Microbial Cell Factories最新文献

{"title":"Investigating endophytic fungi of Calotropis procera for novel bioactive compounds: molecular docking and bioactivity insights.","authors":"Sayed M S Abo El-Souad, Mohamed A Dawoud, Mahmoud Ibrahim, Mona M Soliman","doi":"10.1186/s12934-025-02710-3","DOIUrl":"https://doi.org/10.1186/s12934-025-02710-3","url":null,"abstract":"<p><strong>Background: </strong>The rising danger of antibiotic resistance and the increasing burden of cancer worldwide have highlighted the necessity for a constant supply of new antimicrobial drugs and anticancer therapies. Endophytic fungi, recognized as a rich supplier of secondary metabolites with novel bioactivities that have shown promising antimicrobial and anticancer potential, were isolated from the medicinal plant Calotropis procera. Approximately 70 segments from the leaves and stems of the C. procera plant were evaluated for endophytic colonization, resulting in the isolation and identification of five fungal species based on morphological characteristics.</p><p><strong>Results: </strong>A total of five endophytic fungal species were isolated from Calotropis procera and identified, with Aspergillus versicolor exhibiting the highest frequency of occurrence (50%). In contrast, the remaining fungal species were found at a frequency of 25% each. The endophytic fungal filtrates were evaluated for antimicrobial efficacy against seven pathogens, demonstrating significant inhibition zones ranging from 7 to 25 mm. Additionally, the anticancer activity was assessed against two cell lines, MCF-7 and HCT-16, with IC₅₀ ranging from 7.8 to 50.4 µg/mL. Among the isolates, the filtrate of Aspergillus niger (Accession number PQ568010) exhibited the highest antimicrobial and anticancer activities. The crude extract of A. niger was developed to identify the chemical constituents by gas chromatography. The most active component in the extract, as analyzed by ¹H NMR, revealed that 2,2,4,4-tetramethylpentane was the primary compound responsible for these effects, which demonstrated significant inhibitory activity against Staphylococcus aureus and Bacillus cereus, with inhibition zones of 23 mm and 20 mm, respectively. Molecular docking studies were performed against Phenylalanine-tRNA ligase alpha subunit of Bacillus cereus (UniProt ID: Q633N4), GTPase Der of Escherichia coli (UniProt ID: P0A6P5), peptidoglycan-N-acetylglucosamine deacetylase of Listeria monocytogenes (UniProt ID: A0A3Q0NBH7), DNA gyrase subunit B of Salmonella typhimurium (UniProt ID: P0A2I3), Zinc metalloproteinase aureolysin of Staphylococcus aureus (UniProt ID: P81177), Agglutinin-like protein 2 of Candida albicans (UniProt ID: Q9URQ0), serine/threonine-protein kinase of Saccharomyces cerevisiae (UniProt ID: P32600).</p><p><strong>Conclusion: </strong>The study highlights the potential of endophytic fungi Aspergillus niger as a promising source of novel antimicrobial and anticancer agents. The identification of 2,2,4,4-tetramethylpentane as the primary bioactive compound, combined with the molecular docking analyses, provides valuable insights into the mechanisms of action and potential therapeutic applications. These findings underscore the importance of exploring endophytic fungi for the development of new drugs to combat antibiotic resistance and cancer.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"101"},"PeriodicalIF":4.3,"publicationDate":"2025-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12060415/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144022675","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":"Enhancement of spinosad production in Saccharopolyspora spinosa by overexpression of the complete 74-kb spinosyn gene cluster.","authors":"Lu Gan, Zhengyu Zhang, Jingtao Chen, Zhichun Shen, Wujie Chen, Shaoxin Chen, Jiyang Li","doi":"10.1186/s12934-025-02724-x","DOIUrl":"https://doi.org/10.1186/s12934-025-02724-x","url":null,"abstract":"<p><strong>Background: </strong>Spinosad, a secondary metabolite produced by Saccharopolyspora spinosa, is a polyketide macrolide insecticide with low toxicity and environmental friendliness. Owing to the high level of DNA methylation and unclear regulatory mechanisms, gene engineering to increase spinosad production is challenging. Limited improvements in yield have been observed with heterologous expression or partial overexpression of the 74-kb spinosyn gene cluster (spn), and research on the overexpression of the complete spinosyn gene cluster is lacking.