New biotechnology最新文献

{"title":"Enhancing protein productivities in CHO cells through adenosine uptake modulation – Novel insights into cellular growth and productivity regulation","authors":"Sri Ranganayaki Madabhushi ,&nbsp;Tomali Chakravarty ,&nbsp;Tomas Kasza,&nbsp;Malik Padellan,&nbsp;Tariq Bassam Atieh,&nbsp;Balrina Gupta","doi":"10.1016/j.nbt.2024.08.500","DOIUrl":"10.1016/j.nbt.2024.08.500","url":null,"abstract":"<div><p>Maximizing production potential of recombinant proteins such as monoclonal antibodies (mAbs) in Chinese Hamster Ovary (CHO) cells is a key enabler of reducing cost of goods of biologics. In this study, we explored various strategies to utilize adenosine mediated effects in biologics manufacturing processes. Results show that supplementation of adenosine increases specific productivity by up to two-fold while also arresting cell growth. Introducing adenosine in intensified perfusion processes in a biphasic manner significantly enhanced overall productivity. Interestingly, adenosine effect was observed to be dependent on the cell growth state. Using specific receptor antagonists and inhibitors, we identified that ENTs (primarily Slc29a1) mediate the uptake of adenosine in CHO cell cultures. Transcriptomics data showed an inverse correlation between Slc29a1 expression levels and peak viable cell densities. Data suggests that in fed-batch cultures, adenosine can be produced extracellularly. Blocking Slc29a1 using ENT inhibitors such as DZD and DP alone or in combination with CD73 inhibitor, PSB12379, resulted in a twofold increase in peak viable cell densities as well as productivities in fed batch – a novel strategy that can be applied to biologics manufacturing processes. This is the first study that suggests that adenosine production/accumulation in CHO cell cultures can potentially regulate the transition of CHO cells from exponential to stationary phase. We also demonstrate strategies to leverage this regulatory mechanism to maximize the productivity potential of biologics manufacturing processes.</p></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"83 ","pages":"Pages 163-174"},"PeriodicalIF":4.5,"publicationDate":"2024-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S187167842400534X/pdfft?md5=e71c2cbd0d460c758df32d65b0e2917b&pid=1-s2.0-S187167842400534X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141996222","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":"Production and characterization of novel Anti-HIV Fc-fusion proteins in plant-based systems: Nicotiana benthamiana & tobacco BY-2 cell suspension","authors":"Noemi Gutierrez-Valdes ,&nbsp;Francesc Cunyat ,&nbsp;Juliette Balieu ,&nbsp;Marie-Laure Walet-Balieu ,&nbsp;Matthew J. Paul ,&nbsp;Jonas de Groot ,&nbsp;Amaya Blanco-Perera ,&nbsp;Jorge Carrillo ,&nbsp;Patrice Lerouge ,&nbsp;Mariëlle Jansma-van Seters ,&nbsp;Jussi J. Joensuu ,&nbsp;Muriel Bardor ,&nbsp;Julian Ma ,&nbsp;Julià Blanco ,&nbsp;Anneli Ritala","doi":"10.1016/j.nbt.2024.08.499","DOIUrl":"10.1016/j.nbt.2024.08.499","url":null,"abstract":"<div><p>Multifunctional anti-HIV Fc-fusion proteins aim to tackle HIV efficiently through multiple modes of action. Although results have been promising, these recombinant proteins are hard to produce. This study explored the production and characterization of anti-HIV Fc-fusion proteins in plant-based systems, specifically <em>Nicotiana benthamiana</em> plants and tobacco BY-2 cell suspension. Fc-fusion protein expression in plants was optimized by incorporating codon optimization, ER retention signals, and hydrophobin fusion elements. Successful transient protein expression was achieved in <em>N. benthamiana</em>, with notable improvements in expression levels achieved through <em>N</em>-terminal hydrophobin fusion and ER retention signals. Stable expression in tobacco BY-2 resulted in varying accumulation levels being at highest 2.2.mg/g DW. The inclusion of hydrophobin significantly enhanced accumulation, providing potential benefits for downstream processing. Mass spectrometry analysis confirmed the presence of the ER retention signal and of <em>N</em>-glycans. Functional characterization revealed strong binding to CD64 and CD16a receptors, the latter being important for antibody-dependent cellular cytotoxicity (ADCC). Interaction with HIV antigens indicated potential neutralization capabilities. In conclusion, this research highlights the potential of plant-based systems for producing functional anti-HIV Fc-fusion proteins, offering a promising avenue for the development of these novel HIV therapies.