Journal of Industrial Microbiology & Biotechnology最新文献

{"title":"Microbial biosynthesis of rare cannabinoids.","authors":"Chunsheng Yan, Ikechukwu C Okorafor, Colin W Johnson, Kendall N Houk, Neil K Garg, Yi Tang","doi":"10.1093/jimb/kuaf013","DOIUrl":"10.1093/jimb/kuaf013","url":null,"abstract":"<p><p>∆9-tetrahydrocannabinol (∆9-THC) and cannabidiol are the most abundant natural cannabinoids isolated from the different cultivars of the Cannabis plant. Other natural ∆9-THC analogs, especially those with different alkyl chain substitutions, display different and potent bioactivity. However, these rare cannabinoids are typically isolated in minuscule amounts and are difficult to synthesize. Targeted microbial biosynthesis can therefore be an attractive route to access such molecules. Here, we report the development of a Saccharomyces cerevisiae host to biosynthesize 2 rare cannabinoids from simple sugars. The yeast host is engineered to accumulate excess geranyl pyrophosphate, to overexpress a fungal pathway to 2,4-dihydroxy-6-alkyl-benzoic acids, as well as the downstream UbiA-prenyltransferase and ∆9-tetrahydrocannabinolic acid synthase. Two rare cannabinoid acids, the C1-substituted ∆9-tetrahydrocannabiorcolic acid (∼16 mg/L) and the C7-substituted ∆9-tetrahydrocannabiphorolic acid (∼5 mg/L) were obtained from this host; the latter was thermally decarboxylated to give ∆9-tetrahydrocannabiphorol. Given the diversity of fungal biosynthetic gene clusters that can produce resorcylic acids, this microbial platform offers the potential to produce other rare and new-to-nature cannabinoids. One Sentence Summary:  Saccharomyces cerevisiae as a host to produce rare cannabinoids.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12134893/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143996141","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biochemical production of bio-based terephthalic acid from lignocellulosic biomass for CO2 emission reduction.","authors":"Seiji Nakagame, Takuma Kawao, Daiki Narita, Akira Yamamura, Takeshi Noda","doi":"10.1093/jimb/kuaf030","DOIUrl":"10.1093/jimb/kuaf030","url":null,"abstract":"<p><p>Terephthalic acid, a key precursor for polyester production, is traditionally derived from fossil resources, contributing to global warming. Although numerous studies have explored the production of terephthalic acid from biomass polysaccharides, energy-intensive chemical processes are predominantly employed. In this study, we have developed a biochemical process to produce bio-based terephthalic acid from lignocellulosic biomass for reducing CO2 emissions. Among the tested six underutilized lignocellulosic biomasses, hardwood, with high cellulose contents of 52.8%, was subjected to alkaline pretreatment (165 °C for 2.5 hr) to eliminate lignin, and the resulting water-insoluble fraction was hydrolyzed by cellulases to yield monosaccharides. These monosaccharides were then biochemically transformed into terephthalic acid via p-tolualdehyde using two microorganisms, Phlebia sp. (a p-tolualdehyde-producing strain) and Comamonas testosteroni DSM6577. Comamonas testosteroni DSM6577 was used to oxidize the side chains of p-tolualdehyde into carboxylic acid to obtain terephthalic acid. This report describes the successful production of small amounts of bio-based terephthalic acid from lignocellulosic biomass. One-Sentence Summary: This paper shows that terephthalic acid, a key precursor for polyester production, can be produced from lignocellulosic biomass by microbial fermentation, which would contribute to reduce CO2 emission compared with other previous processes.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12514950/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145137526","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bacteria-powered self-healing concrete: Breakthroughs, challenges, and future prospects.","authors":"Ibrahim M Elgendy, Nehal E Elkaliny, Hoda M Saleh, Gehad O Darwish, Mervt M Almostafa, Kamel Metwally, Galal Yahya, Yehia A-G Mahmoud","doi":"10.1093/jimb/kuae051","DOIUrl":"10.1093/jimb/kuae051","url":null,"abstract":"<p><p>In a world where concrete structures face constant degradation from environmental forces, a revolutionary solution has emerged: bio-self-healing concrete. This innovation involves embedding dormant bacteria within the concrete mix, poised to spring into action when cracks form. As moisture seeps into the cracks, these bacterial agents are activated, consuming nutrients and converting them into calcium carbonate, a natural substance that fills and repairs the fractures, restoring the material's integrity. This fascinating process represents a cutting-edge approach to maintaining concrete infrastructure, turning once-vulnerable materials into self-sustaining systems capable of healing themselves. The ongoing research into bio-self-healing concrete is focused on selecting bacterial strains that can withstand the extreme conditions within concrete, including its highly alkaline environment. The bacteria must also form resilient spores, remaining viable until they are needed for repair. Additionally, the study explores various challenges associated with this technology, such as the cost of production, the bacteria's long-term viability, and their potential environmental impact. Advancements in genetic engineering and smart technology are being explored to enhance these bacterial strains, making them more efficient and robust in their role as microscopic repair agents. This review delves into the potential of bio-self-healing concrete to revolutionize how we approach infrastructure maintenance, offering a glimpse into a future where concrete structures not only endure but actively repair themselves, extending their lifespan and reducing the need for costly repairs.