Pub Date : 2025-02-01DOI: 10.1016/j.jbiotec.2024.12.008
Karthika Balusamy , Balaji V. Rokade , Manuel Bruch , Meg Walsh , William Casey , Federico Cerrone , Bryan Dalton , Patrick Jerome Guiry , Tanja Narančić , Kevin E. O’Connor
We demonstrate the proof of concept of increasing the bioavailability of carbon substrates, derived from plastic waste, for their conversion to the biodegradable polymer polyhydroxyalkanoate [PHA] by bacteria and test various approaches to PHA accumulation through batch, fed batch and continuous culture. Styrene, ethylbenzene, and toluene are produced from the pyrolysis of mixed plastic waste (Kaminsky, 2021; Miandad et al., 2017), but they are volatile and poorly soluble in water making them difficult to work with in aqueous fermentation systems. By chemically converting these aromatic compounds to benzoic acid, and subsequently to its sodium salt, we increased the solubility and reduced the volatility of the substrate supplied to Pseudomonas putida CA-3 to accumulate polyhydroxyalkanoates. 1 L scale batch, fed batch, and continuous fermentations were carried out; the fed batch fermentation resulted in the maximum volumetric PHA productivity of 61.67 ± 7.34 mg L−1 h−1; while batch and continuous, at a dilution rate, d = 0.2 h−1, fermentations resulted in 13.30 ± 0.01 and 4.06 ± 0.01 mg L−1 h−1 of PHA respectively.
{"title":"Converting multiple hydrophobic aromatic plastic monomers into a single water-soluble substrate to increase bioavailability for the synthesis of polyhydroxyalkanoates by bacteria using batch, fed batch and continuous cultivation","authors":"Karthika Balusamy , Balaji V. Rokade , Manuel Bruch , Meg Walsh , William Casey , Federico Cerrone , Bryan Dalton , Patrick Jerome Guiry , Tanja Narančić , Kevin E. O’Connor","doi":"10.1016/j.jbiotec.2024.12.008","DOIUrl":"10.1016/j.jbiotec.2024.12.008","url":null,"abstract":"<div><div>We demonstrate the proof of concept of increasing the bioavailability of carbon substrates, derived from plastic waste, for their conversion to the biodegradable polymer polyhydroxyalkanoate [PHA] by bacteria and test various approaches to PHA accumulation through batch, fed batch and continuous culture. Styrene, ethylbenzene, and toluene are produced from the pyrolysis of mixed plastic waste (Kaminsky, 2021; Miandad et al., 2017), but they are volatile and poorly soluble in water making them difficult to work with in aqueous fermentation systems. By chemically converting these aromatic compounds to benzoic acid, and subsequently to its sodium salt, we increased the solubility and reduced the volatility of the substrate supplied to <em>Pseudomonas putida</em> CA-3 to accumulate polyhydroxyalkanoates. 1 L scale batch, fed batch, and continuous fermentations were carried out; the fed batch fermentation resulted in the maximum volumetric PHA productivity of 61.67 ± 7.34 mg L<sup>−1</sup> h<sup>−1</sup>; while batch and continuous, at a dilution rate, d = 0.2 h<sup>−1</sup>, fermentations resulted in 13.30 ± 0.01 and 4.06 ± 0.01 mg L<sup>−1</sup> h<sup>−1</sup> of PHA respectively.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"398 ","pages":"Pages 193-201"},"PeriodicalIF":4.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142894667","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.jbiotec.2024.12.013
Guoli Wang , Xiqin Liang , Zhenke Wu, Bengui Fan, Jun Wang, Qiusheng Zheng, Defang Li, Tianyue An
Aromatic amino acids and their derivatives are high value chemicals widely used in food, pharmaceutical and feed industries. Current preparation methods for aromatic amino acid products are fraught with limitations. In this study, the efficient biosynthesis of aromatic amino acid compound tyrosol was investigated by epigenetic modification-based regulation and optimization of the biosynthetic pathway of aromatic amino acids. The production of tyrosol was significantly improved by the overexpression of m6A modification writer Ime4 and reader Pho92, and the positive regulator Gcr2. Introduction of Bbxfpk and deletion of Gpp1 further improved tyrosol production. Then the feedback inhibition of the shikimate pathway was relieved by the mutants Aro4K229L and Aro7G141S. The final tyrosol producing engineered strain was constructed by the deletion of PHA2, replacement of the native promoter of ARO10 with the strong promoter PGK1p, and introduction of tyrosine decarboxylase PcAAS. In the background of m6A modification regulation, this strain ultimately produced 954.69 ± 43.72 mg/L of tyrosol, promoted by 61.7-fold in shake-flask fermentation.
