Biotechnology Journal最新文献_第9页

Development of a Human Recombinant Collagen for Vat Polymerization-Based Bioprinting 开发用于基于大桶聚合的生物打印的人类重组胶原蛋白。

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2024-10-09 DOI: 10.1002/biot.202400393

Domenic Schlauch, Jan Peter Ebbecke, Johanna Meyer, Tabea Marie Fleischhammer, Hamidreza Pirmahboub, Lutz Kloke, Selin Kara, Antonina Lavrentieva, Iliyana Pepelanova

{"title":"Development of a Human Recombinant Collagen for Vat Polymerization-Based Bioprinting","authors":"Domenic Schlauch, Jan Peter Ebbecke, Johanna Meyer, Tabea Marie Fleischhammer, Hamidreza Pirmahboub, Lutz Kloke, Selin Kara, Antonina Lavrentieva, Iliyana Pepelanova","doi":"10.1002/biot.202400393","DOIUrl":"10.1002/biot.202400393","url":null,"abstract":"In light-based 3D-bioprinting, gelatin methacrylate (GelMA) is one of the most widely used materials, as it supports cell attachment, and shows good biocompatibility and degradability in vivo. However, as an animal-derived material, it also causes safety concerns when used in medical applications. Gelatin is a partial hydrolysate of collagen, containing high amounts of hydroxyproline. This causes the material to form a thermally induced gel at ambient temperatures, a behavior also observed in GelMA. This temperature-dependent gelation requires precise temperature control during the bioprinting process to prevent the gelation of the material. To avoid safety concerns associated with animal-derived materials and reduce potential issues caused by thermal gelation, a recombinant human alpha-1 collagen I fragment was expressed in Komagataella phaffii without hydroxylation. The resulting protein was successfully modified with methacryloyl groups and underwent rapid photopolymerization upon ultraviolet light exposure. The developed material exhibited slightly slower polymerization and lower storage modulus compared to GelMA, while it showed higher stretchability. However, unlike the latter, the material did not undergo physical gelation at ambient temperatures, but only when cooled down to below 10°C, a characteristic that has not been described for comparable materials so far. This gelation was not caused by the formation of triple-helical structures, as shown by the absence of the characteristic peak at 220 nm in CD spectra. Moreover, the developed recombinant material facilitated cell adherence with high cell viability after crosslinking via light to a 3D structure. Furthermore, desired geometries could be easily printed on a stereolithographic bioprinter.","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 10","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400393","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386597","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Single-Batch Expression of an Experimental Recombinant Snakebite Antivenom Based on an Oligoclonal Mixture of Human Monoclonal Antibodies 基于人类单克隆抗体寡克隆混合物的实验性重组蛇咬伤抗蛇毒血清的单批次表达。

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2024-10-09 DOI: 10.1002/biot.202400348

Anna C. Adams, Lise M. Grav, Shirin Ahmadi, Camilla Holst Dahl, Anne Ljungars, Andreas H. Laustsen, Lars K. Nielsen

{"title":"Single-Batch Expression of an Experimental Recombinant Snakebite Antivenom Based on an Oligoclonal Mixture of Human Monoclonal Antibodies","authors":"Anna C. Adams, Lise M. Grav, Shirin Ahmadi, Camilla Holst Dahl, Anne Ljungars, Andreas H. Laustsen, Lars K. Nielsen","doi":"10.1002/biot.202400348","DOIUrl":"10.1002/biot.202400348","url":null,"abstract":"Oligoclonal antibodies, which are carefully defined mixtures of monoclonal antibodies, are valuable for the treatment of complex diseases, such as infectionss and cancer. In addition to these areas of medicine, they could be utilized for the treatment of snakebite envenoming, where recombinantly produced monoclonal human antibodies could overcome many of the drawbacks accompanying traditional antivenoms. However, producing multiple individual batches of monoclonal antibodies in an industrial setting is associated with significant costs. Therefore, it is attractive to produce oligoclonal antibodies by mixing multiple antibody-producing cell lines in a single batch to have only one upstream and downstream process. In this study, we selected four antibodies that target different toxins found in the venoms of various elapid snake species, such as mambas and cobras, and generated stable antibody-producing cell lines. Upon co-cultivation, we found the cell line ratios to be stable over 7 days. The purified oligoclonal antibody cocktail contained the anticipated antibody concentrations and bound to the target toxins as expected. These results thus provide a proof of concept for the strategy of mixing multiple cell lines in a single batch to manufacture tailored antivenoms recombinantly, which could be utilized for the treatment of snakebite envenoming and in other fields where oligoclonal antibody mixtures could find utility.","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 10","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400348","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386603","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Issue Information: Biotechnology Journal 10/2024 发行信息：生物技术期刊 10/2024