</p></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"83 ","pages":"Pages 142-154"},"PeriodicalIF":4.5,"publicationDate":"2024-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1871678424005338/pdfft?md5=cec6162c7540395dab7ca896a0f0d232&pid=1-s2.0-S1871678424005338-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141982841","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":"Large-scale cultivation of Synechocystis sp. PCC6803 for the production of Poly(3-hydroxybutyrate) and its potential applications in the manufacturing of bulk and medical prototypes","authors":"Elbaraa Elghazy ,&nbsp;Syed Mohammad Daniel Syed Mohamed ,&nbsp;Kamonchanok Wianglor ,&nbsp;Santosh Tetali ,&nbsp;Mahendra Raut ,&nbsp;Ipsita Roy ,&nbsp;Jagroop Pandhal","doi":"10.1016/j.nbt.2024.08.497","DOIUrl":"10.1016/j.nbt.2024.08.497","url":null,"abstract":"<div><p>Polyhydroxyalkanoates (PHAs) are biopolymers produced by microorganisms under nutrient limiting conditions and in the presence of excess carbon source. PHAs have gained popularity as a sustainable alternative to traditional plastics. However, large scale production of PHAs is economically challenging due to the relatively high costs of organic carbon. Alternative options include using organisms capable of phototrophic or mixotrophic growth. This study aimed at the production of poly(3-hydroxybutyrate) P(3HB), a type of PHA, at pilot scale using the freshwater cyanobacterium <em>Synechocystis</em> sp. PCC6803. First, to identify optimal conditions for P(3HB) production from <em>Synechocystis</em> sp. PCC6803, different supplemental carbon source concentrations and salinity levels were tested at laboratory scale. The addition of 4 g/L acetate with no added NaCl led to P(3HB) accumulation of 10.7 % dry cell weight on the 28th day of cultivation. Although acetate additions were replicated in an outdoor 400 L serpentine photobioreactor, P(3HB) content was lower, implying uncontrolled conditions impact on biopolymer production efficiency. An optimized P(3HB) extraction methodology was developed to remove pigments, and the biopolymer was characterized and subjected to 3D printing (fused deposition modelling) to confirm its processability. This study thus successfully led to the large-scale production of P(3HB) using sustainable and environmentally friendly cyanobacterial fermentation.</p></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"83 ","pages":"Pages 133-141"},"PeriodicalIF":4.5,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1871678424005314/pdfft?md5=781a9272126ec786cca98da0c99ca0d5&pid=1-s2.0-S1871678424005314-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141917199","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":"Escherichia coli-based biorefining process yields optically pure lactic acid from fermented second-generation feedstocks","authors":"Anna Visentin ,&nbsp;Cormac D. Murphy ,&nbsp;Merlin Alvarado-Morales ,&nbsp;Irini Angelidaki ,&nbsp;Joseph B. Sweeney","doi":"10.1016/j.nbt.2024.08.498","DOIUrl":"10.1016/j.nbt.2024.08.498","url":null,"abstract":"<div><p>Within the circular bioeconomy the production of optically pure LA from 2nd generation feedstocks would be ideal but it is very challenging. In this paper genetically engineered <em>Escherichia coli</em> strains were created to resolve racemic LA solutions synthesised and produced from the fermentation of organic waste or ensiled grass. Refining LA racemic mixtures into either a D- or L-LA was achieved by cells being able to consume one LA isomer as a sole carbon and energy source while not being able to consume the other. A D-LA refining strain JSP0005 was grown on fermented source-sorted organic household waste and different grass silage leachates, which are 2nd generation feedstocks containing up to 33 g/L lactic acid racemate. In all growth experiments, L-LA was completely removed leaving D-LA as the only LA stereoisomer, i.e. resulting in optically pure D-LA, which also increased by as much as 248.6 % from its starting concentration, corresponding to 38 g/L. The strains resulting from this study are a promising first step towards a microbial based LA biorefining process.