Biotechnology Journal最新文献

{"title":"Evaluation of NAD+ precursors for improved metabolism and productivity of antibody-producing CHO cell","authors":"Hye-Jin Han,&nbsp;Hagyeong Kim,&nbsp;Hyun Gyu Yu,&nbsp;Jong Uk Park,&nbsp;Joo Hee Bae,&nbsp;Ji Hwan Lee,&nbsp;Jong Kwang Hong,&nbsp;Jong Youn Baik","doi":"10.1002/biot.202400311","DOIUrl":"10.1002/biot.202400311","url":null,"abstract":"<p>In the previous study, the culture medium was treated with nicotinamide adenine dinucleotide (NAD⁺) under the hypothesis that NAD⁺ regeneration is a major factor causing excessive lactate accumulation in Chinese hamster ovary (CHO) cells. The NAD⁺ treatment improved metabolism by not only reducing the Warburg effect but also enhancing oxidative phosphorylation, leading to enhanced antibody production. Building on this, four NAD⁺ precursors – nicotinamide mononucleotide (NMN), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide (NAM) – were tested to elevate intracellular NAD+ levels more economically. First, the ability of CHO cells to utilize both the salvage and Preiss-Handler pathways for NAD⁺ biosynthesis was verified, and then the effect of NAD⁺ precursors on CHO cell cultures was evaluated. These precursors increased intracellular NAD⁺ levels by up to 70.6% compared to the non-treated group. Culture analysis confirmed that all the precursors induced metabolic changes and that NMN, NA, and NR improved productivity akin to NAD⁺ treatment, with comparable integral viable cell density. Despite the positive effects such as the increase in the specific productivity and changes in cellular glucose metabolism, none of the precursors surpassed direct NAD⁺ treatment in antibody titer, presumably due to the reduction in nucleoside availability, as evidenced by the decrease in ATP levels in the NAD⁺ precursor-treated groups. These results underscore the complexity of cellular metabolism as well as the necessity for further investigation to optimize NAD⁺ precursor treatment strategies, potentially with the supplementation of nucleoside precursors. Our findings suggest a feasible approach for improving CHO cell culture performances by using NAD⁺ precursors as medium and feed components for the biopharmaceutical production.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 8","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400311","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142015716","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}

{"title":"Facilitating secretory expression of apple seed β-glucosidase in Komagataella phaffii for the efficient preparation of salidroside","authors":"Xin-Yi Lu,&nbsp;Ming-Yuan Lai,&nbsp;Peng Qin,&nbsp;Yu-Cong Zheng,&nbsp;Jia-Yi Liao,&nbsp;Zhi-Jun Zhang,&nbsp;Jian-He Xu,&nbsp;Hui-Lei Yu","doi":"10.1002/biot.202400347","DOIUrl":"10.1002/biot.202400347","url":null,"abstract":"<p>Plant-derived β-glucosidases hold promise for glycoside biosynthesis via reverse hydrolysis because of their excellent glucose tolerance and robust stability. However, their poor heterologous expression hinders the development of large-scale production and applications. In this study, we overexpressed apple seed β-glucosidase (ASG II) in <i>Komagataella phaffii</i> and enhanced its production from 289 to 4322 U L⁻¹ through expression cassette engineering and protein engineering. Upon scaling up to a 5-L high cell-density fermentation, the resultant mutant ASG II_V80A achieved a maximum protein concentration and activity in the secreted supernatant of 2.3 g L⁻¹ and 41.4 kU L⁻¹, respectively. The preparative biosynthesis of salidroside by ASG II_V80A exhibited a high space-time yield of 33.1 g L⁻¹ d⁻¹, which is so far the highest level by plant-derived β-glucosidase. Our work addresses the long-standing challenge of the heterologous expression of plant-derived β-glucosidase in microorganisms and presents new avenues for the efficient production of salidroside and other natural glycosides.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 8","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142015717","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}