{"title":"Improved biosynthesis of tyrosol by epigenetic modification-based regulation and metabolic engineering in Saccharomyces cerevisiae","authors":"Guoli Wang , Xiqin Liang , Zhenke Wu, Bengui Fan, Jun Wang, Qiusheng Zheng, Defang Li, Tianyue An","doi":"10.1016/j.jbiotec.2024.12.013","DOIUrl":"10.1016/j.jbiotec.2024.12.013","url":null,"abstract":"<div><div>Aromatic amino acids and their derivatives are high value chemicals widely used in food, pharmaceutical and feed industries. Current preparation methods for aromatic amino acid products are fraught with limitations. In this study, the efficient biosynthesis of aromatic amino acid compound tyrosol was investigated by epigenetic modification-based regulation and optimization of the biosynthetic pathway of aromatic amino acids. The production of tyrosol was significantly improved by the overexpression of m<sup>6</sup>A modification writer Ime4 and reader Pho92, and the positive regulator Gcr2. Introduction of Bbxfpk and deletion of Gpp1 further improved tyrosol production. Then the feedback inhibition of the shikimate pathway was relieved by the mutants Aro4<sup>K229L</sup> and Aro7<sup>G141S</sup>. The final tyrosol producing engineered strain was constructed by the deletion of <em>PHA2</em>, replacement of the native promoter of <em>ARO10</em> with the strong promoter PGK1p, and introduction of tyrosine decarboxylase PcAAS. In the background of m<sup>6</sup>A modification regulation, this strain ultimately produced 954.69 ± 43.72 mg/L of tyrosol, promoted by 61.7-fold in shake-flask fermentation.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"398 ","pages":"Pages 175-182"},"PeriodicalIF":4.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142921807","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.jbiotec.2025.01.002
Marcelo M. Ruas-Filho , Ana Maria Mazotto , Anderson S. Pinheiro
The search for new non-animal textile materials has increased yearly as environmental awareness and veganism continue to spread, driving the development of greener fabrics. Concurrently, β-keratin, a fibrous, resistant, and insoluble protein shows great potential for producing innovative biomaterials. However, β-keratin is naturally abundant in animal feathers. Therefore, the recombinant production of β-keratin from Gallus gallus feathers was proposed using a strategy of parallel expression in different vectors. Statistical tools of experimental design were employed to improve the production of soluble biosynthetic keratin. It was shown that β-keratins fused to His6MBP had better performance regarding soluble expression. In addition, the optimized regions for the values of induction temperature, induction time, and induction absorbance were obtained. As a result, a yield of 185.3 ± 1.4 mg/L of soluble His6MBP-Chr2.FK4 was achieved, representing the highest yield reported to date.