</p><p><strong>Results: </strong>The plasmid pCM265-spn was constructed using CRISPR/Cas9-mediated Transformation-Associated Recombination cloning to enable the overexpression of the complete spn gene cluster in Sa. spinosa. The engineered strain Sa. spinosa-spn achieved a 124% increase in spinosad yield (693 mg/L) compared to the wild type (309 mg/L). The overexpression of the spn gene cluster also delayed spore formation and reduced hyphal compartmentalization by influencing the transcription of related genes (bldD, ssgA, whiA, whiB, and fstZ). Transcriptional analysis revealed significant upregulation of genes in the spn gene cluster, thereby enhancing secondary metabolism. Additionally, optimization of the fermentation medium through response surface methodology further increased spinosad production to 920 mg/L.</p><p><strong>Conclusions: </strong>This study is the first to successfully overexpress the complete spn gene cluster in Sa. spinosa, significantly enhancing spinosad production. These findings have significance for further optimization of spinosad biosynthesis.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"102"},"PeriodicalIF":4.3,"publicationDate":"2025-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12060398/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144001904","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":"Advances and trends for astaxanthin synthesis in Phaffia rhodozyma.","authors":"Jiajun Sun, Zhaokun Zhang, Le Gao, Fan Yang","doi":"10.1186/s12934-025-02704-1","DOIUrl":"https://doi.org/10.1186/s12934-025-02704-1","url":null,"abstract":"<p><p>Astaxanthin, a carotenoid endowed with potent antioxidant capacity, exhibits considerable application prospects in nutraceuticals, pharmaceuticals, and cosmetics. In contrast to the chemical synthesis method, the biosynthesis of astaxanthin is undoubtedly a greener and more environmentally friendly production approach. In this review, we comprehensively review the biosynthetic pathways and multiple strategies for astaxanthin synthesis in Phaffia rhodozyma. Some biotechnology advancements for increasing the yield of astaxanthin in Phaffia rhodozyma encompass mutagenesis breeding, genetic modification, and optimizing fermentation conditions, thereby opening up new avenues for its application in functional foods and feed. Nevertheless, the yield of product synthesis is constrained by the host metabolic stoichiometry. Besides breaking the threshold of astaxanthin production and alleviating the impact of astaxanthin accumulation on cell growth, a comprehensive comprehension of multiple interconnected metabolic pathways and complex regulatory mechanisms is indispensable for significantly enhancing astaxanthin production. This review presents some prospects of integrating digital concepts into astaxanthin production to aid in overcoming current challenges.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"100"},"PeriodicalIF":4.3,"publicationDate":"2025-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12057283/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144018467","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":"Nutritional optimization for bioprocess production of staphyloxanthin from Staphylococcus aureus with response surface methodology: promising anticancer scaffold targeting EGFR inhibition.","authors":"Ahmed M Nosair, Ahmed A Abdelaziz, Amal M Abo-Kamer, Lamiaa A Al-Madboly, Mahmoud H Farghali","doi":"10.1186/s12934-025-02717-w","DOIUrl":"https://doi.org/10.1186/s12934-025-02717-w","url":null,"abstract":"<p><strong>Background: </strong>Staphyloxanthin (STX) is a secondary metabolite pigment associated with membrane structures, recognized for its significant antioxidant properties. It plays a crucial role in combating reactive oxygen species (ROS), positioning it as a promising and effective alternative in cancer treatment. This study focused on enhancing the production of STX pigment by employing statistical optimization of media components, alongside the evaluation of its safety and anticancer properties.