Microbial Cell Factories最新文献

{"title":"Expression and characterization of cold-adapted xylanase Xyl-L in Pichia pastoris for xylooligosaccharide (XOS) preparation.","authors":"Sebastián Rodríguez, Carolina González, José Pablo Reyes-Godoy, Brigitte Gasser, Barbara Andrews, Juan A Asenjo","doi":"10.1186/s12934-025-02690-4","DOIUrl":"10.1186/s12934-025-02690-4","url":null,"abstract":"<p><strong>Background: </strong>Xylan, the second most abundant polysaccharide in plant biomass, requires endoxylanases for its hydrolysis into xylooligosaccharides (XOS). Xylanases have been widely used in industries such as animal feed, bakery, juice production, and paper pulp. Recently, XOS have gained attention for their health benefits, including improved digestion, reduced cholesterol, and antioxidant effects. The cold-adapted GH10 xylanase of Antarctic origin Xyl-L was previously expressed in Escherichia coli, showing promising low-temperature activity. However, Pichia pastoris is currently a preferred host for industrial xylanase production due to its ability to express complex proteins and secrete them into the culture medium. This study explored the expression of Xyl-L in P. pastoris and evaluated its potential for XOS production using common flours as substrates, aiming for applications in the food and nutraceutical industry.</p><p><strong>Results: </strong>Comparison between AOX1 ( <math><msub><mtext>P</mtext> <mrow><mi>A</mi> <mi>O</mi> <mi>X</mi> <mn>1</mn></mrow> </msub> </math> ) and GAP ( <math><msub><mtext>P</mtext> <mrow><mi>GAP</mi></mrow> </msub> </math> ) promoters for recombinant Xyl-L production in P. pastoris showed that the <math><msub><mtext>P</mtext> <mrow><mi>A</mi> <mi>O</mi> <mi>X</mi> <mn>1</mn></mrow> </msub> </math> promoter resulted in higher activity per wet-cell weight. Co-transforming <math><msub><mtext>P</mtext> <mrow><mi>A</mi> <mi>O</mi> <mi>X</mi> <mn>1</mn></mrow> </msub> </math> -Xyl strains with plasmids encoding genes aiding in protein folding (HAC1 or PDI1) did not enhance Xyl-L catalytic activity compared to the parental <math><msub><mtext>P</mtext> <mrow><mi>A</mi> <mi>O</mi> <mi>X</mi> <mn>1</mn></mrow> </msub> </math> strain. Thus, <math><msub><mtext>P</mtext> <mrow><mi>A</mi> <mi>O</mi> <mi>X</mi> <mn>1</mn></mrow> </msub> </math> -Xyl was cultivated in 3 L bioreactors in fed-batch cultures; it is presumed that the enzyme is produced with glycosylations within its structure, given its migration within the SDS-PAGE gels. The produced Xyl-L was purified from the culture supernatant, resulting in peak xylanase activity after 90 h, with specific activity of 5.10 ± 0.21 U/mg, at pH 7.5 and <math><msup><mn>25</mn> <mo>∘</mo></msup> </math> C, using beechwood xylan. It also showed a Km of 3.5 mg/mL and a kcat of 9.16 <math><msup><mtext>s</mtext> <mrow><mo>-</mo> <mn>1</mn></mrow> </msup> </math> . Xyl-L maintained over 80% of relative activity between pH 5.6 <math><mo>-</mo></math> 8.6 and <math><mrow><mn>37</mn> <mo>-</mo> <msup><mn>44</mn> <mo>∘</mo></msup> </mrow> </math> C, and was activated by <math><msub><mtext>CaCl</mtext> <mn>2</mn></msub> </math> and <math><msub><mtext>MgCl</mtext> <mn>2</mn></msub> </math> , but inhibited by <math><msub><mtext>MnCl</mtext> <mn>2</mn></msub> </math> . Xyl-L was tested using several flours (whole wheat, rye, oatmeal and all-purpose) as substrates, where XOS wit","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"82"},"PeriodicalIF":4.3,"publicationDate":"2025-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11971854/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143788659","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":"Determining metabolic mechanism linking phospholipids and docosahexaenoic acid through phosphatidylcholine synthesis by phosphocholine cytidylyltransferase (CCT) overexpression in Schizochytrium sp.","authors":"Xiaowen Cui, Tingting Chen, Yizhen Meng, Xueshan Pan, Ruizhe Wu, Yinghua Lu, Chuanyi Yao, Xihuang Lin, Xueping Ling","doi":"10.1186/s12934-025-02703-2","DOIUrl":"10.1186/s12934-025-02703-2","url":null,"abstract":"<p><p>The polyunsaturated fatty acid (PUFA) metabolism of Schizochytrium, an excellent oil-producing microorganism, is closely related to phosphatidylcholine (PC) synthesis, which favors the migration and accumulation of docosahexaenoic acid (DHA). Phosphocholine cytidylyltransferase (CCT), a key enzyme involved in PC synthesis, profoundly impacts lipid metabolism in plants; however, few studies have focused on CCT in microorganisms. We investigated the effects of CCT overexpression on lipid metabolism in Schizochytrium sp. CCT overexpression slightly inhibited cell growth, but significantly promoted total lipid synthesis. Compared to the wild-type strain, PUFA content and DHA production in the CCT-overexpressing strain (SR21-CCT) increased by about 49% and 46%, respectively. Analysis of phospholipids and quantitative real-time PCR revealed that CCT overexpression enhanced phospholipid synthesis, especially by strengthening glycerophosphorylcholine acylation and de novo PC synthesis pathways, which promote DHA esterification to PC and DHA accumulation in triacylglycerols. This study helps decipher the mechanism correlating phospholipid metabolism and DHA production.