</p></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"83 ","pages":"Pages 155-162"},"PeriodicalIF":4.5,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1871678424005326/pdfft?md5=03ba51ccfd8d3b4e06beaa45cb38b136&pid=1-s2.0-S1871678424005326-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141917198","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 and anti-inflammatory properties of Lactiplantibacillus plantarum MKTJ24 isolated from an artisanal fermented fish of North-east India","authors":"Tulsi K. Joishy ,&nbsp;Anupam Bhattacharya ,&nbsp;Chingtham Thanil Singh ,&nbsp;Ashis K. Mukherjee ,&nbsp;Mojibur R. Khan","doi":"10.1016/j.nbt.2024.07.005","DOIUrl":"10.1016/j.nbt.2024.07.005","url":null,"abstract":"<div><p>The study aimed to isolate and characterize lactic acid bacteria from various traditional fermented fish products from North East India, including <em>Xindol</em>, <em>Hentak,</em> and <em>Ngari,</em> which hold significant dietary importance for the indigenous tribes. Additionally, the study sought to examine their untargeted metabolomic profiles. A total of 43 strains of <em>Bacillus</em>, <em>Priestia, Staphylococcus, Pediococcus,</em> and <em>Lactiplantibacillus</em> were isolated, characterized by 16 S rRNA gene and tested for probiotic properties. Five strains passed pH and bile salt tests with strain dependent antimicrobial activity, which exhibited moderate autoaggregation and hydrophobicity properties. <em>Lactiplantibacillus plantarum</em> MKTJ24 exhibited the highest hydrophobicity (42 %), which was further confirmed by adhesion assay in HT-29 cell lines (100 %). <em>Lactiplantibacillus plantarum</em> MKTJ24 treatment in LPS-stimulated HT-29 cells up-regulated expression of mucin genes compared to LPS-treated cells. Treatment of RAW 264.7 cells with <em>Lactiplantibacillus plantarum</em> MKTJ24 decreased LPS-induced reactive oxygen species (ROS) and nitric oxide (NO) productions. Further, genome analysis of <em>Lactiplantibacillus plantarum</em> MKTJ24 revealed the presence of several probiotic markers and immunomodulatory genes. The genome was found to harbor plantaricin operon involved in bacteriocin production. A pangenome analysis using all the publicly available <em>L. plantarum</em> genomes specifically isolated from fermented fish products identified 120 unique genes in <em>Lactiplantibacillus plantarum</em> MKTJ24. Metabolomic analysis indicated dominance of ascorbic acids, pentafluropropionate, cyclopropaneacetic acid, florobenzylamine, and furanone in <em>Xindol</em>. This study suggests that <em>Lactiplantibacillus plantarum</em> MKTJ24 has potential probiotic and immunomodulatory properties that could be used in processing traditional fermented fish products on an industrial scale to improve their quality and enhance functional properties.</p></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"83 ","pages":"Pages 121-132"},"PeriodicalIF":4.5,"publicationDate":"2024-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1871678424000347/pdfft?md5=cd246ee91625e95e0cdfecae42952f2f&pid=1-s2.0-S1871678424000347-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141902486","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":"Cell-based glycoengineering of extracellular vesicles through precise genome editing","authors":"Weihua Tian,&nbsp;Chiara Zagami,&nbsp;Jiasi Chen,&nbsp;Anne Louise Blomberg,&nbsp;Laura Salse Guiu,&nbsp;Sarah Line Skovbakke,&nbsp;Steffen Goletz","doi":"10.1016/j.nbt.2024.07.004","DOIUrl":"10.1016/j.nbt.2024.07.004","url":null,"abstract":"<div><p>Engineering of extracellular vesicles (EVs) towards more efficient targeting and uptake to specific cells has large potentials for their application as therapeutics. Carbohydrates play key roles in various biological interactions and are essential for EV biology. The extent to which glycan modification of EVs can be achieved through genetic glycoengineering of their parental cells has not been explored yet. Here we introduce targeted glycan modification of EVs through cell-based glycoengineering via modification of various enzymes in the glycosylation machinery. In a “simple cell” strategy, we modified major glycosylation pathways by knocking-out (KO) essential genes for N-glycosylation (<em>MGAT1</em>), O-GalNAc glycosylation (<em>C1GALT1C1</em>), glycosphingolipids (<em>B4GALT5/6</em>), glycosaminoglycans (<em>B4GALT7</em>) and sialylation (<em>GNE</em>) involved in the elongation or biosynthesis of the glycans in HEK293F cells. The gene editing led to corresponding glycan changes on the cells as demonstrated by differential lectin staining. Small EVs (sEVs) isolated from the cells showed overall corresponding glycan changes, but also some unexpected differences to their parental cell including enrichment preference for certain glycan structures and absence of other glycan types. The genetic glycoengineering did not significantly impact sEVs production, size distribution, or syntenin-1 biomarker expression, while a clonal influence on sEVs production yields was observed. Our findings demonstrate the successful implementation of sEVs glycoengineering via genetic modification of the parental cell and a stable source for generation of glycoengineered sEVs. The utilization of glycoengineered sEVs offers a promising opportunity to study the role of glycosylation in EV biology, as well as to facilitate the optimization of sEVs for therapeutic purposes.