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2024-10-09 DOI: 10.1002/biot.202470101

引用次数: 0

25SrRNA Methyltransferase CgBMT5 From Candida glycerinogenes Improves Tolerance and Fermentation Performance of Saccharomyces cerevisiae and Yarrowia lipolytica From Undetoxified Cellulose Hydrolysate 来自甘油腺念珠菌的 25SrRNA 甲基转移酶 CgBMT5 提高了酿酒酵母和脂肪分解酵母对未解毒纤维素水解物的耐受性和发酵性能。

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2024-10-09 DOI: 10.1002/biot.202400397

Xinyao Lu, Xiaoqing Hao, Wen Lv, Bin Zhuge, Hong Zong

{"title":"25SrRNA Methyltransferase CgBMT5 From Candida glycerinogenes Improves Tolerance and Fermentation Performance of Saccharomyces cerevisiae and Yarrowia lipolytica From Undetoxified Cellulose Hydrolysate","authors":"Xinyao Lu, Xiaoqing Hao, Wen Lv, Bin Zhuge, Hong Zong","doi":"10.1002/biot.202400397","DOIUrl":"10.1002/biot.202400397","url":null,"abstract":"<div>\u0000 \u0000 The hydrolysis of cellulose generates inhibitors like acetate, suppressing fermentation performance. Here, 25SrRNA methyltransferase CgBMT5 from stress-tolerant yeast Candida glycerinogenes was used as an anti-stress gene element in Saccharomyces cerevisiae and Yarrowia lipolytica. Expression of CgBMT5 in S. cerevisiae increased cell tolerance to acetate, high osmolarity, and heat stress and rescued the delay in cell growth under acetate stress. Ethanol productivity was improved from 0.52 g·(L/h) to 0.69 g·(L/h). CgBMT5 improved GFP expression. The transcription factor ARG81 binds to the promoter of CgBMT5. CgBMT5 upregulated HOG1, GPD1, HAA1, and PMA1 and reduced ROS level, thereby improving cell resistance to acetate. CgBMT5 also improved resistance of Y. lipolytica Po1g to multiple-stress. The lipid titer was improved by 37% in the typical medium. Y. lipolytica-CgBMT5 produced 94 mg/L lipid in the undetoxified cellulose hydrolysate.\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 10","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386593","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Selecting Endogenous Promoters for Improving Biosynthesis of Squalene in Schizochytrium sp. 选择内源启动子以改善裂殖子藻类角鲨烯的生物合成

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2024-10-09 DOI: 10.1002/biot.202400237

Fang-Tong Nong, Zi-Xu Zhang, Lu-Wei Xu, Fei Du, Wang Ma, Guang Yang, Xiao-Man Sun