{"title":"Semi-rational engineering of D-allulose 3-epimerase for simultaneously improving the catalytic activity and thermostability based on D-allulose biosensor","authors":"Zijie Li,&nbsp;Yangfan Hu,&nbsp;Cheng Yu,&nbsp;Kangqing Fei,&nbsp;Liqun Shen,&nbsp;Yishi Liu,&nbsp;Hideki Nakanishi","doi":"10.1002/biot.202400280","DOIUrl":"10.1002/biot.202400280","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Background</h3>\u0000 \u0000 <p>D-Allulose is one of the most well-known rare sugars widely used in food, cosmetics, and pharmaceutical industries. The most popular method for D-allulose production is the conversion from D-fructose catalyzed by D-allulose 3-epimerase (DAEase). To address the general problem of low catalytic efficiency and poor thermostability of wild-type DAEase, D-allulose biosensor was adopted in this study to develop a convenient and efficient method for high-throughput screening of DAEase variants.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>The catalytic activity and thermostability of DAEase from <i>Caballeronia insecticola</i> were simultaneously improved by semi-rational molecular modification. Compared with the wild-type enzyme, DAEase^S37N/F157Y variant exhibited 14.7% improvement in the catalytic activity and the half-time value (<i>t</i>_1/2) at 65°C increased from 1.60 to 27.56 h by 17.23-fold. To our delight, the conversion rate of D-allulose was 33.6% from 500-g L⁻¹ D-fructose in 1 h by <i>Bacillus subtilis</i> WB800 whole cells expressing this DAEase variant. Furthermore, the practicability of cell immobilization was evaluated and more than 80% relative activity of the immobilized cells was maintained from the second to seventh cycle.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusion</h3>\u0000 \u0000 <p>All these results indicated that the DAEase^S37N/F157Y variant would be a potential candidate for the industrial production of D-allulose.</p>\u0000 </section>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 8","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142015721","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}

{"title":"Microfabricated dynamic brain organoid cocultures to assess the effects of surface geometry on assembloid formation","authors":"Camille Cassel de Camps,&nbsp;Sabra Rostami,&nbsp;Vanessa Xu,&nbsp;Chen Li,&nbsp;Paula Lépine,&nbsp;Thomas M. Durcan,&nbsp;Christopher Moraes","doi":"10.1002/biot.202400070","DOIUrl":"10.1002/biot.202400070","url":null,"abstract":"<p>Organoids have emerged as valuable tools for the study of development and disease. Assembloids are formed by integrating multiple organoid types to create more complex models. However, the process by which organoids integrate to form assembloids remains unclear and may play an important role in the resulting organoid structure. Here, a microfluidic platform is developed that allows separate culture of distinct organoid types and provides the capacity to partially control the geometry of the resulting organoid surfaces. Removal of a microfabricated barrier then allows the shaped and positioned organoids to interact and form an assembloid. When midbrain and unguided brain organoids were allowed to assemble with a defined spacing between them, axonal projections from midbrain organoids and cell migration out of unguided organoids were observed and quantitatively measured as the two types of organoids fused together. Axonal projection directions were statistically biased toward other midbrain organoids, and unguided organoid surface geometry was found to affect cell invasion. This platform provides a tool to observe cellular interactions between organoid surfaces that are spaced apart in a controlled manner, and may ultimately have value in exploring neuronal migration, axon targeting, and assembloid formation mechanisms.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 8","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400070","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142015719","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}

{"title":"Outside Front Cover: (Biotechnology Journal 6/2024)","authors":"","doi":"10.1002/biot.202470072","DOIUrl":"https://doi.org/10.1002/biot.202470072","url":null,"abstract":"<p>The cover image is based on the Research Article <i>Unlocking the formate utilization of wild-type Yarrowia lipolytica through adaptive laboratory evolution</i> by Qian Chen et al., https://doi.org/10.1002/biot.202400290.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure></p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 6","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202470072","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141994255","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}

