge.</p><p>We here present the corrected versions of the protologues for the four new <i>Candidatus</i> genera and species proposed in Section 3.7 of the original paper. ‘<i>Candidatus</i> Thermotepidanaerobacter aceticum’ have been changed to ‘<i>Candidatus</i> Thermotepidanaerobacter aceticus’ and ‘<i>Candidatus</i> Thermodarwinisyntropha acetovorans’ have been changed to‘<i>Candidatus</i> Thermodarwinisyntropha acetivorans’. Numbers of figures and tables and references cited below refer to that paper. Further information about the taxa can be found in the original publication and its supporting information. In Figure 3, the updated taxonomic names are included in this corrigendum. Furthermore, there was a spelling mistake of the phylum <i>Thermotogota</i>. It was previously spelled as <i>Thermotoga</i>, which is the genus name. Moreover, the original name of <i>Metanoculleus thermohydrogenotrophicum</i> was given in the databases (GTDB/NCBI), while the name later has been updated to‘<i>Candidatus</i> Methanoculleus thermohydrogenitrophicus’. Figure 3 has been updated accordingly.</p><p>We apologize for this error.</p><p>The corrected Section 3.7 should read as:</p><p>\u0000 <b>3.7 Description of New Genus/Species</b>\u0000 </p><p>The genomes presented below are novel species with the ability to produce or consume acetate via WLP/GSRP, which motivated a further analysis to reveal information on their genomic potential and classification as ‘Candidatus’ species. The analysis included both an investigation of general genome characteristics and phylogeny as well as analysis involving carbon and energy metabolism (Table S4).</p><p><b>‘<i>Candidatus</i> Thermotepidanaerobacter’ gen. nov</b>.</p><p>Thermotepidanaerobacter (Ther.mo.te.pid.an.ae.ro.bac'ter. Gr. masc. adj. <i>thermos</i>, hot; N.L. masc. n. <i>Tepidanaerobacter</i>, a bacterial genus name; N.L. masc. n. ‘<i>Candidatus</i> Thermotepidanaerobacter’, a hot-loving <i>Tepidanaerobacter</i>).</p><p>The properties of the genus are as the properties of the only species describ","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 8","pages":""},"PeriodicalIF":5.2,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://enviromicro-journals.onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70217","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144832608","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 of 1,4-Butanediol and Adipic Acid Metabolism in Pseudomonas taiwanensis for Upcycling to Aromatic Compounds","authors":"Leonie Op de Hipt,&nbsp;Yannic S. Ackermann,&nbsp;Hannah de Jong,&nbsp;Tino Polen,&nbsp;Benedikt Wynands,&nbsp;Nick Wierckx","doi":"10.1111/1751-7915.70205","DOIUrl":"10.1111/1751-7915.70205","url":null,"abstract":"<p>The overwhelming amount of plastic produced is an unprecedented challenge for humanity due to the lack of end-of-life solutions for heterogeneous plastic wastes. One possibility is feedstock recycling of mixed plastics and complex polymers with subsequent biological funnelling and upcycling. Major depolymerisation products of common plastics such as polyurethanes, polyesters and polyamides include aliphatic dicarboxylic acids or diols such as adipic acid (AA) and 1,4-butanediol (BDO), which can be metabolised by engineered <i>Pseudomonas putida</i> strains. However, the spectrum of upcycled compounds that can be produced from these monomers is still limited. Therefore, we extended the substrate spectrum of an aromatics-overproducing <i>Pseudomonas taiwanensis</i> strain to AA and BDO. Adaptive laboratory evolution (ALE) followed by genome sequencing was used to identify and reverse engineer key growth-enabling mutations. In this context, we observed a conflict between the dual objectives of fast growth on AA and efficient aromatics production, which materialised in the form of mutations in the ribosomal protein-encoding gene <i>rpmE.</i> These mutations promote faster growth on AA at the cost of aromatics production. In contrast to <i>P. putida</i> KT2440, knockout of the repressor gene <i>psrA</i> regulating expression of genes involved in <i>β</i>-oxidation had no positive effect on growth of <i>P. taiwanensis</i> on AA. Evolution for growth on BDO revealed several point mutations that affect expression of multiple oxidoreductases, with an identified key role for the dehydrogenase encoded by PVLB_10545. This dehydrogenase likely catalyses the initial oxidation of BDO, thus substituting for PedE, which is present in <i>P. putida</i> but absent in <i>P. taiwanensis</i>. Integration of <i>RpcTAL</i> into the Tn7 site enabled <i>de novo</i> production of 4-coumarate with a yield of 14.4% ± 0.1% (Cmol/Cmol) from BDO and 11.5% ± 0.3% (Cmol/Cmol) from AA. Thereby, the potential of these <i>P. taiwanensis</i> strains for upcycling plastic hydrolysates to value-added compounds was successfully demonstrated.