{"title":"High-yield soluble production of recombinant β-keratin from Gallus gallus feathers using an experimental design approach","authors":"Marcelo M. Ruas-Filho , Ana Maria Mazotto , Anderson S. Pinheiro","doi":"10.1016/j.jbiotec.2025.01.002","DOIUrl":"10.1016/j.jbiotec.2025.01.002","url":null,"abstract":"<div><div>The search for new non-animal textile materials has increased yearly as environmental awareness and veganism continue to spread, driving the development of greener fabrics. Concurrently, β-keratin, a fibrous, resistant, and insoluble protein shows great potential for producing innovative biomaterials. However, β-keratin is naturally abundant in animal feathers. Therefore, the recombinant production of β-keratin from G<em>allus gallus</em> feathers was proposed using a strategy of parallel expression in different vectors. Statistical tools of experimental design were employed to improve the production of soluble biosynthetic keratin. It was shown that β-keratins fused to His<sub>6</sub>MBP had better performance regarding soluble expression. In addition, the optimized regions for the values of induction temperature, induction time, and induction absorbance were obtained. As a result, a yield of 185.3 ± 1.4 mg/L of soluble His<sub>6</sub>MBP-Chr2.FK4 was achieved, representing the highest yield reported to date.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"398 ","pages":"Pages 215-223"},"PeriodicalIF":4.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142970739","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The immune system is regulated by dendritic cells (DCs), which are highly specialized cells for presenting antigens. They are thought of as natural sentinels that start the immune response triggered by naive T cells against invasive infections. DCs participate in the initial stage of muscle damage in conjunction with monocytes, macrophages, and myogenic cells. The goal of this study was to determine whether DCs might mitigate tissue damage and aid in the regeneration of the gastrocnemius muscle following envenomation with Bothrops jararacussu venom (BjV). Mature bone marrow dendritic cells (BMDCs) were used to treat mice in an experimental envenomation model with BjV by activation with lipopolysaccharide (LPS). BMDCs were injected into the gastrocnemius muscle at the same site of the BjV injury, in a single dose, 3 h after envenomation, and envenoming effects were observed at different periods for 7 days. In both untreated (NT) and treated (T) groups tissue necrosis, leukocyte influx, and hemorrhage at the injury site were observed. Results showed an increase in serum and tissue CK as well as IL-6, TNF-α, and IL-1β release in the first hours after envenoming. In contrast, after treatment with BMDCs results obtained demonstrated an attenuated local effect with a small leukocyte influx, decreased or non-existent necrosis and hemorrhage, as well as a reduction in both serum and tissue CK levels as well as cytokine release and, consequently, the onset of a moderate regenerative process. The present study's findings concluded that BjV causes a severe inflammatory reaction at the site of injury and that treating envenoming with BMDCs in the muscle was crucial for minimizing damage to the muscle and the inflammatory reaction and promoting the early onset of the tissue repair process.
{"title":"Bone marrow-derived dendritic cells play a role in attenuating inflammation on Bothrops jararacussu venom muscle damage","authors":"N.M. Nery , A.A. Ferreira e Ferreira , H.M. Santana , S.N. Serrath , V.P. Reis , M.V. Paloschi , M.D.S. Silva , J.G.S. Magalhães , L.F. Cruz , T.Y. Shibayama , S.S. Setubal. , J.P. Zuliani","doi":"10.1016/j.jbiotec.2024.11.014","DOIUrl":"10.1016/j.jbiotec.2024.11.014","url":null,"abstract":"<div><div>The immune system is regulated by dendritic cells (DCs), which are highly specialized cells for presenting antigens. They are thought of as natural sentinels that start the immune response triggered by naive T cells against invasive infections. DCs participate in the initial stage of muscle damage in conjunction with monocytes, macrophages, and myogenic cells. The goal of this study was to determine whether DCs might mitigate tissue damage and aid in the regeneration of the gastrocnemius muscle following envenomation with <em>Bothrops jararacussu</em> venom (BjV). Mature bone marrow dendritic cells (BMDCs) were used to treat mice in an experimental envenomation model with BjV by activation with lipopolysaccharide (LPS). BMDCs were injected into the gastrocnemius muscle at the same site of the BjV injury, in a single dose, 3 h after envenomation, and envenoming effects were observed at different periods for 7 days. In both untreated (NT) and treated (T) groups tissue necrosis, leukocyte influx, and hemorrhage at the injury site were observed. Results showed an increase in serum and tissue CK as well as IL-6, TNF-α, and IL-1β release in the first hours after envenoming. In contrast, after treatment with BMDCs results obtained demonstrated an attenuated local effect with a small leukocyte influx, decreased or non-existent necrosis and hemorrhage, as well as a reduction in both serum and tissue CK levels as well as cytokine release and, consequently, the onset of a moderate regenerative process. The present study's findings concluded that BjV causes a severe inflammatory reaction at the site of injury and that treating envenoming with BMDCs in the muscle was crucial for minimizing damage to the muscle and the inflammatory reaction and promoting the early onset of the tissue repair process.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"398 ","pages":"Pages 29-40"},"PeriodicalIF":4.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142769260","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.jbiotec.2025.01.001