{"title":"Truncation of a novel C-terminal domain of a β-glucanase improves its thermal stability and specific activity","authors":"Anastasia Klemanska,&nbsp;Kelly Dwyer,&nbsp;Gary Walsh","doi":"10.1002/biot.202400245","DOIUrl":"10.1002/biot.202400245","url":null,"abstract":"<p>Enzymes that degrade β-glucan play important roles in various industries, including those related to brewing, animal feed, and health care. Csph16A, an endo-β-1,3(4)-glucanase encoded by a gene from the halotolerant, xerotolerant, and radiotrophic black fungus <i>Cladosporium sphaerospermum</i>, was cloned and expressed in <i>Pichia pastoris</i>. Two isoforms (Csph16A.1 and Csph16A.2) are produced, arising from differential glycosylation. The proteins were predicted to contain a catalytic Lam16A domain, along with a C-terminal domain (CTD) of unknown function which exhibits minimal secondary structure. Employing PCR-mediated gene truncation, the CTD of Csph16A was excised to assess its functional impact on the enzyme and determine potential alterations in biotechnologically relevant characteristics. The truncated mutant, Csph16A-ΔC, exhibited significantly enhanced thermal stability at 50°C, with D-values 14.8 and 23.5 times greater than those of Csph16A.1 and Csph16A.2, respectively. Moreover, Csph16A-ΔC demonstrated a 20%–25% increase in halotolerance at 1.25 and 1.5 M NaCl, respectively, compared to the full-length enzymes. Notably, specific activity against cereal β-glucan, lichenan, and curdlan was increased by up to 238%. This study represents the first characterization of a glucanase from the stress-tolerant fungus <i>C. sphaerospermum</i> and the first report of a halotolerant and engineered endo-β-1,3(4)-glucanase. Additionally, it sheds light on a group of endo-β-1,3(4)-glucanases from Antarctic rock-inhabiting black fungi harboring a Lam16A catalytic domain and a novel CTD of unknown function.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 8","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400245","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141905186","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}

{"title":"3D-printed titanium scaffolds loaded with gelatin hydrogel containing strontium-doped silver nanoparticles promote osteoblast differentiation and antibacterial activity for bone tissue engineering","authors":"Ramprasad Anushikaa,&nbsp;S. Shree Ganesh,&nbsp;Venkadesan Sri Swetha Victoria,&nbsp;Abinaya Shanmugavadivu,&nbsp;Krishnaraj Lavanya,&nbsp;Sundaravadhanan Lekhavadhani,&nbsp;Nagarajan Selvamurugan","doi":"10.1002/biot.202400288","DOIUrl":"10.1002/biot.202400288","url":null,"abstract":"<p>Bone tissue engineering offers a promising alternative to stimulate the regeneration of damaged tissue, overcoming the limitations of conventional autografts and allografts. Recently, titanium alloy (Ti) implants have garnered significant attention for treating critical-sized bone defects, especially with the advancement of 3D printing technology. Although Ti alloys have impressive versatility, their lack of cellular adhesion, osteogenic and antibacterial properties are significant factors that contribute to their failure. Hence, to overcome these obstacles, this study aimed to incorporate osteoinductive and antibacterial cue-loaded hydrogels into 3D-printed Ti (3D-Ti) scaffolds. 3D-Ti scaffolds were synthesized using the direct metal laser sintering method and loaded with a gelatin (Gel) hydrogel containing strontium-doped silver nanoparticles (Sr-Ag NPs). Compared with Ag NPs, Sr-doped Ag NPs increased the expression of Runx2 mRNA, which is a key bone transcription factor. We subjected the bioactive 3D-hybrid scaffolds (3D-Ti/Gel/Sr-Ag NPs) to physicochemical and material characterization, followed by cytocompatibility and osteogenic evaluation. The microporous and macroporous topographies of the scaffolds with Sr-Ag NPs showed increased Runx2 expression and matrix mineralization, with potent antibacterial properties. Therefore, the 3D-Ti scaffolds incorporated with Sr-Ag NP-loaded Gel hydrogels favored osteoblast differentiation and antibacterial activity, indicating their potential for orthopedic applications.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 8","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141900248","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}

{"title":"Use of tRNA gene barriers improves stability of transgene expression in CHO cells","authors":"Rebecca E. Sizer,&nbsp;Richard M. Ingram,&nbsp;Caroline Swan,&nbsp;Emma K. Biggs,&nbsp;Leon P. Pybus,&nbsp;Robert J. White","doi":"10.1002/biot.202400196","DOIUrl":"10.1002/biot.202400196","url":null,"abstract":"<p>Instability of transgene expression is a major challenge for the biopharmaceutical industry, which can impact yields and regulatory approval. Some tRNA genes (tDNAs) can resist epigenetic silencing, the principal mechanism of expression instability, and protect adjacent genes against the spread of repressive heterochromatin. We have taken two naturally occurring clusters of human tDNAs and tested their ability to reduce epigenetic silencing of transgenes integrated into the genome of Chinese hamster ovary (CHO) cells. We find sustained improvements in productivity both in adherent CHO-K1 cells and in an industrially relevant CHO-DG44 expression system (Apollo X, FUJIFILM Diosynth Biotechnologies). We conclude that specific tDNA clusters offer potential to mitigate the widespread problem of production instability.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 8","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400196","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141900251","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}