{"title":"Selecting Endogenous Promoters for Improving Biosynthesis of Squalene in Schizochytrium sp.","authors":"Fang-Tong Nong, Zi-Xu Zhang, Lu-Wei Xu, Fei Du, Wang Ma, Guang Yang, Xiao-Man Sun","doi":"10.1002/biot.202400237","DOIUrl":"10.1002/biot.202400237","url":null,"abstract":"<div>\u0000 \u0000 Squalene (C30H50) is an acyclic triterpenoid compound renowned for its myriad physiological functions, such as anticancer and antioxidative properties, rendering it invaluable in both the food and pharmaceutical sectors. Due to the natural resource constraints, microbial fermentation has emerged as a prominent trend. Schizochytrium sp., known to harbor the intact mevalonate acid (MVA) pathway, possesses the inherent capability to biosynthesize squalene. However, there is a dearth of reported key genes in both the MVA and the squalene synthesis pathways, along with the associated promoter elements for their modification. This study commenced by cloning and characterizing 13 endogenous promoters derived from transcriptome sequencing data. Subsequently, five promoters exhibiting varying expression intensities were chosen from the aforementioned pool to facilitate the overexpression of the squalene synthase gene squalene synthetase (SQS), pivotal in the MVA pathway. Ultimately, a transformed strain designated as SQS-3626, exhibiting squalene production 2.8 times greater than that of the wild-type strain, was identified. Finally, the optimization of nitrogen source concentrations and trace element contents in the fermentation medium was conducted. Following 120 h of fed-batch fermentation, the accumulated final squalene yield in the transformed strain SQS-3626 reached 2.2 g/L.\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 10","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386602","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Concentration-Dependent Effects of MXene Nanocomposite-Loaded Carboxymethyl Cellulose on Wound Healing MXene 纳米复合材料负载羧甲基纤维素对伤口愈合的浓度依赖性影响

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2024-10-09 DOI: 10.1002/biot.202400448

Salma Nasser, Mohamed Abd Elkodous, Rasha Tawfik, Hossam Tohamy, Mahmoud El-Kammar, Samir Nouh, Hoda Elkhenany

{"title":"Concentration-Dependent Effects of MXene Nanocomposite-Loaded Carboxymethyl Cellulose on Wound Healing","authors":"Salma Nasser, Mohamed Abd Elkodous, Rasha Tawfik, Hossam Tohamy, Mahmoud El-Kammar, Samir Nouh, Hoda Elkhenany","doi":"10.1002/biot.202400448","DOIUrl":"10.1002/biot.202400448","url":null,"abstract":"<div>\u0000 \u0000 Nanoparticles (NPs) have emerged as a promising solution for many biomedical applications. Although not all particles have antimicrobial or regenerative properties, certain NPs show promise in enhancing wound healing by promoting tissue regeneration, reducing inflammation, and preventing infection. Integrating various NPs can further enhance these effects. Herein, the zinc oxide (ZnO)-MXene-Ag nanocomposite was prepared, and the conjugation of its three components was confirmed through scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) mapping analysis. In vitro analysis using the agar well diffusion technique demonstrated that ZnO-MXene-Ag nanocomposite exhibited high antimicrobial efficacy, significantly inhibiting Escherichia coli, Salmonella, and Candida albicans, and showing enhanced potency when combined with tetracycline, resulting in a 2.6-fold increase against Staphylococcus and a 2.4-fold increase against Pseudomonas. The efficacy of nanocomposite-loaded carboxymethyl cellulose (CMC) gel on wound healing was investigated using varying concentrations (0, 1, 5, and 10 mg/mL). Wound healing was monitored over 21 days, with results indicating that wounds treated with 1 mg/mL ZnO-MXene-Ag gel exhibited superior healing compared to the control group (0 mg/mL), with significant improvements noted from Day 3 onward. Conversely, higher concentrations (10 mg/mL) resulted in reduced healing efficiency, particularly notable on Day 15. In conclusion, the ZnO-MXene-Ag nanocomposite-loaded CMC gel is a promising agent for enhanced wound healing and antimicrobial applications. These findings highlight the importance of optimizing NP concentration to maximize therapeutic benefits while minimizing potential cytotoxicity.\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 10","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386596","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Explainable Machine Learning Models to Predict Gibbs–Donnan Effect During Ultrafiltration and Diafiltration of High-Concentration Monoclonal Antibody Formulations 预测高浓度单克隆抗体制剂超滤和渗滤过程中吉布斯-多南效应的可解释机器学习模型。

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2024-10-09 DOI: 10.1002/biot.202400212