</p><p><strong>Results: </strong>A total of 59 Staphylococcus aureus isolates were screened and quantitatively estimated for STX production. The best pigment-producing isolate was identified based on molecular phylogenetic analysis as S. aureus A2, with accession number PP197164. A Box-Wilson central composite design was employed to evaluate the intricate interactions among six variables affecting the pigment yield. The most optimal conditions resulted in the highest production of STX of OD₄₅₆ = 0.328, which is approximately 1.5-fold greater than the yield (OD₄₅₆ = 0.215) obtained from OFAT optimization. The final response surface model fitting the data achieved a R² of 0.8748. STX exhibited marked cytotoxicity against the A549 NSCLC cell line with IC50 of 57.3 µg/mL, a safe dose in normal Vero cells. The anticancer activity of STX was predominantly mediated by the apoptotic pathway, as confirmed by confocal microscopy, the annexin V-FITC apoptosis assay, and the overexpression of caspase-3. Moreover, STX disrupted cell cycle at pre-G1 and G0/G1 phases in lung cancer. Intriguingly, STX exhibited its antitumor activity through reducing the EGFR expression. The molecular docking study revealed the potential binding interactions and affinities within the active sites of both wild-type and mutant EGFR.</p><p><strong>Conclusion: </strong>The bioprocess for optimized production, combined with the biological profiling and low cytotoxicity, substantiates the potential application of STX pigment in combating lung cancer.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"99"},"PeriodicalIF":4.3,"publicationDate":"2025-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12054202/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143972210","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":"Stable protease from Bacillus licheniformis-MA1 strain: statistical production optimization, kinetic and thermodynamic characterization, and application in silver recovery from used X-ray films.","authors":"Mohamed A A Abdella, Samia A Ahmed","doi":"10.1186/s12934-025-02706-z","DOIUrl":"https://doi.org/10.1186/s12934-025-02706-z","url":null,"abstract":"<p><strong>Background: </strong>Alkaline proteases are useful enzymes for various industrial applications as bio-additives in detergents and in the recovery of silver from used X-ray films. Therefore, many strategies were used to increase enzyme production and reduce production costs by using microbial cultures, using agro-industrial waste, and improving growth conditions via statistical methods. The enzyme kinetics and thermodynamics were studied as well as its ability to recover silver was also evaluated.</p><p><strong>Results: </strong>An alkaline protease suitable for industrial applications was produced by Bacillus licheniformis strain-MA1. The ability of B. licheniformis strain-MA1 to produce protease was optimized using multi-factorial designs (Plackett-Burman and Box-Behnken). Optimization process improved enzyme production by 9.6-fold over that obtained from the original medium. Highest alkaline protease production was reached after 72 h at pH 7.0, 35 °C, and 150 rpm. The protease was maximally active at 50 °C and pH 9.0 with high thermal and pH stability. The protease showed high catalytic efficiency and high affinity toward substrate with low activation energy (Ea). In addition, the thermodynamic parameters of protease enzyme (enthalpy, free energy, and entropy) were also investigated and showed its superior thermal stability. At 70 °C the thermal deactivation constant (kd) was 4.75-fold higher than that at 50 °C. The higher t_0.5, D-values, and activation energy for thermal denaturation (Ed) of the protease indicated its higher thermal stability and thus its potential application in industrial processes. The compatibility of the protease with laundry detergents at 40 °C was higher than at 50 °C. In the presence of EDTA, the protease enzyme retained 93.6% of its activity. Furthermore, the crude enzyme successfully hydrolyzed the gelatin layer from X-ray films waste after 1 h enabling recycling and reuse.</p><p><strong>Conclusions: </strong>Stable alkaline protease from B. licheniformis strain-MA1 was suitable for some industrial aspects as a bio-additive in detergents and capable of recovering silver from used X-ray.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"98"},"PeriodicalIF":4.3,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12053861/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144018931","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 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":"https://doi.org/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}