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 8","pages":""},"PeriodicalIF":5.2,"publicationDate":"2025-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://enviromicro-journals.onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70205","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144814565","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 of 1,4-Butanediol and Adipic Acid Metabolism in Pseudomonas taiwanensis for Upcycling to Aromatic Compounds","authors":"Leonie Op de Hipt,&nbsp;Yannic S. Ackermann,&nbsp;Hannah de Jong,&nbsp;Tino Polen,&nbsp;Benedikt Wynands,&nbsp;Nick Wierckx","doi":"10.1111/1751-7915.70205","DOIUrl":"10.1111/1751-7915.70205","url":null,"abstract":"<p>The overwhelming amount of plastic produced is an unprecedented challenge for humanity due to the lack of end-of-life solutions for heterogeneous plastic wastes. One possibility is feedstock recycling of mixed plastics and complex polymers with subsequent biological funnelling and upcycling. Major depolymerisation products of common plastics such as polyurethanes, polyesters and polyamides include aliphatic dicarboxylic acids or diols such as adipic acid (AA) and 1,4-butanediol (BDO), which can be metabolised by engineered <i>Pseudomonas putida</i> strains. However, the spectrum of upcycled compounds that can be produced from these monomers is still limited. Therefore, we extended the substrate spectrum of an aromatics-overproducing <i>Pseudomonas taiwanensis</i> strain to AA and BDO. Adaptive laboratory evolution (ALE) followed by genome sequencing was used to identify and reverse engineer key growth-enabling mutations. In this context, we observed a conflict between the dual objectives of fast growth on AA and efficient aromatics production, which materialised in the form of mutations in the ribosomal protein-encoding gene <i>rpmE.</i> These mutations promote faster growth on AA at the cost of aromatics production. In contrast to <i>P. putida</i> KT2440, knockout of the repressor gene <i>psrA</i> regulating expression of genes involved in <i>β</i>-oxidation had no positive effect on growth of <i>P. taiwanensis</i> on AA. Evolution for growth on BDO revealed several point mutations that affect expression of multiple oxidoreductases, with an identified key role for the dehydrogenase encoded by PVLB_10545. This dehydrogenase likely catalyses the initial oxidation of BDO, thus substituting for PedE, which is present in <i>P. putida</i> but absent in <i>P. taiwanensis</i>. Integration of <i>RpcTAL</i> into the Tn7 site enabled <i>de novo</i> production of 4-coumarate with a yield of 14.4% ± 0.1% (Cmol/Cmol) from BDO and 11.5% ± 0.3% (Cmol/Cmol) from AA. Thereby, the potential of these <i>P. taiwanensis</i> strains for upcycling plastic hydrolysates to value-added compounds was successfully demonstrated.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 8","pages":""},"PeriodicalIF":5.2,"publicationDate":"2025-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://enviromicro-journals.onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70205","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144814783","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":"Evaluation of Engineering Potential in Undomesticated Microbes With VECTOR","authors":"Riley Williamson,&nbsp;Nicholas Dusek,&nbsp;Eglantina Lopez-Echartea,&nbsp;Megan K. Townsend Ramsett,&nbsp;Barney A. Geddes","doi":"10.1111/1751-7915.70215","DOIUrl":"10.1111/1751-7915.70215","url":null,"abstract":"<p>Genetic engineering research has predominantly focused on well-characterised organisms like <i>Escherichia coli</i> and <i>Bacillus subtilis</i>, with methods that often fail to translate to other microorganisms. This limitation presents a significant challenge, particularly given the increasing isolation of large microbial collections through high-throughput culturomics. In response, we developed a scalable, high-throughput pipeline to evaluate the engineerability of diverse microbial community members we named VECTOR (Versatile Engineering and Characterisation of Transferable Origins and Resistance). We utilised a library of vectors with the Bacterial Expression Vector Archive (BEVA) architecture that included combinations of three antibiotic resistance genes and three broad host-range origins of replication (pBBR1, RK2 and RSF1010) or the restricted host-range R6K with an integrative mariner transposon. We tagged each vector with green fluorescent protein and a unique nucleotide barcode. The resulting plasmids were delivered en masse to libraries of undomesticated microbes from plant microbiomes in workflows designed to evaluate their ability to be engineered. Utilising OD₆₀₀ and relative fluorescence measurements, we were able to monitor genetic cargo transfer in real time, indicating successfully engineered strains. Next-generation sequencing of plasmid molecular barcodes allowed us to identify specific vector architectures that worked well in particular bacterial strains from a large community. Modifications to the procedure facilitated isolation of engineered microbes.