</p><p><strong>One-sentence summary: </strong>Bio-self-healing concrete utilizes bacteria that activate upon crack formation to repair structures by producing calcium carbonate, offering a sustainable solution to prolong the lifespan of concrete infrastructure.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11730074/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142824235","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Pseudoalteromonas agarivorans-derived novel ulvan lyase of polysaccharide lyase family 40: Potential application of ulvan and partially hydrolyzed products in cosmetic industry.","authors":"Navindu Dinara Gajanayaka, Eunyoung Jo, Minthari Sakethanika Bandara, Svini Dileepa Marasinghe, Sachithra Amarin Hettiarachchi, Sithumini Wijewickrama, Gun-Hoo Park, Chulhong Oh, Youngdeuk Lee","doi":"10.1093/jimb/kuaf004","DOIUrl":"10.1093/jimb/kuaf004","url":null,"abstract":"<p><p>Ulvan is a complex sulfated polysaccharide in the cell walls of green algae with extensive applications in food, pharmaceutical, and agricultural industries, prompting extensive studies on ulvan, its oligosaccharides, monosaccharides, and cost-effective depolymerization methods. Our primary objectives were to investigate novel ulvan-utilizing marine bacteria, perform recombinant engineering of genes responsible for ulvan depolymerization, and determine their potential industrial applications. Samples were collected from Jeju Island, which is a South Korean region with significant excessive green algal growth, especially that of Ulva species. The marine bacterium Pseudoalteromonas agarivorans efficiently uses ulvan as its primary carbon source, indicating its potential for ulvan degradation. Through whole-genome sequencing the paul40 gene, which is a polysaccharide lyase family 40 (PL40) member, was identified and subsequently engineered into the pET-16b vector for expression as a His-tagged 95 kDa fusion protein. The ulvan depolymerization process was evaluated and confirmed using various analytical techniques including dinitrosalicylic acid assay, thin-layer chromatography, and gel permeation chromatography. Optimal enzyme activity occurred at 35°C, pH 8.0 in phosphate buffer, and 2.5 mM of NaCl. Furthermore, enzyme characterization and specific activity measurements were performed. This study is the first to report hyaluronidase and elastase inhibition by ulvan and its derivatives along with the characterization of an ulvan lyase enzyme from the PL40 family.</p><p><strong>One-sentence summary: </strong>This study reports the identification and recombinant expression of a novel ulvan-degrading enzyme from Pseudoalteromonas agarivorans, demonstrating its potential for cosmetic industrial applications by revealing ulvan's and partially hydrolyzed ulvan's hyaluronidase and elastase inhibition properties.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11835017/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370648","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biocide-resistant Pseudomonas oleovorans isolated from water-based coatings used in construction.","authors":"Muatasem Latif Ali, Lionel Ferrieres, Tuulia Hyötyläinen, Jana Jass","doi":"10.1093/jimb/kuaf015","DOIUrl":"10.1093/jimb/kuaf015","url":null,"abstract":"<p><p>Biocides are crucial in industrial applications to minimize microbial growth and prevent product spoilage. Water-based construction coatings are susceptible to microbial contamination during manufacturing and storage and this adversely impacts product properties, reduces shelf-life, and leads to substantial commercial losses. The future trend to lower the biocide concentrations in water-based coatings raises concerns about the emergence of biocide-resistant microbes. This study aims to identify and characterize the biocide-resistant microbe isolated from construction water-based coating materials to better understand its mechanisms of resistance. A total of 63 samples were collected from spoiled products, raw materials, and water from a manufacturing facility, and Pseudomonas oleovorans P4A were identified in all biocides-treated samples. A comparison between a P. oleovorans reference strain, 1045, and the P4A isolate revealed distinct colony morphology, growth rate and sensitivity to biocides and antibiotics. The P4A isolate was threefold more resistant to 