Microbial Cell Factories最新文献

{"title":"Comparative analysis of pre-treatment strategies and bacterial strain efficiency for improvement of feather hydrolysis.","authors":"Clémentine Isembart, Boris Zimmermann, Josipa Matić, Cristian Bolaño Losada, Nils K Afseth, Achim Kohler, Svein J Horn, Vincent Eijsink, Piotr Chylenski, Volha Shapaval","doi":"10.1186/s12934-025-02743-8","DOIUrl":"10.1186/s12934-025-02743-8","url":null,"abstract":"<p><strong>Background: </strong>Feathers are a major by-product of the poultry industry, which poses an environmental challenge due to the recalcitrant structure of keratin, making them resistant to degradation. Traditional methods of feather handling, like conversion to feather meal, are energy-intensive and have limited efficiency. Biotechnological approaches, particularly microbial hydrolysis, offer a novel and more sustainable alternative for keratin degradation. This study evaluated feather hydrolysis by two bacterial strains, newly characterized cold-adapted Arthrobacter oryzae (BIM B-1663) and Bacillus licheniformis (CCM 2145^T), known as a keratin degrader, under various feather pre-treatment conditions, including washing, autoclaving, drying, and grinding.</p><p><strong>Results: </strong>Both bacterial strains were able to degrade pretreated feathers with a degradation efficiency of 75 to 90%, resulting in high ratios of nitrogen to carbon in the hydrolysates. B. licheniformis confirmed its enzymatic capabilities with high levels of general and specific protease activity and furthermore presented enriched amounts of amino acids of industrial interest. A. oryzae showed a much higher keratinase/protease activity ratio, demonstrating high specificity and efficiency of its enzymes. Autoclaving emerged as the most important determinant of microbial degradation efficiency and influenced the composition (peptide pattern, amino acid content, and chemical composition assessed through FTIR) of the resulting hydrolysates. Feather drying, although not improving microbial degradation efficiencies, had a considerable impact on hydrolysate composition.</p><p><strong>Conclusions: </strong>The results show that both tested bacterial strains can efficiently degrade autoclaved feathers but use distinct enzymatic strategies to do so. Enriched profiles in amino acids and high nitrogen content in the hydrolysates also advocate for the benefits of microbial feather hydrolysis over an enzymatic one. To the authors' knowledge this study is the first to report a comprehensive evaluation of the impact of various feather pre-treatment methods on the efficiency of subsequent microbial feather hydrolysis and is the first one to report enrichment in phenylalanine, lysine, and tyrosine secreted by B. licheniformis.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"118"},"PeriodicalIF":4.3,"publicationDate":"2025-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12093666/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144111375","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":"Optimized expression of oxazolomycins in engineered Streptomyces longshengensis and their activity evaluation.","authors":"Huiying Sun, Xiang Liu, Junyue Li, Yang Xu, Yue Li, Yuqing Tian, Huarong Tan, Jihui Zhang","doi":"10.1186/s12934-025-02726-9","DOIUrl":"10.1186/s12934-025-02726-9","url":null,"abstract":"<p><strong>Background: </strong>To cope with the growing number of severe diseases and intractable pathogens, drug innovation in both chemical structures and pharmacological efficiency has become an imperative global mission. Oxazolomycins are a unique family of polyketide-polypeptide antibiotics from Streptomyces with diverse functional groups in their structures, conferring them multifarious activities. But further development into clinical applications has been hindered for decades for many reasons. Among them, the yield improvement is a critical basis for activity evaluation and drug-like property optimization. This study aims to enhance the production of oxazolomycins in Streptomyces longshengensis through metabolic engineering and evaluate their bioactivity against clinically relevant pathogens.</p><p><strong>Results: </strong>Co-transcriptional analyses suggested that two operons (the transcriptional unit from gene oxaG to oxaB, and that from gene oxaH to oxaQ) could be included in the oxazolomycin biosynthetic gene cluster (oxa BGC) of S. longshengensis. So a strategy was designed to replace the native promoter regions between oxaG and oxaH with constitutive promoters P_neo and P_kasO* following functional module evaluation. In the resultant strain (SL_OE), the production of oxazolomycin component Toxa5 was increased to 4-fold of that in the wild-type strain. Accordingly, the transcription of all related genes in oxa was clearly promoted. SL_OE was then subjected to sublethal dose of gentamicin to induce mutagenesis for optimizing the genetic background, generating a resistant mutant SL_ROE. With the introduction of transporter genes (ozmS and oxaA) into SL_ROE, 175 mg/L of Toxa5 was achieved, representing the highest yield in shake-flask fermentation to the best of our knowledge. Finally, the purified Toxa5 showed significant inhibition on the growth of clinically important Gram-negative pathogenic bacterium, Pseudomonas aeruginosa, and the biofilm formation of Bacillus subtilis. Intriguingly, an unprecedented antioxidant activity was also demonstrated.