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"81"},"PeriodicalIF":4.3,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11969949/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143788696","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":"Diversity and epimedium biotransformation potential of cultivable endophytic fungi associated with Epimedium brevicornum Maxim in the Qinling Mountains, China.","authors":"Saijian Ma, Chao An, Wenjiao Xue, Chen Liu, Hao Ding, Qiwen Zhang, Xinwei Shi, Jingjing Zhang, Yao Liu, Jingjing Shao","doi":"10.1186/s12934-025-02698-w","DOIUrl":"10.1186/s12934-025-02698-w","url":null,"abstract":"<p><strong>Background: </strong>The use of biocatalysis technology to manufacture rare natural products can solve the contradiction between the market demand for rare natural products in large health industry fields and the protection and sustainable development of wildlife resources. However, the currently available research on fungal endophytes, which are great potential resources for glycoside hydrolase biocatalysts, is still insufficient. In this study, endophytic fungi from Epimedium brevicornum Maxim. were isolated in the Qinling Mountains, identified and tested for their potential to biotransform epimedium extracts into minor epimedium flavonoids.</p><p><strong>Results: </strong>A total of 84 representative morphotype strains were isolated and identified via ITS rDNA sequence analyses and were grouped into 32 taxa. The Shannon‒Wiener index (H', 3.089) indicated that E. brevicornum Maxim. harboured abundant fungal resources. Ten strains showed strong β-glucosidase activity and exhibited the ability to biotransform major epimedium flavonoids into deglycosylated minor epimedium flavonoids, such as baohuoside I and icaritin, via various glycoside-hydrolysing pathways. Among these strains, strains 8509 and F8889, which were initially characterized as Aspergillus ochraceus and A. protuberus, have the potential for further development in the biotransformation of epimedium extracts into minor epimedium flavonoids because of their excellent biosafety, enzyme activity, and enzymatic characteristics. The enzyme activity of the crude enzyme obtained by freeze-drying from the F8509 fermentation broth supernatant reached 78.24 ± 2.48 U/g. Further research revealed that major glycosylated flavonoids from 100 g/L epimedium extracts were bio-transformed completely into minor deglycosylated flavonoids in 90 min after the addition of 1 g/L crude enzyme. In addition, the liquid phase separation conditions were optimized, and ethyl alcohol and water were ultimately used as the mobile phase for efficient separation of the conversion products at equal flow degrees.</p><p><strong>Conclusions: </strong>This study not only identified a series of candidates for the biotransformation of minor epimedium flavonoids but also provided an efficient purification method. More importantly, this study also demonstrated the important value of endophytes in the biotransformation of rare natural products.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"80"},"PeriodicalIF":4.3,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11966834/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143780509","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":"Introduction of human m⁶Am methyltransferase PCIF1 facilitates the biosynthesis of terpenoids in Saccharomyces cerevisiae.","authors":"Guoli Wang, Mingkai Li, Bengui Fan, Xiqin Liang, Jun Wang, Yanbing Shi, Qiusheng Zheng, Defang Li, Tianyue An","doi":"10.1186/s12934-025-02701-4","DOIUrl":"10.1186/s12934-025-02701-4","url":null,"abstract":"<p><strong>Background: </strong>The application of synthetic biology techniques has been recognized as an efficient alternative for the biosynthesis of high-value natural products, and various metabolic engineering strategies have been employed to develop microbial cell factories. However, exploration of more efficient metabolic pathway optimization strategies is still required to further improve the producing potential of microbial cell factories to meet the industrial requirements.</p><p><strong>Results: </strong>In this study, we found that the introduction of human N6,2'-O-dimethyladenosine (m⁶Am) methyltransferase PCIF1 into Saccharomyces cerevisiae significantly promoted the biosynthesis of squalene, increased by 2.3-fold. Transcriptome analysis revealed that PCIF1 upregulated genes associated with glycolysis and acetyl-CoA biosynthesis pathways, and also activated the cell wall integrity mitogen-activated protein kinase (MAPK) pathway to improve the cell wall stress response. Importantly, PCIF1 expression notably enhanced squalene and sesquiterpenoid longifolene production in engineered yeast strains, with 2.3-fold and 1.4-fold higher increase, respectively.