5-chloro-2-methyl-isothiazolin-3-one and 1.5-fold more resistant to benzothiazolinone (BIT) compared to the reference strain. Conversely, it was 1.4-fold more sensitive to methylisothiazolinone (MIT) compared to the reference strain. No cross-resistance to antibiotics was observed. Metabolomic analysis using liquid chromatography combined with high-resolution mass spectrometry of lipids and polar metabolites showed that P4A had a relatively higher amount of lipids, while 1045 had a relatively higher amount of polar metabolites identified. A significant difference in lipid composition, specifically in diacylglycerol, phosphatidic acid, phosphatidylcholine, and phosphatidylserine was observed between P. oleovorans strains 1045 and P4A. These distinctions highlight increased lipid metabolism in P. oleovorans P4A and this may contribute to its adaptation to biocides. Microbial resistance can directly affect the effectiveness of these products, leading to an increased need for frequent maintenance and replacement, safety concerns, and environmental implications. One-Sentence Summary: Biocide-resistant Pseudomonas oleovorans isolate exhibited reduced growth rate and increased lipid levels relative to the reference strain.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12231568/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144505959","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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 Streptomyces coelicolor for heterologous expression of the thiopeptide GE2270A-A cautionary tale.","authors":"Francesco Del Carratore, Erik K R Hanko, Kamila Schmidt, Oksana Bilyk, Suhui Ye Huang, Marianna Iorio, Mercedes Pérez-Bonilla, Rosario Pérez-Redondo, Michelle Rudden, Emmanuele Severi, Arianna Tocchetti, Margherita Sosio, Emily J Johnson, Timothy Kirkwood, Dominic R Whittall, Alkisti Manousaki, Olga Genilloud, Antonio Rodríguez-García, Gavin H Thomas, Stefano Donadio, Rainer Breitling, Eriko Takano","doi":"10.1093/jimb/kuaf019","DOIUrl":"10.1093/jimb/kuaf019","url":null,"abstract":"<p><p>The thiopeptide GE2270A is a clinically relevant, ribosomally synthesised, and post-translationally modified peptide naturally produced by Planobispora rosea. Due to the genetically intractable nature of P. rosea, heterologous expression is considered a possible route to yield improvement. In this study, we focused on improving GE2270A production through heterologous expression of the biosynthetic gene cluster (BGC) in the model organism Streptomyces coelicolor M1146. A statistically significant yield improvement was obtained in the S. coelicolor system through the data-driven rational engineering of the BGC, including the introduction of additional copies of key biosynthetic and regulatory genes. However, despite our best efforts, the highest production level observed in the strains generated in this study is 12× lower than published titres achieved in the natural producer and 50× lower than published titres obtained using Nonomuraea ATCC 39727 as expression host. These results suggest that, while using the most genetically amenable strain as host can be the right choice when exploring different BGC designs, the choice of the most suitable host has a major effect on the achievable yield and should be carefully considered. The analysis of the multiomics data obtained in this study suggests an important role of PbtX in GE2270A biosynthesis and provides insights into the differences in production metabolic profiles between the different strains. One Sentence Summary: Data-driven rational engineering of Streptomyces coelicolor for heterologous production of the thiopeptide antibiotic GE2270A resulted in increased production but encountered unexpected challenges compared to production in the natural producer or the alternative host Nonomuraea ATCC 39727.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12309360/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144659396","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Novel supplementation of Fe3O4-doped green carbonized nanoparticles on hydrogenases genes and microbial biodiversity for enhancing biohydrogen yield in dark fermentation microbial electrohydrogenesis cells.","authors":"Hikmatullah Ahmadi, Anam Jalil, Sohail Khan, Irfan Ali Phulpoto, Zhang Chengyu, Zhisheng Yu","doi":"10.1093/jimb/kuaf016","DOIUrl":"10.1093/jimb/kuaf016","url":null,"abstract":"<p><p>Achieving high-purity biohydrogen (Bio-H₂) production necessitates the suppression of hydrogenotrophic methanogens, as their activity can impede hydrogen yield. Various inoculum pretreatments have been employed to suppress methane-producing microorganisms; however, these methods can negatively impact the enzymatic activity of hydrogen-producing microorganisms, thereby reducing hydrogen production. To address this challenge, this research investigates a novel approach to enhance Bio-H₂ production by activating microbial enzymes using magnetite Fe₃O4-doped carbonized nanoparticles (NPs) derived from vegetable leaves (VLCFe₃O4-NPs) within a coupled dark fermentation-microbial