Qiaolei Zhu , Bingyu Zhao , Nairu Ji , Yunping Zhu , Xinyu Shi
Melanin with antioxidant and antibacterial properties can be used in food, cosmetics, biotechnology, and other fields, but its insolubility become a main challenge hindering for its application. In this study, water-soluble melanin produced by the novel species Streptomyces vilmorinianum YP1 was characterized using scanning electron microscopy (SEM), UVvisible spectroscopy (with an absorption peak at 220 nm), and Fourier transform infrared (FTIR) spectroscopy. The glycosyltransferase gene ORF14 was knocked out, which improved the production of water-soluble melanin by inhibiting competitive pathway. In order to further enhance production of melanin, PlackettBurman and response surface methodology statistical design was employed to screen for key factors and determine the optimal combination. The maximum melanin production (4.00 g/L) was obtained under the conditions: amylodextrine concentration of 40 g/L, soya peptone concentration of 7 g/L, tryptone concentration of 5 g/L, NaCl concentration of 5.4 g/L, pH of 6.7 and temperature of 36 °C for 180 h. The physicochemical properties and bioactivity of melanin were further investigated, revealing that melanin had a good stability across a pH range of 4–12, antioxidant (with a survival rate of over 85 %), and resistance to reducing agents (with a survival rate of over 99 %). The results underscored that S. vilmorinianum YP1 is a promising candidate for water-soluble melanin production.
{"title":"Effects of ORF14 gene on melanin expression, fermentation conditions and properties of melanin production in modified strains","authors":"Qiaolei Zhu , Bingyu Zhao , Nairu Ji , Yunping Zhu , Xinyu Shi","doi":"10.1016/j.jbiotec.2025.01.001","DOIUrl":"10.1016/j.jbiotec.2025.01.001","url":null,"abstract":"<div><div>Melanin with antioxidant and antibacterial properties can be used in food, cosmetics, biotechnology, and other fields, but its insolubility become a main challenge hindering for its application. In this study, water-soluble melanin produced by the novel species <em>Streptomyces vilmorinianum</em> YP1 was characterized using scanning electron microscopy (SEM), UV<img>visible spectroscopy (with an absorption peak at 220 nm), and Fourier transform infrared (FTIR) spectroscopy. The glycosyltransferase gene <em>ORF14</em> was knocked out, which improved the production of water-soluble melanin by inhibiting competitive pathway. In order to further enhance production of melanin, Plackett<img>Burman and response surface methodology statistical design was employed to screen for key factors and determine the optimal combination. The maximum melanin production (4.00 g/L) was obtained under the conditions: amylodextrine concentration of 40 g/L, soya peptone concentration of 7 g/L, tryptone concentration of 5 g/L, NaCl concentration of 5.4 g/L, pH of 6.7 and temperature of 36 °C for 180 h. The physicochemical properties and bioactivity of melanin were further investigated, revealing that melanin had a good stability across a pH range of 4–12, antioxidant (with a survival rate of over 85 %), and resistance to reducing agents (with a survival rate of over 99 %). The results underscored that <em>S. vilmorinianum</em> YP1 is a promising candidate for water-soluble melanin production.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"398 ","pages":"Pages 224-234"},"PeriodicalIF":4.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142970131","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.jbiotec.2024.12.010
Roberta Anjos de Jesus , Ivani Meneses Costa , Katlin Ivon Barrios Eguiluz , Giancarlo Richard Salazar-Banda
Efficiently managing agricultural waste while innovating to derive value-added products is a significant challenge in the 21st century. In recent decades, these by-products have been increasingly explored as alternative sources for materials such as biosilica. Biosilica is renowned for its high surface area, biocompatibility, chemical stability, and modifiable surface, which makes it suitable for various applications. Additionally, the biomineralization process—biosilicification—in living organisms like diatoms offers an eco-friendly pathway for silica production. Despite the potential applications of biosilica, research on its use in sensor technology remains limited. This review aims to address this gap by covering the primary methodologies for extracting silica from biomass, discussing key techniques for its characterization, and highlighting its potential for functionalization in diverse applications. Special emphasis is given to the utility of diatom-derived biosilicas in developing sensors for detecting gaseous molecules and biomolecules.