</p><p><strong>Conclusion: </strong>This study presents a PCIF1-based metabolic engineering strategy that could serve as an effective approach for the optimization of terpene biosynthesis in yeast cell factories.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"78"},"PeriodicalIF":4.3,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11963462/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143772995","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":"Enhanced Natamycin production in Streptomyces gilvosporeus through phosphate tolerance screening and transcriptome-based analysis of high-yielding mechanisms.","authors":"Liang Wang, Wen Xiao, Ting Qiu, Hongjian Zhang, Jianhua Zhang, Xusheng Chen","doi":"10.1186/s12934-025-02696-y","DOIUrl":"10.1186/s12934-025-02696-y","url":null,"abstract":"<p><strong>Background: </strong>Natamycin is a natural antibiotic with broad-spectrum antifungal activity, widely used in food preservation, medicine, and biological control. However, the relatively low biosynthetic capacity of producing strains limits further industrialization and broader applications of natamycin. Due to the complexity of cellular metabolism, evolutionary engineering is required for developing strains with enhanced natamycin biosynthetic capacity.</p><p><strong>Results: </strong>Here, protoplast fusion combined with phosphate tolerance screening was employed for the first time to enhance natamycin production of Streptomyces gilvosporeus. A high-yielding strain, GR-2, was obtained, with natamycin production twice that of the original strain. Transcriptomic analysis revealed that the natamycin biosynthetic gene cluster and several primary metabolic pathways were significantly upregulated in GR-2, likely contributing to its high production performance. Further experiments, including amino acid addition and reverse engineering, confirmed that branched-chain amino acid, nitrogen, and phosphate metabolism play crucial roles in promoting natamycin production. Silencing of the phosphate metabolism transcriptional regulators PhoP and PhoR led to a decreased expression of natamycin biosynthetic genes and significantly reduced natamycin production, highlighting the key role of these regulators in S. gilvosporeus. Based on omics data, co-expression of phoP and phoR in GR-2 resulted in the engineered strain GR2-P3, which exhibited a 25% increase in natamycin production in shake flasks. In a 5 L fermenter, GR2-P3 achieved a natamycin production of 12.2 ± 0.6 g·L⁻¹, the highest yield reported for S. gilvosporeus to date.</p><p><strong>Conclusions: </strong>Our findings suggest that the high production performance of GR-2 is primarily due to the upregulation of the natamycin biosynthetic gene cluster and genes related to precursor supply. Increasing the intracellular supply of valine and glutamate significantly enhanced natamycin production. Additionally, the natamycin biosynthetic gene cluster is likely positively regulated by PhoP and PhoR. Our work presents a novel strategy for strain screening and evolution to improve natamycin production and identifies novel molecular targets for metabolic engineering.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"79"},"PeriodicalIF":4.3,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11963449/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143772887","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":"In vitro and preclinical evaluation of the antifungal activity of 6-methoxy-1 H-indole-2-carboxylic acid produced by Bacillus toyonensis strain OQ071612 formulated as nanosponge hydrogel.","authors":"Sayed E El-Sayed, Neveen A Abdelaziz, Ghadir S El-Housseiny, Khaled M Aboshanab","doi":"10.1186/s12934-025-02688-y","DOIUrl":"10.1186/s12934-025-02688-y","url":null,"abstract":"<p><strong>Background: </strong>In a previous study, 6-methoxy-1 H-indole-2-carboxylic acid (MICA) was isolated from the culture broth of Bacillus toyonensis strain OQ071612 soil isolate in our laboratory, and it demonstrated promising antifungal activities. The current study was designed to create a nanosponge (NS)-hydrogel (HG)-containing MICA followed by in vitro and preclinical evaluation for potential clinical use in the topical treatment of mycotic infections.</p><p><strong>Results: </strong>The enhanced NS formula was created using the Box Behnken Design (BBD), with independent process parameters including polyvinyl alcohol percentage (w/v%), homogenization time, speed and polymer: linker ratio. Dependent parameters were particle size (PS), polydispersity index (PDI), and entrapment efficiency percent (EE%). A hydrogel was formulated from the NS. In vitro drug release data indicated that the hydrogel best matched Higuchi's kinetic release model. The formulated NS-HG was stable and when compared to fluconazole, it exhibited increased antimycotic activity against C. albicans. An in vivo investigation revealed that MICA-NS-HG enhanced survival rates, wound gap repair, wound reduction, and inflammation inhibition. Masson's trichrome staining and histological analyses revealed increased collagen deposition and improved healing. Moreover, MICA hydrogel exhibited 1.5-fold greater permeability through rat skin compared to the control, 1% isoconazole.