</p><p><strong>Conclusions: </strong>An oxazolomycin high-producing system of S. longshengensis was established by employing genetic engineering strategies to facilitate the bioactivity exploitation. Oxazolomycin Toxa5 showed interesting inhibitory effects against multiple Gram-negative and -positive pathogens as well as antioxidant capacity, indicating its great potential in clinical applications. The findings provide an efficient strategy for the overproduction and activity evaluation of oxazolomycins.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"114"},"PeriodicalIF":4.3,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12090451/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144111391","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a set of bacterial engineered glycoconjugates as novel serogroup-specific antigens for the serodiagnosis of Escherichia coli O26, O111, O103 and O45 infections associated to hemolytic uremic syndrome.","authors":"Ana J Caillava, Luciano J Melli, Malena Landoni, Stella Maris Landivar, Isabel Chinen, Alicia S Couto, Marta Rivas, Juan E Ugalde, Diego J Comerci, Andrés E Ciocchini","doi":"10.1186/s12934-025-02694-0","DOIUrl":"10.1186/s12934-025-02694-0","url":null,"abstract":"<p><p>Hemolytic uremic syndrome associated to Shiga toxin-producing Escherichia coli infection (STEC-HUS) is a life-threatening condition characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney failure. Among STEC, E. coli O157:H7 is the dominant serotype related with human disease worldwide; however, a subset of STEC non-O157 serotypes -named the \"Big-Six\"- that include the E. coli serogroups O145, O121, O26, O111, O103 and O45 became of a great concern for their potential to cause HUS. Previously, we have demonstrated that serological tests based on bacterial engineered glycoconjugates developed by exploiting the Campylobacter jejuni N-glycosylation machinery, notably increases the association rate of HUS to O157, O145 and O121 STEC infections. In this work, we developed the recombinant glycoproteins O26-AcrA, O111-AcrA, O103-AcrA and O45-AcrA by co-expressing in E. coli the gene cluster required for the synthesis of the O polysaccharide corresponding to each serogroup, the C. jejuni oligosaccharyltransferase (OTase) PglB, and the carrier protein AcrA. The glycans attached to AcrA in the produced and purified glycoconjugates were characterized by mass spectrometry. The glycoconjugates were evaluated as antigens for detection of IgM antibodies against the O polysaccharide of the lipopolysaccharide of O26, O111 and O103 STEC strains in human serum samples. Our results demonstrate that O26-AcrA, O111-AcrA and O103-AcrA allow a clear discrimination between negative and positive samples obtained from patients with HUS associated to O26, O111 and O103 STEC infections. Additionally, these novel antigens are serospecific allowing E. coli serogroup identification which may contribute to the epidemiological surveillance of STEC-HUS patients and their contacts.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"116"},"PeriodicalIF":4.3,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12093740/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144111376","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":"pTripleTREP - A vector for tightly controlled expression and purification of virulence factors in Staphylococcus aureus.","authors":"Hannes Wolfgramm, Christopher Saade, Marco Harms, Larissa M Busch, Josephine Lange, Maximilian Schedlowski, Kristin Surmann, Manuela Gesell Salazar, Christian Hentschker, Leif Steil, Stephan Michalik, Uwe Völker, Alexander Reder","doi":"10.1186/s12934-025-02736-7","DOIUrl":"10.1186/s12934-025-02736-7","url":null,"abstract":"<p><strong>Background: </strong>Recombinant proteins facilitate and contribute to detailed studies of the virulence mechanisms and pathophysiology of the major human pathogen Staphylococcus aureus. Of particular interest are secreted virulence factors. However, due to their potential toxicity and specific post-translational processing, virulence factors are difficult targets for heterologous protein production. Purified proteins with native conformation and adequate purity can therefore often only be achieved by elaborate multi-step purification workflows. While homologous expression in S. aureus theoretically offers a promising alternative in this regard, its application remains limited due to the lack of systems that ensure both tightly controlled expression and subsequent efficient purification.