{"title":"The role of biosilica and its potential for sensing technologies: A review","authors":"Roberta Anjos de Jesus , Ivani Meneses Costa , Katlin Ivon Barrios Eguiluz , Giancarlo Richard Salazar-Banda","doi":"10.1016/j.jbiotec.2024.12.010","DOIUrl":"10.1016/j.jbiotec.2024.12.010","url":null,"abstract":"<div><div>Efficiently managing agricultural waste while innovating to derive value-added products is a significant challenge in the 21st century. In recent decades, these by-products have been increasingly explored as alternative sources for materials such as biosilica. Biosilica is renowned for its high surface area, biocompatibility, chemical stability, and modifiable surface, which makes it suitable for various applications. Additionally, the biomineralization process—biosilicification—in living organisms like diatoms offers an eco-friendly pathway for silica production. Despite the potential applications of biosilica, research on its use in sensor technology remains limited. This review aims to address this gap by covering the primary methodologies for extracting silica from biomass, discussing key techniques for its characterization, and highlighting its potential for functionalization in diverse applications. Special emphasis is given to the utility of diatom-derived biosilicas in developing sensors for detecting gaseous molecules and biomolecules.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"398 ","pages":"Pages 158-174"},"PeriodicalIF":4.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142894674","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.jbiotec.2024.11.021
Can Guo , Zhiqiang Bin , Pengjie Zhang , Jing Tang , Lianqing Wang , Yefu Chen , Dongguang Xiao , Xuewu Guo
RNA (Ribonucleic Acid) is an essential component of organisms and is widely used in the food and pharmaceutical industries. Saccharomyces cerevisiae, recognized as a safe strain, is widely used for RNA production. In this study, the S. cerevisiae W303–1a was used as a starting strain and molecular modifications were made to the functional ncRNA-SRG1 to evaluate the effect on RNA production. At the same time, its transcriptionally associated helper genes (Spt2, Spt6 and Cha4) were overexpressed and the culture medium was supplemented with serine to induce SRG1 transcription, to increase SRG1 transcription levels and investigate its effect on intracellular RNA levels. The results showed that the intracellular RNA content of the recombinant strain W303–1a-SRG1 was 10.27 %, an increase of 11.15 % compared to the starting strain (W303–1a, with an intracellular RNA content of 9.24 %). On this basis, a gene co-overexpression strain-W303–1a-SRG1-Spt6 was constructed. Simultaneously, the addition of 2 % serine strategy was used to increase the transcription level of SRG1 and RNA content of the recombinant strain. The intracellular RNA of the recombinant strain reached 11.41 %, an increase of 23.38 % compared to the starting strain (W303–1a, without serine supplementation). In addition, the growth performance of the strain was assessed by measuring the SRG1 transcription level in the strain and plotting the growth curve. Therefore, we found that improving the transcription level of ncRNA can be used as a new idea to construct S. cerevisiae with high RNA content, which provides a strong help for subsequent research in related fields. This work provides a new strategy for increasing the nucleic acid content of S. cerevisiae.