</p><p><strong>Conclusion: </strong>The NS-HG formulation is a viable vehicle for better and more effective topical release of MICA. These findings represent a significant advancement in the formulation of MICA derived from naturally occurring soil bacteria.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"77"},"PeriodicalIF":4.3,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11959791/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143764495","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 Saccharomyces boulardii for enhanced surface display capacity.","authors":"Luping Xu, Xingjian Bai, Deokyeol Jeong, Dahye Lee, Fransheska Semidey, Chenhai Li, Eun Joong Oh","doi":"10.1186/s12934-025-02702-3","DOIUrl":"10.1186/s12934-025-02702-3","url":null,"abstract":"<p><p>Saccharomyces boulardii (Sb) has gained significant attention for its potential therapeutic application as a probiotic yeast strain. Current approaches often leverage its secretion and display capabilities to deliver therapeutic agents aimed at alleviating intestinal disorders. However, relatively few studies have focused on optimizing its display efficiency. In this study, we evaluated two surface display systems, Aga2- and Sed1-based, for use in Sb by systematically modifying display cassette components and the host strain. Initially, both systems were tested in Saccharomyces cerevisiae (Sc) and Sb to validate their design. Sc consistently outperformed Sb in both display expression and efficiency, highlighting the need for further optimization in Sb. To enhance the display efficiency in Sb, we investigated specific modifications to the display cassette, including the use of linker sequences for Aga2 and variations in anchor length for Sed1. These experiments identified key factors influencing display performance. Subsequently, we engineered a modified Sb strain, LIP02, by overexpressing AGA1 and deleting cell wall-related genes (CCW12, CCW14, and FYV5). These modifications were expected to expand the available docking sites for the protein of interest (POI) and improve overall protein secretion and display efficiency. As a result, the modified strain exhibited a significant enhancement in display capacity compared to the wild-type Sb strain. Furthermore, genome integration of the display cassette in LIP02 enhanced both stability and expression compared to plasmid-based systems. Importantly, the functionality of β-glucosidase displayed on LIP02 was preserved, as demonstrated by improved enzymatic activity and robust growth on cellobiose as the sole carbon source. These findings establish LIP02 as a superior host for surface display applications in Sb, offering a more stable and efficient platform for the expression of therapeutic proteins and other functional biomolecules.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"76"},"PeriodicalIF":4.3,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11959792/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143764494","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 in synthesizing plant-derived isoflavones and their precursors with multiple pharmacological activities using engineered yeasts.","authors":"Wenhui Niu, Jingxian Zhang, Lingbo Qu, Xiao-Jun Ji, Yongjun Wei","doi":"10.1186/s12934-025-02692-2","DOIUrl":"https://doi.org/10.1186/s12934-025-02692-2","url":null,"abstract":"<p><p>Isoflavones such as daidzein and genistein are naturally occurring compounds found in plants such as legumes. They have diverse pharmacological activities, making them valuable in the food, pharmaceutical, and cosmetic industries. Currently, isoflavones are mainly obtained through the extraction of plant biomass. Chemical synthesis is challenging for most isoflavones due to the complexity of their structures. The limited supply of isoflavones cannot meet the market demands. Advances in synthetic biology have provided a sustainable and efficient solution for the production of isoflavones, with yeasts often serving as the microbial chassis for biosynthesis. This review summarizes the pharmacological properties of specific isoflavones, their biosynthetic pathways, and the technical strategies used in engineered yeasts for isoflavone production. In addition, the development of synthetic biology and state-of-the-art biotechnological strategies for the environmentally friendly production of bioactive isoflavones is discussed.