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 8","pages":""},"PeriodicalIF":5.2,"publicationDate":"2025-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://enviromicro-journals.onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70215","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144811203","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":"Evaluation of Engineering Potential in Undomesticated Microbes With VECTOR","authors":"Riley Williamson,&nbsp;Nicholas Dusek,&nbsp;Eglantina Lopez-Echartea,&nbsp;Megan K. Townsend Ramsett,&nbsp;Barney A. Geddes","doi":"10.1111/1751-7915.70215","DOIUrl":"https://doi.org/10.1111/1751-7915.70215","url":null,"abstract":"<p>Genetic engineering research has predominantly focused on well-characterised organisms like <i>Escherichia coli</i> and <i>Bacillus subtilis</i>, with methods that often fail to translate to other microorganisms. This limitation presents a significant challenge, particularly given the increasing isolation of large microbial collections through high-throughput culturomics. In response, we developed a scalable, high-throughput pipeline to evaluate the engineerability of diverse microbial community members we named VECTOR (Versatile Engineering and Characterisation of Transferable Origins and Resistance). We utilised a library of vectors with the Bacterial Expression Vector Archive (BEVA) architecture that included combinations of three antibiotic resistance genes and three broad host-range origins of replication (pBBR1, RK2 and RSF1010) or the restricted host-range R6K with an integrative mariner transposon. We tagged each vector with green fluorescent protein and a unique nucleotide barcode. The resulting plasmids were delivered en masse to libraries of undomesticated microbes from plant microbiomes in workflows designed to evaluate their ability to be engineered. Utilising OD₆₀₀ and relative fluorescence measurements, we were able to monitor genetic cargo transfer in real time, indicating successfully engineered strains. Next-generation sequencing of plasmid molecular barcodes allowed us to identify specific vector architectures that worked well in particular bacterial strains from a large community. Modifications to the procedure facilitated isolation of engineered microbes.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 8","pages":""},"PeriodicalIF":5.2,"publicationDate":"2025-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://enviromicro-journals.onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70215","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144811206","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":"Reverse-Engineered Gas-Fermenting Acetogen Strains Recover Enhanced Phenotypes From Autotrophic Adaptive Laboratory Evolution","authors":"Henri Ingelman,&nbsp;Kurshedaktar Majibullah Shaikh,&nbsp;Kaspar Valgepea","doi":"10.1111/1751-7915.70208","DOIUrl":"https://doi.org/10.1111/1751-7915.70208","url":null,"abstract":"<p>Gas-fermenting acetogens, such as <i>Clostridium autoethanogenum</i>, have emerged as promising biocatalysts capable of converting CO and CO₂-containing gases into fuels and chemicals relevant for a circular economy. However, the functionalities of the majority of genes in acetogens remain uncharacterised, hindering the development of acetogen cell factories through targeted genetic engineering. We previously identified gene targets through adaptive laboratory evolution (ALE) that potentially realise enhanced autotrophic phenotypes in <i>C. autoethanogenum</i>. In this study, we deleted one of the targets—CLAU_0471 (proposed amino acid permease)—with high mutation occurrence in ALE isolates and extensively characterised the autotrophic growth of strain RE3 in batch bottle and bioreactor continuous cultures. In addition, we characterised two previously reverse-engineered strains RE1 (deletion of CLAU_3129; putative sporulation transcriptional activator Spo0A) and RE2 (SNP in CLAU_1957; proposed two-component transcriptional regulator winged helix family). Strikingly, the strains recovered the superior phenotypes of ALE isolates, including faster autotrophic growth, no need for yeast extract, and robustness in bioreactor operation (e.g., low sensitivity to gas ramping, high biomass, and dilution rates). Notably, RE3 exhibited elevated 2,3-butanediol production, while RE1 performed similarly to the best-performing previously characterised ALE isolate LAbrini. The three reverse-engineered strains showed similarities in proteome expression, and bioinformatic analyses suggest that the targeted genes may be involved in overlapping regulatory networks. Our work provides insights into genotype–phenotype relationships for a better understanding of the metabolism of an industrially relevant acetogen.