Chyi-Shin Chen, Seiryu Ujiie, Reina Tanibata, Takuo Kawase, Shohei Kobayashi

{"title":"Explainable Machine Learning Models to Predict Gibbs–Donnan Effect During Ultrafiltration and Diafiltration of High-Concentration Monoclonal Antibody Formulations","authors":"Chyi-Shin Chen, Seiryu Ujiie, Reina Tanibata, Takuo Kawase, Shohei Kobayashi","doi":"10.1002/biot.202400212","DOIUrl":"10.1002/biot.202400212","url":null,"abstract":"Evaluating the Gibbs–Donnan and volume exclusion effects during protein ultrafiltration and diafiltration (UF/DF) is crucial in biopharmaceutical process development to precisely control the concentration of the drug substance in the final formulation. Understanding the interactions between formulation excipients and proteins under these conditions requires a domain-specific knowledge of molecular-level phenomena. This study developed gradient boosted tree models to predict the Gibbs–Donnan and volume exclusion effects for amino acids and therapeutic monoclonal antibodies using simple molecular descriptors. The models’ predictions were interpreted by information gain and Shapley additive explanation (SHAP) values to understand the modes of action of the antibodies and excipients and to validate the models. The results translated feature effects in machine learning to real-world molecular interactions, which were cross-referenced with existing scientific literature for verification. The models were validated in pilot-scale manufacturing runs of two antibody products requiring high levels of concentration. By only requiring a molecule's biophysicochemical descriptors and process conditions, the proposed models provide an in silico alternative to conventional UF/DF experiments to accelerate process development and boost process understanding of the underlying molecular mechanisms through rational interpretation of the models.","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 10","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400212","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386599","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Genome-Scale Metabolic Modeling of Halomonas elongata 153B Explains Polyhydroxyalkanoate and Ectoine Biosynthesis in Hypersaline Environments Halomonas elongata 153B 的基因组尺度代谢模型解释了低盐环境中多羟基烷酸和外辛的生物合成。

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2024-10-09 DOI: 10.1002/biot.202400267

Blaise Manga Enuh, Pınar Aytar Çelik, Claudio Angione

{"title":"Genome-Scale Metabolic Modeling of Halomonas elongata 153B Explains Polyhydroxyalkanoate and Ectoine Biosynthesis in Hypersaline Environments","authors":"Blaise Manga Enuh, Pınar Aytar Çelik, Claudio Angione","doi":"10.1002/biot.202400267","DOIUrl":"10.1002/biot.202400267","url":null,"abstract":"Halomonas elongata thrives in hypersaline environments producing polyhydroxyalkanoates (PHAs) and osmoprotectants such as ectoine. Despite its biotechnological importance, several aspects of the dynamics of its metabolism remain elusive. Here, we construct and validate a genome-scale metabolic network model for H. elongata 153B. Then, we investigate the flux distribution dynamics during optimal growth, ectoine, and PHA biosynthesis using statistical methods, and a pipeline based on shadow prices. Lastly, we use optimization algorithms to uncover novel engineering targets to increase PHA production. The resulting model (iEB1239) includes 1534 metabolites, 2314 reactions, and 1239 genes. iEB1239 can reproduce growth on several carbon sources and predict growth on previously unreported ones. It also reproduces biochemical phenotypes related to Oad and Ppc gene functions in ectoine biosynthesis. A flux distribution analysis during optimal ectoine and PHA biosynthesis shows decreased energy production through oxidative phosphorylation. Furthermore, our analysis unveils a diverse spectrum of metabolic alterations that extend beyond mere flux changes to encompass heightened precursor production for ectoine and PHA synthesis. Crucially, these findings capture other metabolic changes linked to adaptation in hypersaline environments. Bottlenecks in the glycolysis and fatty acid metabolism pathways are identified, in addition to PhaC, which has been shown to increase PHA production when overexpressed. Overall, our pipeline demonstrates the potential of genome-scale metabolic models in combination with statistical approaches to obtain insights into the metabolism of H. elongata. Our platform can be exploited for researching environmental adaptation, and for designing and optimizing metabolic engineering strategies for bioproduct synthesis.","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 10","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400267","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386600","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Enhancing the expression of terminal deoxynucleotidyl transferases by N-terminal truncation 通过 N 端截断增强末端脱氧核苷酸转移酶的表达