</p></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"83 ","pages":"Pages 101-109"},"PeriodicalIF":4.5,"publicationDate":"2024-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1871678424000335/pdfft?md5=ce5729946fff08ebd9973deb08456252&pid=1-s2.0-S1871678424000335-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141850956","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":"Discovery of a new class of bacterial heme-containing CC cleaving oxygenases","authors":"Ni Nyoman Purwani ,&nbsp;Henriette J. Rozeboom ,&nbsp;Vivian P. Willers ,&nbsp;Hein J. Wijma ,&nbsp;Marco W. Fraaije","doi":"10.1016/j.nbt.2024.07.002","DOIUrl":"10.1016/j.nbt.2024.07.002","url":null,"abstract":"<div><p>Previously, some bacteria were shown to harbour enzymes capable of catalysing the oxidative cleavage of the double bond of <em>t</em>-anethole and related compounds. The cofactor dependence of these enzymes remained enigmatic due to a lack of biochemical information. We report on catalytic and structural details of a representative of this group of oxidative enzymes: <em>t</em>-anethole oxygenase from <em>Stenotrophomonas maltophilia</em> (TAO_Sm). The bacterial enzyme could be recombinantly expressed and purified, enabling a detailed biochemical study that has settled the dispute on its cofactor dependence. We have established that TAO_Sm contains a tightly bound b-type heme and merely depends on dioxygen for catalysis. It was found to accept <em>t</em>-anethole, isoeugenol and O-methyl isoeugenol as substrates, all being converted into the corresponding aromatic aldehydes without the need of any cofactor regeneration. The elucidated crystal structure of TAO_Sm has revealed that it contains a unique active site architecture that is conserved for this distinct class of heme-containing bacterial oxygenases. Similar to other hemoproteins, TAO_Sm has a histidine (His121) as proximal ligand. Yet, unique for TAOs, an arginine (Arg89) is located at the distal axial position. Site directed mutagenesis confirmed crucial roles for these heme-liganding residues and other residues that form the substrate binding pocket. In conclusion, the results reported here reveal a new class of bacterial heme-containing oxygenases that can be used for the cleavage of alkene double bonds, analogous to ozonolysis in organic chemistry.</p></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"83 ","pages":"Pages 82-90"},"PeriodicalIF":4.5,"publicationDate":"2024-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1871678424000311/pdfft?md5=0424220bab546d011e27e91b592a90ef&pid=1-s2.0-S1871678424000311-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141760075","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":"Enhancing phenolic and lipid compound production in oat bran via acid pretreatment and solid-state fermentation with Aspergillus niger","authors":"Silvia Amalia NEMES ,&nbsp;Anca Corina FĂRCAS ,&nbsp;Floricuta RANGA ,&nbsp;Bernadette-Emoke TELEKY ,&nbsp;Lavinia Florina CĂLINOIU ,&nbsp;Francisc Vasile DULF ,&nbsp;Dan Cristian VODNAR","doi":"10.1016/j.nbt.2024.07.003","DOIUrl":"10.1016/j.nbt.2024.07.003","url":null,"abstract":"<div><p>Oat (<em>Avena sativa</em>) processing generates a large amount of by-products, especially oat bran. These by-products are excellent sources of bioactive compounds such as polyphenols and essential fatty acids. Therefore, enhancing the extraction of these bioactive substances and incorporating them into the human diet is critical. This study investigates the effect of acid pretreatment on the solid-state fermentation of oat bran with <em>Aspergillus niger</em>, with an emphasis on the bioaccessibility of phenolic acids and lipid profile. The results showed a considerable increase in reducing sugars following acid pretreatment. On the sixth day, there was a notable increase in the total phenolic content, reaching 58.114 ± 0.09 mg GAE/g DW, and the vanillic acid level significantly rose to 77.419 ± 0.27 μg/g DW. The lipid profile study revealed changes ranging from 4.66 % in the control to 7.33 % on the sixth day of SSF. Aside from biochemical alterations, antioxidant activity measurement using the DPPH technique demonstrated the maximum scavenging activity on day 4 (83.33 %). This study highlights acid pretreatment's role in enhancing bioactive compound accessibility in solid-state fermentation and its importance for functional food development.