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 8","pages":""},"PeriodicalIF":5.2,"publicationDate":"2025-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://enviromicro-journals.onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70208","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144810938","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":"Reverse-Engineered Gas-Fermenting Acetogen Strains Recover Enhanced Phenotypes From Autotrophic Adaptive Laboratory Evolution","authors":"Henri Ingelman,&nbsp;Kurshedaktar Majibullah Shaikh,&nbsp;Kaspar Valgepea","doi":"10.1111/1751-7915.70208","DOIUrl":"10.1111/1751-7915.70208","url":null,"abstract":"<p>Gas-fermenting acetogens, such as <i>Clostridium autoethanogenum</i>, have emerged as promising biocatalysts capable of converting CO and CO₂-containing gases into fuels and chemicals relevant for a circular economy. However, the functionalities of the majority of genes in acetogens remain uncharacterised, hindering the development of acetogen cell factories through targeted genetic engineering. We previously identified gene targets through adaptive laboratory evolution (ALE) that potentially realise enhanced autotrophic phenotypes in <i>C. autoethanogenum</i>. In this study, we deleted one of the targets—CLAU_0471 (proposed amino acid permease)—with high mutation occurrence in ALE isolates and extensively characterised the autotrophic growth of strain RE3 in batch bottle and bioreactor continuous cultures. In addition, we characterised two previously reverse-engineered strains RE1 (deletion of CLAU_3129; putative sporulation transcriptional activator Spo0A) and RE2 (SNP in CLAU_1957; proposed two-component transcriptional regulator winged helix family). Strikingly, the strains recovered the superior phenotypes of ALE isolates, including faster autotrophic growth, no need for yeast extract, and robustness in bioreactor operation (e.g., low sensitivity to gas ramping, high biomass, and dilution rates). Notably, RE3 exhibited elevated 2,3-butanediol production, while RE1 performed similarly to the best-performing previously characterised ALE isolate LAbrini. The three reverse-engineered strains showed similarities in proteome expression, and bioinformatic analyses suggest that the targeted genes may be involved in overlapping regulatory networks. Our work provides insights into genotype–phenotype relationships for a better understanding of the metabolism of an industrially relevant acetogen.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 8","pages":""},"PeriodicalIF":5.2,"publicationDate":"2025-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://enviromicro-journals.onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70208","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144810937","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":"Lipid Metabolism Optimisation in Ashbya gossypii to Produce Microbial Oils From Xylose-Rich Feedstocks","authors":"Javier-Fernando Montero-Bullón,&nbsp;Javier Martín-González,&nbsp;Gloria Muñoz-Fernández,&nbsp;Rubén M. Buey,&nbsp;Alberto Jiménez","doi":"10.1111/1751-7915.70209","DOIUrl":"10.1111/1751-7915.70209","url":null,"abstract":"<p>The development of lipogenic biocatalysts with enhanced ability for agro-waste bioconversion represents a critical challenge in microbial biotechnology, requiring the metabolic optimisation of different oil-producing microorganisms. <i>Ashbya gossypii</i>, although not classified as an oleaginous microorganism, exhibits a significant capacity for intracellular lipid accumulation and can efficiently metabolise various low-cost substrates, including xylose-rich feedstocks. Indeed, previous works have shown metabolically engineered strains of <i>A. gossypii</i> reaching between 20% and 40% of the cell dry weight in lipid content when using xylose as the primary carbon source. Building on these findings, this study employs a multigenic optimisation strategy to further enhance lipid production by increasing the lipogenic supply of both acetyl-CoA and NADPH, while simultaneously abolishing β-oxidation, in a strain engineered for efficient xylose utilisation. The optimised strain achieved approximately 60% lipid content when cultivated on mixed sugar formulations designed to simulate xylose-rich feedstocks. These results underscore the potential of <i>A. gossypii</i> as a promising microbial platform for sustainable lipid production from renewable resources, reinforcing its utility in biotechnological applications for bio-based chemical and biofuel production.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 8","pages":""},"PeriodicalIF":5.2,"publicationDate":"2025-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://enviromicro-journals.onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70209","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145128863","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":"The TonB-Dependent Transport System Facilitates the Uptake of Inorganic Metal Mediators in Pseudomonas putida KT2440 in a Bioelectrochemical System","authors":"Anna Weimer,&nbsp;Jens Krömer,&nbsp;Bin Lai,&nbsp;Christoph Wittmann","doi":"10.1111/1751-7915.70206","DOIUrl":"https://doi.org/10.1111/1751-7915.70206","url":null,"abstract":"<p>Mediator-based extracellular electron transfer (EET) in a bioelectrochemical system is a unique approach to regulate the microbial redox and energy metabolism of <i>Pseudomonas putida</i> KT2440, which enables a new-to-nature high product yield under