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"75"},"PeriodicalIF":4.3,"publicationDate":"2025-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11954244/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143743664","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":"Low-biomass pyruvate production with engineered Vibrio natriegens is accompanied by parapyruvate formation.","authors":"Maurice Hädrich, Clarissa Scheuchenegger, Sören-Tobias Vital, Christoph Gunkel, Susanne Müller, Josef Hoff, Jennifer Borger, Erich Glawischnig, Felix Thoma, Bastian Blombach","doi":"10.1186/s12934-025-02693-1","DOIUrl":"10.1186/s12934-025-02693-1","url":null,"abstract":"<p><strong>Background: </strong>Pyruvate is a precursor for various compounds in the chemical, drug, and food industries and is therefore an attractive target molecule for microbial production processes. The fast-growing bacterium Vibrio natriegens excels with its specific substrate uptake rate as an unconventional chassis for industrial biotechnology. Here, we aim to exploit the traits of V. natriegens for pyruvate production in fermentations with low biomass concentrations.</p><p><strong>Results: </strong>We inactivated the pyruvate dehydrogenase complex in V. natriegens Δvnp12, which harbors deletions of the prophage regions vnp12. The resulting strain V. natriegens Δvnp12 ΔaceE was unable to grow in minimal medium with glucose unless supplemented with acetate. In shaking flasks, the strain showed a growth rate of 1.16 ± 0.03 h^- 1 and produced 4.0 ± 0.3 g_Pyr L^- 1 within 5 h. We optimized the parameters in an aerobic fermentation process and applied a constant maintenance feed of 0.24 g_Ac h^- 1 which resulted in a maximal biomass concentration of only 6.6 ± 0.4 g_CDW L^- 1 and yielded highly active resting cells with a glucose uptake rate (q_S) of 3.5 ± 0.2 g_Glc g_CDW^-1 h^- 1. V. natriegens Δvnp12 ΔaceE produced 41.0 ± 1.8 g_Pyr L^- 1 with a volumetric productivity of 4.1 ± 0.2 g_Pyr L^- 1 h^- 1. Carbon balancing disclosed a gap of 30%, which we identified partly as parapyruvate. Deletion of ligK encoding the HMG/CHA aldolase in V. natriegens Δvnp12 ΔaceE did not impact biomass formation but plasmid-based overexpression of ligK negatively affected growth and led to a 3-fold higher parapyruvate concentration in the culture broth. Notably, we also identified parapyruvate in supernatants of a pyruvate-producing Corynebacterium glutamicum strain. Cell-free bioreactor experiments mimicking the biological process also resulted in parapyruvate formation, pointing to a chemical reaction contributing to its synthesis.</p><p><strong>Conclusions: </strong>We engineered metabolically highly active resting cells of V. natriegens producing pyruvate with high productivity at a low biomass concentration. However, we also found that pyruvate production is accompanied by parapyruvate formation in V. natriegens as well as in a pyruvate producing C. glutamicum strain. Parapyruvate formation seems to be a result of chemical pyruvate conversion and might be supported biochemically by an aldolase reaction.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"73"},"PeriodicalIF":4.3,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11951559/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143730465","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 rapid and efficient strategy for combinatorial repression of multiple genes in Escherichia coli.","authors":"Yi Zheng, Yuxia Mo, Yingbo Yuan, Tianyuan Su, Qingsheng Qi","doi":"10.1186/s12934-025-02697-x","DOIUrl":"10.1186/s12934-025-02697-x","url":null,"abstract":"<p><strong>Background: </strong>The regulation of multiple gene expression is pivotal for metabolic engineering. Although CRISPR interference (CRISPRi) has been extensively utilized for multi-gene regulation, the construction of numerous single-guide RNA (sgRNA) expression plasmids for combinatorial regulation remains a significant challenge.</p><p><strong>Results: </strong>In this study, we developed a combinatorial repression system for multiple genes by optimizing the expression of multi-sgRNA with various inducible promoters in Escherichia coli. We designed a modified Golden Gate Assembly method to rapidly construct the sgRNA expression plasmid p3gRNA-LTA. By optimizing both the promoter and the sgRNA handle sequence, we substantially mitigated undesired repression caused by the leaky expression of sgRNA. This method facilitates the rapid assessment of the effects of various inhibitory combinations on three genes by simply adding different inducers. Using the biosynthesis of N-acetylneuraminic acid (NeuAc) as an example, we found that the optimal combinatorial inhibition of the pta, ptsI, and pykA genes resulted in a 2.4-fold increase in NeuAc yield compared to the control.</p><p><strong>Conclusion: </strong>We anticipate that our combinatorial repression system will greatly simplify the regulation of multiple genes and facilitate the fine-tuning of metabolic flow in the engineered strains.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"74"},"PeriodicalIF":4.3,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11951683/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143730464","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}