Electrohydrogenesis system. Characterization results revealed that VLCFe₃O4-NPs exhibited cubic and spherical morphologies, with a small diameter of 1 ± 100 nm and a mean crystallite size of 38.1 nm, indicating high purity. Fermentation tests investigated the impact of different nanoparticle dosages on Bio-H₂ generation, hydrogenase gene expression (Fe-Fe and Ni-Fe), and microbial biodiversity. Bio-H₂ production significantly improved with 500 mg/L VLCFe₃O4-NPs, yielding 1.2-fold more than the control group, while even a low dose of 25 mg/L resulted in a 0.22-fold increase. Relative gene expression analysis using qPCR and the 2-ΔΔCT method demonstrated a 30-fold increase in Cbei 1773 (Fe-Fe hydrogenase) and a 23-fold increase in hucL (Ni-Fe hydrogenase) gene expression, along with an increase in 16S rDNA. Additionally, the abundance of biohydrogen-producing bacteria, Clostridium_sensu_stricto_1 and Clostridium_sensu_stricto_11, increased by 14.3% and 11.1%, respectively, compared to 4.9% and 3.9% in the control group. This research indicates that VLCFe₃O4-NPs offer an eco-friendly solution for boosting biohydrogen production within microbial electrohydrogenesis cells with dark fermentation systems, thereby supporting sustainable bioenergy generation. One-sentence summary: Green carbonized nanoparticles Fe3O4-doped have been shown to turn on the genes of bacteria (Fe-Fe and Ne-Fe) and increase the biodiversity of microbes, both of which are important for biohydrogen production.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12259281/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144511990","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Towards sustainable agarwood production: integrating microbial interactions, anatomical changes, and metabolite biosynthesis.","authors":"Yashirdisai Sampasivam, Khalisah Khairina Razman, Nor Syakila Mohd Mazlan, Kamalrul Azlan Azizan, Yogesh K Ahlawat, Roohaida Othman","doi":"10.1093/jimb/kuaf025","DOIUrl":"10.1093/jimb/kuaf025","url":null,"abstract":"<p><p>Agarwood is a highly valuable non-timber forest product mainly derived from the Aquilaria genus, widely traded in the perfumery, religious items, and traditional medicine industries. Naturally, agarwood forms within the xylem as part of the tree's defense mechanism against environmental stressors and microbial infection. The escalating demand for agarwood has led to the overexploitation of Aquilaria species, with some now classified as critically endangered. Despite advancements in artificial induction methods for sustainable agarwood supply, the intricate links between physiological and molecular mechanisms governing its formation remain poorly understood. This review addresses these knowledge gaps by examining the interplay between morphological changes in xylem structure during tylose formation and molecular alterations, particularly the biosynthesis of 2-(2-phenylethyl)chromones (PECs), key compounds in agarwood. Additionally, it integrates findings from multi-omics approaches including genomics, transcriptomics, proteomics, and metagenomics to reveal how secondary metabolite biosynthesis, including PECs and terpenes, is regulated across various Aquilaria species, regions, and induction techniques. The role of microbial communities, particularly endophytes such as Fusarium, in regulating agarwood formation is also discussed, emphasizing their involvement in both natural and artificial induction strategies. Furthermore, this review explores the role of reactive oxygen species in mediating morphological and biochemical defense responses, alongside the functions of transcription factors (TFs), protein kinases, and signaling molecules in balancing defense and growth. However, the crosstalk between key genes such as chalcone synthases, MAPK, cytochromes, NADPH oxidases, TFs, and miRNAs require further study to fully understand the complex defense mechanisms in Aquilaria trees. Overall, this review aims to bridge the current knowledge gaps by linking morphological and biochemical changes in agarwood formation, particularly PEC biosynthesis, while proposing metabolite engineering using microbial hosts as a promising tool for sustainable and technology-driven agarwood production. One-Sentence Summary: This review explores the physiological and molecular processes behind agarwood formation in Aquilaria malaccensis, highlighting the roles of tyloses, microbial interactions, secondary metabolite biosynthesis particularly 2-(2-phenylethyl)chromones and the integration of biotechnology for sustainable production and metabolic engineering.