anaerobic conditions. Previous studies identified respiratory complex III in the inner membrane as a key redox protein involved in mediator (ferricyanide) interactions, but the exact mechanism through which the mediator crosses the outer membrane to extract electrons from membrane-bound redox proteins and transfer them to the anode remains unclear. In this study, we demonstrated the critical role of the TonB-dependent system, a widespread transportation system in gram-negative bacteria, in the mediator-based EET process. Transcriptomic analyses revealed significant upregulation of TonB-dependent receptors in response to ferricyanide exposure, suggesting their involvement in mediator uptake. Deletion of the TonB complex resulted in <i>a</i> &gt; 50% decrease in the mediator reduction rate and current output, confirming the role of the TonB-dependent system in mediator transport. Additionally, increasing passive diffusion through the overexpression of the general porin OprF increased cell permeability and the mediator reduction rate, but it failed to compensate for the absence of TonB-dependent transport. These findings suggest that both systems act in a complementary manner: the TonB-dependent system is likely the primary mechanism for periplasmic mediator uptake, whereas OprF is likely involved mainly in mediator efflux. Further bioelectrochemical system experiments demonstrated that, with a functional TonB-dependent system, OprF overexpression increased current output, glucose consumption, and 2-ketogluconate production, suggesting a viable strategy for enhancing the efficacy of mediator-based EET. This work reveals the major mediator transport mechanism in <i>P. putida</i> and deepens the understanding of the mediator-based EET pathway, laying the basis for future rational engineering of EET kinetics and facilitating the integration of mediator-based electron transfer into industrial biotechnology to push its process boundaries.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 8","pages":""},"PeriodicalIF":5.2,"publicationDate":"2025-08-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70206","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144767777","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":"The TonB-Dependent Transport System Facilitates the Uptake of Inorganic Metal Mediators in Pseudomonas putida KT2440 in a Bioelectrochemical System","authors":"Anna Weimer,&nbsp;Jens Krömer,&nbsp;Bin Lai,&nbsp;Christoph Wittmann","doi":"10.1111/1751-7915.70206","DOIUrl":"10.1111/1751-7915.70206","url":null,"abstract":"<p>Mediator-based extracellular electron transfer (EET) in a bioelectrochemical system is a unique approach to regulate the microbial redox and energy metabolism of <i>Pseudomonas putida</i> KT2440, which enables a new-to-nature high product yield under anaerobic conditions. Previous studies identified respiratory complex III in the inner membrane as a key redox protein involved in mediator (ferricyanide) interactions, but the exact mechanism through which the mediator crosses the outer membrane to extract electrons from membrane-bound redox proteins and transfer them to the anode remains unclear. In this study, we demonstrated the critical role of the TonB-dependent system, a widespread transportation system in gram-negative bacteria, in the mediator-based EET process. Transcriptomic analyses revealed significant upregulation of TonB-dependent receptors in response to ferricyanide exposure, suggesting their involvement in mediator uptake. Deletion of the TonB complex resulted in <i>a</i> &gt; 50% decrease in the mediator reduction rate and current output, confirming the role of the TonB-dependent system in mediator transport. Additionally, increasing passive diffusion through the overexpression of the general porin OprF increased cell permeability and the mediator reduction rate, but it failed to compensate for the absence of TonB-dependent transport. These findings suggest that both systems act in a complementary manner: the TonB-dependent system is likely the primary mechanism for periplasmic mediator uptake, whereas OprF is likely involved mainly in mediator efflux. Further bioelectrochemical system experiments demonstrated that, with a functional TonB-dependent system, OprF overexpression increased current output, glucose consumption, and 2-ketogluconate production, suggesting a viable strategy for enhancing the efficacy of mediator-based EET. This work reveals the major mediator transport mechanism in <i>P. putida</i> and deepens the understanding of the mediator-based EET pathway, laying the basis for future rational engineering of EET kinetics and facilitating the integration of mediator-based electron transfer into industrial biotechnology to push its process boundaries.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 8","pages":""},"PeriodicalIF":5.2,"publicationDate":"2025-08-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70206","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144767776","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}