{"title":"Efficient production of RNA in Saccharomyces cerevisiae through inducing high level transcription of functional ncRNA-SRG1","authors":"Can Guo , Zhiqiang Bin , Pengjie Zhang , Jing Tang , Lianqing Wang , Yefu Chen , Dongguang Xiao , Xuewu Guo","doi":"10.1016/j.jbiotec.2024.11.021","DOIUrl":"10.1016/j.jbiotec.2024.11.021","url":null,"abstract":"<div><div>RNA (Ribonucleic Acid) is an essential component of organisms and is widely used in the food and pharmaceutical industries. <em>Saccharomyces cerevisiae</em>, recognized as a safe strain, is widely used for RNA production. In this study, the <em>S. cerevisiae</em> W303–1a was used as a starting strain and molecular modifications were made to the functional ncRNA-SRG1 to evaluate the effect on RNA production. At the same time, its transcriptionally associated helper genes (<em>Spt2</em>, <em>Spt6</em> and <em>Cha4</em>) were overexpressed and the culture medium was supplemented with serine to induce <em>SRG1</em> transcription, to increase <em>SRG1</em> transcription levels and investigate its effect on intracellular RNA levels. The results showed that the intracellular RNA content of the recombinant strain W303–1a-SRG1 was 10.27 %, an increase of 11.15 % compared to the starting strain (W303–1a, with an intracellular RNA content of 9.24 %). On this basis, a gene co-overexpression strain-W303–1a-SRG1-Spt6 was constructed. Simultaneously, the addition of 2 % serine strategy was used to increase the transcription level of <em>SRG1</em> and RNA content of the recombinant strain. The intracellular RNA of the recombinant strain reached 11.41 %, an increase of 23.38 % compared to the starting strain (W303–1a, without serine supplementation). In addition, the growth performance of the strain was assessed by measuring the <em>SRG1</em> transcription level in the strain and plotting the growth curve. Therefore, we found that improving the transcription level of ncRNA can be used as a new idea to construct <em>S. cerevisiae</em> with high RNA content, which provides a strong help for subsequent research in related fields. This work provides a new strategy for increasing the nucleic acid content of <em>S. cerevisiae</em>.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"398 ","pages":"Pages 66-75"},"PeriodicalIF":4.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142785725","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.jbiotec.2024.12.004
Min Li, Zhi Li, Lei Wei, Ge Bai, Saijin Wei, Linbin Zhou, Hanguang Li
In order to improve the production and quality of lipid of D. intermedius Z8 under the furfural stress environment, different exogenous γ-aminobutyric acid (GABA) addition strategies and their reasons for improving the fermentation performance were investigated. For this purpose, the effect of different concentrations of furfural on the production of biomass and lipid in D. intermedius Z8 was researched. The result shows that the growth of D. intermedius Z8 nearly stopped when 1.0 g/L furfural was adopted. However, when 1.5 mmol/L GABA was used, the highest biomass and lipid production of 4.56 g/L and 1.83 g/L were obtained, respectively, which were 36.53 % and 61.95 % higher compared to the control group (the normal development BG11 medium). Additionally, the changes in microalgal cell morphology were analyzed using the scanning electron microscope (SEM) technology, and the results suggest that GABA addition could significantly mitigate the toxic effects of excessive furfural by maintaining the integrity of the cell. Simultaneously, the activities of key enzymes involved in fatty acid synthesis, such as nitrate reductase (NR), phosphatidic acid phosphatase (PAP), acetyl CoA carboxylase (ACC), and fatty acid synthase (FAS), were enhanced under the optimal condition, and thus improving lipid production. According to the results in this study, the exogenous GABA addition strategy is a simple and effective approach to improve microbial tolerance and enhance its fermentation performance.