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12375897/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144957093","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Exploration of alternative microfiltration modalities for the harvest and clarification of diverse recombinant proteins from high-density E. coli culture and lysate using hollow fibre, flat sheet cassette, and vibro membrane filtration technologies.","authors":"Jennifer Reid, Joyce Ni, Airong Chen, Patricia Gomes, Andrew Szto, Analyn Yu, Angela Luo, Belinda Kong, Calvin Adams, Neveathan Jeyachandran, Anumta Amir, Xavier Teixeira, Tao Yuan, Cédric Charretier","doi":"10.1093/jimb/kuaf008","DOIUrl":"10.1093/jimb/kuaf008","url":null,"abstract":"<p><p>Industrial bioprocess optimization has significantly increased the productivity of biomass and biologics in upstream production. Such process improvement in fermentation often translates to challenges in recovering intracellularly expressed recombinant proteins due to increased matrix complexity, resulting in a higher performance burden in midstream. Tangential flow filtration (TFF) is a popular industry standard for buffer exchange and protein separation from cellular debris. However, due to variations in the physicochemical properties of recombinant proteins, solutions for E. coli-based protein clarification remain challenging and often necessitate extensive exploration and process optimization. With growing options in filtration-based technologies, the identification of a near-universal clarification platform is desirable to accelerate bioprocess development overall. In this study, three TFF modalities, hollow fibre (HF), flat-sheet cassette (CAS), and vibro membrane filtration (VMF), were assessed in parallel to evaluate their clarification performance for three E. coli recombinant proteins with different biochemical properties. Reverse phase liquid chromatography data showed target protein recovery was uniformly higher for VMF than HF at equivalent loading. Cell density and lysate protein load were comparable for HF and VMF, and lower for CAS. These results support the choice of VMF and HF as easily optimized and operated TFF modalities for clarification of recombinant protein from complex crude bacterial matrix, where either can be efficiently performed with ease and minimum supervision. Both TFF applications were successfully demonstrated in primary cell harvest, cell wash and cell lysate clarification, for E. coli-based recombinant proteins.</p><p><strong>One-sentence summary: </strong>High-density E. coli microfiltration and lysate clarification were tested for three diverse recombinant proteins, where hollow fibre and vibro membrane filtration outperformed flat sheet cassette in terms of process time, suspended solid loading, and target protein recovery.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":" ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12022607/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143788516","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Thermophilic site-specific recombination system for rapid insertion of heterologous DNA into the Clostridium thermocellum chromosome.","authors":"Nandhini Ashok, Yasemin Kaygusuz, Heidi S Schindel, Sarah Thurmon, Carrie A Eckert, Adam M Guss","doi":"10.1093/jimb/kuaf023","DOIUrl":"https://doi.org/10.1093/jimb/kuaf023","url":null,"abstract":"<p><p>Clostridium thermocellum is an anaerobic thermophile capable of producing ethanol and other commodity chemicals from lignocellulosic biomass. The insertion of heterologous DNA into the C. thermocellum chromosome is currently achieved via a time-consuming homologous recombination process, where a single stable insertion can take 2-4 weeks or more to construct. In this work, we developed a thermostable version of the Serine recombinase Assisted Genome Engineering (tSAGE) approach for gene insertion in C. thermocellum utilizing a site-specific recombinase from Geobacillus sp. Y412MC61, enabling quick and easy insertion of DNA into the chromosome for accelerated genetic tool screening and heterologous gene expression. Using tSAGE, chromosomal insertion of plasmid DNA occurred at a maximum transformation efficiency of 5 × 103 CFU/µg, which is comparable to the transformation efficiency of a replicating control plasmid in C. thermocellum. Using tSAGE, we chromosomally integrated and characterized 17 reporter genes, 15 homologous and 31 heterologous constitutive promoters of varying strengths, 4 inducible promoters, and 5 riboswitches in C. thermocellum. We also determined that a 6-7 nucleotide gap between the ribosome binding site (RBS) and the start codon is optimal for high expression by employing a library of superfolder green fluorescent protein expression constructs driven by our strongest tested promoter (Pclo1313_1194) with different distances between the RBS and start codon. The tools developed here will aid in accelerating C. thermocellum strain engineering for producing sustainable fuels and chemicals directly from plant biomass. One-Sentence Summary: A highly efficient site-specific recombination system was created for Clostridium thermocellum, which enabled the rapid characterization of a large collection of genetic parts for controlled gene expression.</p>","PeriodicalId":16092,"journal":{"name":"Journal of Industrial Microbiology & Biotechnology","volume":"52 ","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12371841/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144957136","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}