</p><p><strong>Results: </strong>To bridge this gap, we present pTripleTREP as a versatile expression vector for S. aureus, which enables the homologous expression and purification of staphylococcal virulence factors. It features a strong SigA-dependent staphylococcal promoter overlapped by three tetracycline responsive elements (TRE), which ensures tight repression under control conditions and high expression levels upon induction of the target gene. This allowed very precise controlled production of the exemplary targets, serine protease-like protein A (SplA) and B (SplB). A simple single-step protein purification workflow using a Twin-Strep-tag and Strep-Tactin^®XT coated magnetic beads yielded endotoxin-free Spl samples with purities above 99%. Thereby, the homologous production host facilitates native secretion and maturation without the need to engineer the target gene sequence. Proper signal peptide cleavage and the corresponding enzymatic activity of the generated protein products were confirmed for SplA and B.</p><p><strong>Conclusion: </strong>The expression vector pTripleTREP adds an important element to the staphylococcal molecular toolbox, facilitating the tightly controlled homologous expression and rapid native purification of secreted staphylococcal virulence factors. The optimised architecture and genetic features of the vector additionally provide a solid background for further applications such as plasmid-based complementation or interaction studies. Thus, pTripleTREP will support research on the role of staphylococcal virulence factors, paving the way for future therapeutic strategies to combat this pathogen.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"115"},"PeriodicalIF":4.3,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12090601/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144111393","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":"Targeted high-level production of chuangxinmycin and its halogenated derivatives with antitubercular activity.","authors":"Xiongfang Zhao, Yuan Lu, Xintong Zhang, Xiumin Zhang, Yu Du, Xingli Han, Yuting Zhu, Wei Yu, Linzhuan Wu, Xingxing Li, Yuanyuan Shi, Tianyu Zhang, Bin Hong","doi":"10.1186/s12934-025-02740-x","DOIUrl":"10.1186/s12934-025-02740-x","url":null,"abstract":"<p><strong>Background: </strong>Chuangxinmycin (CM) is an old antibiotic from Actinoplanes tsinanensis CPCC, 200056, characterized by a dihydrothiopyrano[4,3,2-cd]indole scaffold and potent activity against Mycobacterium tuberculosis. Its congener norchuangxinmycin (NCM), which lacks antibacterial activity against various bacteria, unexpectedly retains antitubercular activity, indicating new mechanisms of action against M. tuberculosis in addition to tryptophan-tRNA synthetase inhibition. However, the variable low productivity and the limited number of active structural analogues represent a significant challenge for the future discovery and development of new anti-tuberculosis drugs involving CM and its derivatives.</p><p><strong>Results: </strong>Based on the elucidation of CM biosynthetic pathway, we employed a stepwise strategy by combining heterologous expression, activator overexpression, promoter optimization and fermentation media screening to achieve directed and high-level production of CM and its congener NCM. The highest yields achieved were 301 mg/L (a 20.1-fold increase) for CM and 117.6 mg/L (a 13.7-fold increase) for NCM. Furthermore, eleven halogenated CM derivatives were obtained through precursor-directed biosynthesis, with six of them being purified and structurally confirmed by HR-MS, HR-MS/MS and NMR. Bioactivity testing against M. tuberculosis H37Rv and clinical isolates of isoniazid/rifampin-resistant M. tuberculosis showed potent activity for 5-F-CM and 7-F-NCM.</p><p><strong>Conclusions: </strong>Synthetic biology techniques are well-suited for the targeted and high-level biosynthesis of CM and its derivatives. This study reports the highest laboratory-level yields of CM and NCM to date. This is the first instance of obtaining CM derivatives by biosynthesis rather than chemical synthesis, and it also marks the first report of halogenated NCM derivatives. High-level production of CM and its diverse analogues will provide a solid material foundation for advancing CM and its derivatives as potential anti-tuberculosis drug candidates.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"113"},"PeriodicalIF":4.3,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12087186/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144102189","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":"Chaperone overexpression boosts heterologous small molecule production in Saccharomyces cerevisiae.","authors":"Andreas M Vestergaard, Wasti Nurani, Paul Cachera, Uffe H Mortensen","doi":"10.1186/s12934-025-02728-7","DOIUrl":"10.1186/s12934-025-02728-7","url":null,"abstract":"<p><strong>Background: </strong>Chaperones play an important role in maintaining cellular proteostasis by mediating protein folding. As a result, chaperone overexpression has been widely used as a tool for enhancing folding and improving production of heterologous proteins in host organisms such as Saccharomyces cerevisiae. In contrast, this strategy has been much less explored for small molecule (SM) production. This is surprising, as SM pathways typically depend on multiple enzymes including large multi-domain synthases or synthetases, which may all benefit from folding assistance to enhance the catalytic power of the pathway.