{"title":"The physiological role of gamma-aminobutyric acid in relieving the effect of furfural inhibitor for improvement the production of lipid in D. intermedius Z8","authors":"Min Li, Zhi Li, Lei Wei, Ge Bai, Saijin Wei, Linbin Zhou, Hanguang Li","doi":"10.1016/j.jbiotec.2024.12.004","DOIUrl":"10.1016/j.jbiotec.2024.12.004","url":null,"abstract":"<div><div>In order to improve the production and quality of lipid of <em>D. intermedius</em> Z<sub>8</sub> under the furfural stress environment, different exogenous γ-aminobutyric acid (GABA) addition strategies and their reasons for improving the fermentation performance were investigated. For this purpose, the effect of different concentrations of furfural on the production of biomass and lipid in <em>D. intermedius</em> Z<sub>8</sub> was researched. The result shows that the growth of <em>D. intermedius</em> Z<sub>8</sub> nearly stopped when 1.0 g/L furfural was adopted. However, when 1.5 mmol/L GABA was used, the highest biomass and lipid production of 4.56 g/L and 1.83 g/L were obtained, respectively, which were 36.53 % and 61.95 % higher compared to the control group (the normal development BG11 medium). Additionally, the changes in microalgal cell morphology were analyzed using the scanning electron microscope (SEM) technology, and the results suggest that GABA addition could significantly mitigate the toxic effects of excessive furfural by maintaining the integrity of the cell. Simultaneously, the activities of key enzymes involved in fatty acid synthesis, such as nitrate reductase (NR), phosphatidic acid phosphatase (PAP), acetyl CoA carboxylase (ACC), and fatty acid synthase (FAS), were enhanced under the optimal condition, and thus improving lipid production. According to the results in this study, the exogenous GABA addition strategy is a simple and effective approach to improve microbial tolerance and enhance its fermentation performance.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"398 ","pages":"Pages 117-126"},"PeriodicalIF":4.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142846650","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.jbiotec.2024.12.011
Manuel Reithofer, Sophie Huber, Reingard Grabherr
Efficient recombinant protein production requires mammalian stable cell lines or often relies on inefficient transfection processes. Baculoviral transduction of mammalian cells (BacMam) offers cost-effective and robust gene transfer and straightforward scalability. The advantages over conventional approaches are, no need of high biosafety level laboratories, efficient transduction of various cell types and transfer of large transgenes into host cells. In our study, we aim to develop a high expression cassette to increase yields of baculoviral transduction. The establishment follows a sequential approach by first identifying the strongest promoter, followed by intron and WPRE sequences as enhancer elements for transcription and translation. The resulting REMBAC-cassette was compared to conventional transfection in suspension and adherent cells. Irrespective of the cell line, transduction reached nearly 100 % efficiency and led to almost 10-fold increases of gene expression levels. We confirmed these results in larger scale with batch and fed-batch cultivations. Finally, expression of different soluble proteins with high degrees of complexity confirmed the versatility of our established cassette. Overall, the REMBAC-cassette incorporated into the BacMam platform is a manifold tool offering advantages over standard transfection, in the scalability, efficiency and gene expression, which results in higher yields, shorter cultivation times and consequently cost-effective production processes.
{"title":"Establishment of the REMBAC-cassette, a rapid, efficient and manifold BacMam tool for recombinant protein expression","authors":"Manuel Reithofer, Sophie Huber, Reingard Grabherr","doi":"10.1016/j.jbiotec.2024.12.011","DOIUrl":"10.1016/j.jbiotec.2024.12.011","url":null,"abstract":"<div><div>Efficient recombinant protein production requires mammalian stable cell lines or often relies on inefficient transfection processes. Baculoviral transduction of mammalian cells (BacMam) offers cost-effective and robust gene transfer and straightforward scalability. The advantages over conventional approaches are, no need of high biosafety level laboratories, efficient transduction of various cell types and transfer of large transgenes into host cells. In our study, we aim to develop a high expression cassette to increase yields of baculoviral transduction. The establishment follows a sequential approach by first identifying the strongest promoter, followed by intron and WPRE sequences as enhancer elements for transcription and translation. The resulting REMBAC-cassette was compared to conventional transfection in suspension and adherent cells. Irrespective of the cell line, transduction reached nearly 100 % efficiency and led to almost 10-fold increases of gene expression levels. We confirmed these results in larger scale with batch and fed-batch cultivations. Finally, expression of different soluble proteins with high degrees of complexity confirmed the versatility of our established cassette. Overall, the REMBAC-cassette incorporated into the BacMam platform is a manifold tool offering advantages over standard transfection, in the scalability, efficiency and gene expression, which results in higher yields, shorter cultivation times and consequently cost-effective production processes.