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2024-09-19 DOI: 10.1002/biot.202400226

An-Na Li, Kun Shi, Bu-Bing Zeng, Jian-He Xu, Hui-Lei Yu

{"title":"Enhancing the expression of terminal deoxynucleotidyl transferases by N-terminal truncation","authors":"An-Na Li, Kun Shi, Bu-Bing Zeng, Jian-He Xu, Hui-Lei Yu","doi":"10.1002/biot.202400226","DOIUrl":"10.1002/biot.202400226","url":null,"abstract":"Terminal deoxynucleotidyl transferase (TdT), a unique DNA polymerase that catalyzes the template-free incorporation of nucleotides into single-stranded DNA, has facilitated the development of various oligonucleotide-based tools and methods, especially in the field of template-free enzymatic DNA synthesis. However, expressing vertebrate-derived TdTs in Escherichia coli complicates purification and increases production costs. In this study, N-terminal truncation of TdTs was performed to improve their expression and stability. The results revealed that N-terminal truncation could enhance the expression level of six TdTs. Among the truncated mutants, N-140-ZaTdT and N-140-CpTdT, with 140 amino acids removed, exhibited an increase in protein expression, which was 9.5- and 23-fold higher than their wild-types, respectively. Importantly, the truncation preserves the catalytic function of TdT. Additionally, the Tm values of N-140-ZaTdT increased by 4.9°C. The improved expression of the truncated mutants makes them more suitable for reducing production costs and advancing enzyme engineering.","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 9","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256061","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

CRISPR/Cas9-based genome engineering in the filamentous fungus Rhizopus oryzae and its application to L-lactic acid production 基于 CRISPR/Cas9 的丝状真菌根瘤菌基因组工程及其在 L-乳酸生产中的应用。

IF 3.2 3区生物学

Biotechnology Journal Pub Date : 2024-09-19 DOI: 10.1002/biot.202400309

Haodong Zhu, Han Wang, Li Wang, Zhiming Zheng

{"title":"CRISPR/Cas9-based genome engineering in the filamentous fungus Rhizopus oryzae and its application to L-lactic acid production","authors":"Haodong Zhu, Han Wang, Li Wang, Zhiming Zheng","doi":"10.1002/biot.202400309","DOIUrl":"10.1002/biot.202400309","url":null,"abstract":"The filamentous fungus Rhizopus oryzae is one of the main industrial strains for the production of a series of important chemicals such as ethanol, lactic acid, and fumaric acid. However, the lack of efficient gene editing tools suitable for R. oryzae makes it difficult to apply technical methods such as metabolic engineering regulation and synthetic biology modification. A CRISPR-Cas9 system suitable for efficient genome editing in R. oryzae was developed. Firstly, four endogenous U6 promoters of R. oryzae were identified and screened with the highest transcriptional activity for application to sgRNA transcription. It was then determined that the U6 promoter mediated CRISPR/Cas9 system has the ability to efficiently edit the genome of R. oryzae through NHEJ and HDR-mediated events. Furthermore, the newly constructed CRISPR-Cas9 dual sgRNAs system can simultaneously disrupt or insert different fragments of the R. oryzae genome. Finally, this CRISPR-Cas9 system was applied to the genome editing of R. oryzae by knocking out pyruvate carboxylase gene (PYC) and pyruvate decarboxylase gene (pdcA) and knocking in phosphofructokinase (pfkB) from Escherichia coli and L-lactate dehydrogenase (L-LDH) from Heyndrickxia coagulans, which resulted in a substantial increase in L-LA production. In summary, this study showed that the CRISPR/Cas9-based genome editing tool is efficient for manipulating genes in R. oryzae.","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 9","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256065","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0