</p><p><strong>Results: </strong>We have established an S. cerevisiae strain library of 68 strains overexpressing endogenous cytosolic chaperones and a mating-based method that allows the chaperone library to be combined with a query strain that contains the pathway of a desirable SM. Using the small molecule aspulvinone E from Aspergillus terreus as a model compound, we screened the chaperone library for chaperones that improve production of aspulvinone E. Screening of the library identified several chaperones and chaperone combinations that improved aspulvinone E production. Specifically, the combined overexpression of YDJ1 and SSA1 was identified as the best hit in our screen. Subsequently, we demonstrated that overexpression of YDJ1 and SSA1 improved aspulvinone E production by 84% in 1.5 mL scale batch fermentations. The observed increase is likely due to higher levels of the MelA synthetase responsible for aspulvinone E synthesis, as overexpression of YDJ1 and SSA1 increases the amounts of fluorescent MelA-mRFP in cells producing this fusion protein.</p><p><strong>Conclusion: </strong>The endogenous cytosolic chaperone overexpression library and mating based screening method presented in this report constitute a tool allowing for fast and efficient identification of specific chaperones and chaperone combinations that benefit production of a given SM in S. cerevisiae-based cell factories.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"112"},"PeriodicalIF":4.3,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12087235/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094210","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":"Combinatorial metabolic engineering of Yarrowia lipolytica for high-level production of the plant-derived diterpenoid sclareol.","authors":"Jiang Chen, Longzheng Huang, Bang-Ce Ye, Ying Zhou","doi":"10.1186/s12934-025-02744-7","DOIUrl":"10.1186/s12934-025-02744-7","url":null,"abstract":"<p><strong>Background: </strong>Sclareol, a diterpene alcohol derived from Salvia sclarea, is primarily used in the synthesis of ambrox, an alternative to the expensive spice ambergris. However, commercial production of sclareol from plant extraction is costly and environmentally problematic, limiting its scalability. Recent advances in synthetic biology have enabled the construction of efficient cell factories for sclareol synthesis, offering a more sustainable solution.</p><p><strong>Results: </strong>In this study, we engineered Yarrowia lipolytica to produce sclareol by integrating genes encoding (13E)-8α-hydroxylabden-15-yl diphosphate synthase (LPPS) and sclareol synthase (SCS). Sclareol titers were further enhanced through the fusion of SsSCS and SsLPPS proteins, as well as multi-copy gene integration. To increase the precursor geranylgeranyl diphosphate (GGPP), we overexpressed various geranylgeranyl diphosphate synthases (GGS1), resulting in significant accumulation of GGPP. Additionally, optimization of the mevalonate pathway, coupled with the downregulation of lipid synthesis and upregulation of lipid degradation, directed more acetyl CoA towards sclareol production.</p><p><strong>Conclusions: </strong>In this study, we reprogrammed the metabolism of Y. lipolytica by combinatorial metabolic engineering with a sclareol titer of 2656.20 ± 91.30 mg/L in shake flasks. Our findings provide a viable strategy for utilizing Y. lipolytica as a microbial cell factory to produce sclareol.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"110"},"PeriodicalIF":4.3,"publicationDate":"2025-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12082891/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144086470","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 bioprocess optimization study to enhance the production of Menaquinone-7 using Bacillus subtilis MM26.","authors":"Maneesha M, Subathra Devi C","doi":"10.1186/s12934-025-02735-8","DOIUrl":"10.1186/s12934-025-02735-8","url":null,"abstract":"<p><strong>Background: </strong>Menaquinone-7 (MK-7) has a vital significance in promoting human health and tackling several global health concerns which makes its production extremely important. MK-7 is not easily accessible at a reasonable cost due to the poor fermentation yields and the existence of several laborious downstream unit processes. Efficient manufacturing methods are essential to meet the global requirements due to the increasing demand in the pharmaceutical and nutraceutical industries. This research study focuses on the enhanced production of MK-7 from Bacillus subtilis MM26 isolated from fermented home-made wine.</p><p><strong>Results: </strong>A suitable MK-7 production medium for Bacillus subtilis MM26 was determined and the yield was found to be 67 ± 0.6 mg/L. The one factor at a time (OFAT) results showed that medium containing lactose, glycine, with a pH 7, a temperature of 37 °C, and an inoculum size of 2.5% (2 × 10⁶ CFU/mL) was optimal synthesis of MK-7. RSM indicated that incubation time, carbon and nitrogen sources were the factors significantly affecting the MK-7 yield. RSM predicted optimal conditions, which yielded a maximum concentration of 442 ± 2.08 mg/L of MK-7.