</p></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"83 ","pages":"Pages 91-100"},"PeriodicalIF":4.5,"publicationDate":"2024-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1871678424000323/pdfft?md5=97a38e6441d84de89fc8217a38b89b75&pid=1-s2.0-S1871678424000323-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141760076","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 HER2-positive cancer therapy using ADAPT6 fused to horseradish peroxidase","authors":"Andreas Wisniewski ,&nbsp;Diana Humer ,&nbsp;Marit Möller ,&nbsp;Sara Kanje ,&nbsp;Oliver Spadiut ,&nbsp;Sophia Hober","doi":"10.1016/j.nbt.2024.07.001","DOIUrl":"10.1016/j.nbt.2024.07.001","url":null,"abstract":"<div><p>Targeted cancer therapy is a promising alternative to the currently established cancer treatments, aiming to selectively kill cancer cells while sparing healthy tissues. Hereby, molecular targeting agents, such as monoclonal antibodies, are used to bind to cancer cell surface markers specifically. Although these agents have shown great clinical success, limitations still remain such as low tumor penetration and off-target effects. To overcome this limitation, novel fusion proteins comprised of the two proteins ADAPT6 and Horseradish Peroxidase (HRP) were engineered. Cancer cell targeting is hereby enabled by the small scaffold protein ADAPT6, engineered to specifically bind to human epidermal growth factor receptor 2 (HER2), a cell surface marker overexpressed in various cancer types, while the enzyme HRP oxidizes the nontoxic prodrug indole-3-acetic acid (IAA) which leads to the formation of free radicals and thereby to cytotoxic effects on cancer cells. The high affinity to HER2, as well as the enzymatic activity of HRP, were still present for the ADAPT6-HRP fusion proteins. Further, <em>in vitro</em> cytotoxicity assay using HER2-positive SKOV-3 cells revealed a clear advantage of the fusion proteins over free HRP by association of the fusion proteins directly to the cancer cells and therefore sustained cell killing. This novel strategy of combining ADAPT6 and HRP represents a promising approach and a viable alternative to antibody conjugation for targeted cancer therapy.</p></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"83 ","pages":"Pages 74-81"},"PeriodicalIF":4.5,"publicationDate":"2024-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S187167842400030X/pdfft?md5=99cd6d720fc11e8a87d470c350947624&pid=1-s2.0-S187167842400030X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141734728","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":"dMAD7 is a promising tool for targeted gene regulation in the methylotrophic yeast Komagataella phaffii","authors":"","doi":"10.1016/j.nbt.2024.06.008","DOIUrl":"10.1016/j.nbt.2024.06.008","url":null,"abstract":"<div><p>The methylotrophic yeast <em>Komagataella phaffii</em> is a popular host system for the pharmaceutical and biotechnological production of recombinant proteins. CRISPR-Cas9 and its derivative CRISPR interference (CRISPRi) offer a promising avenue to further enhance and exploit the full capabilities of this host. MAD7 and its catalytically inactive variant “dead” MAD7 (dMAD7) represent an interesting alternative to established CRISPR-Cas9 systems and are free to use for industrial and academic research. CRISPRi utilizing dMAD7 does not introduce double-strand breaks but only binds to the DNA to regulate gene expression. Here, we report the first use of dMAD7 in <em>K. phaffii</em> to regulate the expression of the enhanced green fluorescent protein (<em>eGFP</em>). A reduction of eGFP fluorescence level (up to 88 %) was achieved in random integration experiments using dMAD7 plasmids. Integration loci/events of investigated strains were assessed through whole genome sequencing. Additionally, RNA-sequencing experiments corroborated the whole genome sequencing results and showed a significantly reduced expression of <em>eGFP</em> in strains containing a dMAD7 plasmid, among others. Our findings conclusively demonstrate the utility of dMAD7 in <em>K. phaffii</em> through successfully regulating <em>eGFP</em> expression.</p></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"83 ","pages":"Pages 110-120"},"PeriodicalIF":4.5,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1871678424000293/pdfft?md5=ab5bf7c1b8723321dd1b9a85decd9d81&pid=1-s2.0-S1871678424000293-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141498557","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}