{"title":"Protein multi-level structure feature-integrated deep learning method for mutational effect prediction","authors":"Ai-Ping Pang,&nbsp;Yongsheng Luo,&nbsp;Junping Zhou,&nbsp;Xue Cai,&nbsp;Lianggang Huang,&nbsp;Bo Zhang,&nbsp;Zhi-Qiang Liu,&nbsp;Yu-Guo Zheng","doi":"10.1002/biot.202400203","DOIUrl":"10.1002/biot.202400203","url":null,"abstract":"<p>Through iterative rounds of mutation and selection, proteins can be engineered to enhance their desired biological functions. Nevertheless, identifying optimal mutation sites for directed evolution remains challenging due to the vastness of the protein sequence landscape and the epistatic mutational effects across residues. To address this challenge, we introduce MLSmut, a deep learning-based approach that leverages multi-level structural features of proteins. MLSmut extracts salient information from protein co-evolution, sequence semantics, and geometric features to predict the mutational effect. Extensive benchmark evaluations on 10 single-site and two multi-site deep mutation scanning datasets demonstrate that MLSmut surpasses existing methods in predicting mutational outcomes. To overcome the limited training data availability, we employ a two-stage training strategy: initial coarse-tuning on a large corpus of unlabeled protein data followed by fine-tuning on a curated dataset of 40−100 experimental measurements. This approach enables our model to achieve satisfactory performance on downstream protein prediction tasks. Importantly, our model holds the potential to predict the mutational effects of any protein sequence. Collectively, these findings suggest that our approach can substantially reduce the reliance on laborious wet lab experiments and deepen our understanding of the intricate relationships between mutations and protein function.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 8","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141900250","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}

{"title":"Engineering a versatile yeast platform for sesquiterpene production from glucose or methanol","authors":"Linhui Gao,&nbsp;Kun Zhang,&nbsp;Yiwei Shen,&nbsp;Peng Cai,&nbsp;Yongjin J. Zhou","doi":"10.1002/biot.202400261","DOIUrl":"10.1002/biot.202400261","url":null,"abstract":"<p>Natural sesquiterpene are valuable compounds with diverse applications in industries, such as cosmetics and energy. Microbial synthesis offers a promising way for sesquiterpene production. Methanol, can be synthesized from CO₂ and solar energy, serves as a sustainable carbon source. However, it is still a challenge to utilize methanol for the synthesis of value-added compounds. <i>Pichia pastoris</i> (syn. <i>Komagataella phaffii</i>), known for its efficient utilization of glucose and methanol, has been widely used in protein synthesis. With advancements in technology, <i>P. pastoris</i> is gradually engineered for chemicals production. Here, we successfully achieved the synthesis of α-bisabolene in <i>P. pastoris</i> with dual carbon sources by expressing the α-bisabolene synthase gene under constitutive promoters. We systematically analyzed the effects of different steps in the mevalonate (MVA) pathway when methanol or glucose was used as the carbon source. Our finding revealed that the sesquiterpene synthase module significantly increased the production when methanol was used. While the metabolic modules <i>MK</i> and <i>PMK</i> greatly improved carbon source utilization, cell growth, and titer when glucose was used. Additionally, we demonstrated the synthesis of β-farnesene from dual carbon source by replacing the α-bisabolene synthase with a β-farnesene synthase. This study establishes a platform strain that is capable to synthesize sesquiterpene from different carbon sources in <i>P. pastoris</i>. Moreover, it paves the way for the development of <i>P. pastoris</i> as a high-efficiency microbial cell factory for producing various chemicals, and lays foundation for large-scale synthesis of high value-added chemicals efficiently from methanol in <i>P. pastoris</i>.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 8","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141900249","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}