</p><p><strong>Conclusions: </strong>The outcomes of this study demonstrated the potential of Bacillus subtilis MM26 in large-scale industrial production of MK-7. The yield of MK-7 was amplified efficiently by integration of OFAT and RSM, paving the way for cost-efficient industrial production.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"109"},"PeriodicalIF":4.3,"publicationDate":"2025-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12082928/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144086467","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 knowledge driven DBTL cycle provides mechanistic insights while optimising dopamine production in Escherichia coli.","authors":"Lorena Hägele, Natalia Trachtmann, Ralf Takors","doi":"10.1186/s12934-025-02729-6","DOIUrl":"10.1186/s12934-025-02729-6","url":null,"abstract":"<p><strong>Background: </strong>Dopamine is a promising organic compound with several key applications in emergency medicine, diagnosis and treatment of cancer, production of lithium anodes, and wastewater treatment. Since studies on in vivo dopamine production are limited, this study demonstrates the development and optimisation of a dopamine production strain by the help of the knowledge driven design-build-test-learn (DBTL) cycle for rational strain engineering.</p><p><strong>Results: </strong>The knowledge driven DBTL cycle, involving upstream in vitro investigation, is an automated workflow that enables both mechanistic understanding and efficient DBTL cycling. Following the in vitro cell lysate studies, the results were translated to the in vivo environment through high-throughput ribosome binding site (RBS) engineering. As a result, we developed a dopamine production strain capable of producing dopamine at concentrations of 69.03 ± 1.2 mg/L which equals 34.34 ± 0.59 mg/g_biomass. Compared to state-of-the-art in vivo dopamine production, our approach improved performance by 2.6 and 6.6-fold, respectively.</p><p><strong>Conclusion: </strong>In essence, a highly efficient dopamine production strain was developed by implementing the knowledge driven DBTL cycle involving upstream in vitro investigation. The fine-tuning of the dopamine pathway by high-throughput RBS engineering clearly demonstrated the impact of GC content in the Shine-Dalgarno sequence on the RBS strength.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"111"},"PeriodicalIF":4.3,"publicationDate":"2025-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12084978/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144086562","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 highly efficient heterologous expression platform to facilitate the production of microbial natural products in Streptomyces.","authors":"Xiuling Wang, Ping Lin, Qiyao Shen, Xueyan Feng, Shouying Xu, Qijun Zhang, Yang Liu, Cailing Ren, Daojing Yong, Qiong Duan, Liujie Huo, Youming Zhang, Gang Li, Jun Fu, Ruijuan Li","doi":"10.1186/s12934-025-02722-z","DOIUrl":"https://doi.org/10.1186/s12934-025-02722-z","url":null,"abstract":"<p><strong>Background: </strong>Heterologous expression in Streptomyces provides a platform for mining natural products (NPs) encoded by cryptic biosynthetic gene clusters (BGCs) of bacteria. The BGCs are first engineered in hosts with robust recombineering systems, such as Escherichia coli, followed by expression in optimized heterologous hosts, such as Streptomyces, with defined metabolic backgrounds.</p><p><strong>Results: </strong>We developed a highly efficient heterologous expression platform, named Micro-HEP (microbial heterologous expression platform), that uses versatile E. coli strains capable of both modification and conjugation transfer of foreign BGCs and optimized chassis Streptomyces strain for expression. The stability of repeat sequences in these E. coli strains was superior to that of the commonly used conjugative transfer system E. coli ET12567 (pUZ8002). For optimizing expression of foreign BGCs, the chassis strain S. coelicolor A3(2)-2023 was generated by deleting four endogenous BGCs followed by introducing multiple recombinase-mediated cassette exchange (RMCE) sites in the S. coelicolor A3(2) chromosome. Additionally, modular RMCE cassettes (Cre-lox, Vika-vox, Dre-rox, and phiBT1-attP) were constructed for integrating BGCs into the chassis strain. Micro-HEP was tested using BGCs for the anti-fibrotic compound xiamenmycin and griseorhodins. Two to four copies of the xim BGC were integrated by RMCE, with increasing copy number associated with increasing yield of xiamenmycin. The grh BGC was also efficiently expressed, and the new compound griseorhodin H was identified.</p><p><strong>Conclusion: </strong>We demonstrated that our Micro-HEP system enables the efficient expression of foreign BGCs, facilitating the discovery of new NPs and increasing yields.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"24 1","pages":"105"},"PeriodicalIF":4.3,"publicationDate":"2025-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12076808/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144078894","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}