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"398 ","pages":"Pages 183-192"},"PeriodicalIF":4.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142927174","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}
Pub Date : 2025-02-01DOI: 10.1016/j.jbiotec.2024.12.006
Gang Luo , Wen Yang , Zijian Geng , Yiyi Cheng , Yingqing Xu , Yimeng Xiao , Jiying Liu
The Pichia pastoris expression system is a favorable platform for production of pharmaceutical proteins. Treatment of strains with N-acetyl-L-cysteine (NAC) has been shown to enhance the yield of recombinant proteins, thereby contributing to a reduction in production costs. However, the specific mechanism of action of NAC remains unclear. Previous research has indicated that glutathione (GSH) and autophagy are involved in the increased production of human serum albumin and porcine follicle-stimulating hormone β (HSA-pFSHβ) by NAC. This study investigated the potential interaction between GSH and autophagy in the production of HSA-pFSHβ. The findings indicated that sulfhydryl-free antioxidants such as melatonin, vitamin C, or vitamin E did not exhibit similar effects to NAC in enhancing HSA-pFSHβ yield. Moreover, NAC was found to enhance HSA-pFSHβ production by modulating GSH metabolism to reduce GSH consumption, increase total GSH levels, as well as glutathione peroxidase (GSH-Px) and glutathione reductase (GR) activities. Additionally, inhibition of autophagy through disruption of autophagy scaffolding proteins Atg1 or Atg11 led to an increase in recombinant HSA-pFSHβ production. Furthermore, NAC significantly decreased the phosphorylation of Slt2, and the absence of the SLT2 gene influenced the effect of NAC on HSA-pFSHβ secretion by modulating mitophagy and GSH metabolism. In conclusion, these results suggest a complex interplay between GSH metabolism and autophagy in the regulation of NAC-induced HSA-pFSHβ secretion.
{"title":"Molecular mechanism of GSH metabolism and autophagy in NAC-promoted recombinant human serum albumin and follicle stimulating hormone beta fusion protein secretion in Pichia pastoris","authors":"Gang Luo , Wen Yang , Zijian Geng , Yiyi Cheng , Yingqing Xu , Yimeng Xiao , Jiying Liu","doi":"10.1016/j.jbiotec.2024.12.006","DOIUrl":"10.1016/j.jbiotec.2024.12.006","url":null,"abstract":"<div><div>The <em>Pichia pastoris</em> expression system is a favorable platform for production of pharmaceutical proteins. Treatment of strains with N-acetyl-L-cysteine (NAC) has been shown to enhance the yield of recombinant proteins, thereby contributing to a reduction in production costs. However, the specific mechanism of action of NAC remains unclear. Previous research has indicated that glutathione (GSH) and autophagy are involved in the increased production of human serum albumin and porcine follicle-stimulating hormone β (HSA-pFSHβ) by NAC. This study investigated the potential interaction between GSH and autophagy in the production of HSA-pFSHβ. The findings indicated that sulfhydryl-free antioxidants such as melatonin, vitamin C, or vitamin E did not exhibit similar effects to NAC in enhancing HSA-pFSHβ yield. Moreover, NAC was found to enhance HSA-pFSHβ production by modulating GSH metabolism to reduce GSH consumption, increase total GSH levels, as well as glutathione peroxidase (GSH-Px) and glutathione reductase (GR) activities. Additionally, inhibition of autophagy through disruption of autophagy scaffolding proteins <em>Atg1</em> or <em>Atg11</em> led to an increase in recombinant HSA-pFSHβ production. Furthermore, NAC significantly decreased the phosphorylation of Slt2, and the absence of the <em>SLT2</em> gene influenced the effect of NAC on HSA-pFSHβ secretion by modulating mitophagy and GSH metabolism. In conclusion, these results suggest a complex interplay between GSH metabolism and autophagy in the regulation of NAC-induced HSA-pFSHβ secretion.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"398 ","pages":"Pages 146-157"},"PeriodicalIF":4.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142877145","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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