Pub Date : 2025-04-18DOI: 10.1016/j.jbiotec.2025.04.010
Fnu Aapjeet , Tiffany Tang , Yixiao Zhang , Aditya Gopalan , Satish Kallappagoudar , Jessica Pan , Fengfei Ma , Sunil S. Shah , Shannon Rivera , Anita Ping-wen Liu , Veronica Juan , Ren Liu
During the development of a recombinant CHO cell line expressing human Interleukin-12 fused to human IgG1 Fc (rhIL-12), we observed a prominent proteolytic cleavage of the rhIL-12 in its p40 subunit between Lys260 and Arg261. Using class-specific protease inhibitors, we concluded that the serine hydrolase family was responsible for the clipping. To identify the specific serine proteases involved, we conducted transcriptomic and proteomic analyses and identified several potential candidates. By performing in-vitro enzyme digestion experiments with these proteases, we determined that matriptase was responsible for the observed p40 clipping. Further confirmation was obtained through the development of matriptase (St14) knockout cell lines in which rhIL-12 clipping was almost completely abolished. Armed with this knowledge, we devised several strategies including increasing culture pH to reduce matriptase activity and rhIL-12 clipping during the manufacturing process.
{"title":"Fragmentation of recombinant human interleukin-12 by matriptase in CHO cell culture","authors":"Fnu Aapjeet , Tiffany Tang , Yixiao Zhang , Aditya Gopalan , Satish Kallappagoudar , Jessica Pan , Fengfei Ma , Sunil S. Shah , Shannon Rivera , Anita Ping-wen Liu , Veronica Juan , Ren Liu","doi":"10.1016/j.jbiotec.2025.04.010","DOIUrl":"10.1016/j.jbiotec.2025.04.010","url":null,"abstract":"<div><div>During the development of a recombinant CHO cell line expressing human Interleukin-12 fused to human IgG1 Fc (rhIL-12), we observed a prominent proteolytic cleavage of the rhIL-12 in its p40 subunit between Lys260 and Arg261. Using class-specific protease inhibitors, we concluded that the serine hydrolase family was responsible for the clipping. To identify the specific serine proteases involved, we conducted transcriptomic and proteomic analyses and identified several potential candidates. By performing in-vitro enzyme digestion experiments with these proteases, we determined that matriptase was responsible for the observed p40 clipping. Further confirmation was obtained through the development of matriptase (S<em>t14</em>) knockout cell lines in which rhIL-12 clipping was almost completely abolished. Armed with this knowledge, we devised several strategies including increasing culture pH to reduce matriptase activity and rhIL-12 clipping during the manufacturing process.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"404 ","pages":"Pages 112-120"},"PeriodicalIF":4.1,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143859004","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 : 2025-04-17DOI: 10.1016/j.jbiotec.2025.04.016
Joneclei Alves Barreto , Matheus Victor Maso Lacôrte e Silva , Danieli Canaver Marin , Michel Brienzo , Ana Paula Jacobus , Jonas Contiero , Jeferson Gross
Adaptive Laboratory Evolution (ALE) is a powerful approach for mining genetic data to engineer industrial microorganisms. This evolution-informed design requires robust genetic tools to incorporate the discovered alleles into target strains. Here, we introduce the EasyGuide CRISPR, a five-plasmid platform that exploits E. coli’s natural recombination system to assemble gRNA plasmids from overlapping PCR fragments. The production of gRNAs and donor DNA is further facilitated by using recombination cassettes generated through PCR with 40–60-mer oligos. With the new CRISPR toolkit, we constructed 22 gene edits in E. coli DH5α, most of which corresponded to alleles mapped in E. coli DH5α and E2348/69 ALE populations selected for sucrose propagation. For DH5α ALE, sucrose consumption was supported by the cscBKA operon expression from a high-copy plasmid. During ALE, plasmid integration into the chromosome, or its copy number reduction due to the pcnB deletion, conferred a 30–35 % fitness gain, as demonstrated by CRISPR-engineered strains. A ∼5 % advantage was also associated with a ∼40.4 kb deletion involving fli operons for flagella assembly. In E2348/69 ALE, inactivation of the hfl system suggested selection pressures for maintaining λ-prophage dormancy (lysogeny). We further enhanced our CRISPR toolkit using yeast for in vivo assembly of donors and expression cassettes, enabling the establishment of polyhydroxybutyrate synthesis from sucrose. Overall, our study highlights the importance of combining ALE with streamlined CRISPR-mediated allele editing to advance microbial production using cost-effective carbon sources.
{"title":"Engineering adaptive alleles for Escherichia coli growth on sucrose using the EasyGuide CRISPR system","authors":"Joneclei Alves Barreto , Matheus Victor Maso Lacôrte e Silva , Danieli Canaver Marin , Michel Brienzo , Ana Paula Jacobus , Jonas Contiero , Jeferson Gross","doi":"10.1016/j.jbiotec.2025.04.016","DOIUrl":"10.1016/j.jbiotec.2025.04.016","url":null,"abstract":"<div><div>Adaptive Laboratory Evolution (ALE) is a powerful approach for mining genetic data to engineer industrial microorganisms. This evolution-informed design requires robust genetic tools to incorporate the discovered alleles into target strains. Here, we introduce the EasyGuide CRISPR, a five-plasmid platform that exploits <em>E. coli</em>’s natural recombination system to assemble gRNA plasmids from overlapping PCR fragments. The production of gRNAs and donor DNA is further facilitated by using recombination cassettes generated through PCR with 40–60-mer oligos. With the new CRISPR toolkit, we constructed 22 gene edits in <em>E. coli</em> DH5α, most of which corresponded to alleles mapped in <em>E. coli</em> DH5α and E2348/69 ALE populations selected for sucrose propagation. For DH5α ALE, sucrose consumption was supported by the <em>cscBKA</em> operon expression from a high-copy plasmid. During ALE, plasmid integration into the chromosome, or its copy number reduction due to the <em>pcnB</em> deletion, conferred a 30–35 % fitness gain, as demonstrated by CRISPR-engineered strains. A ∼5 % advantage was also associated with a ∼40.4 kb deletion involving <em>fli</em> operons for flagella assembly. In E2348/69 ALE, inactivation of the <em>hfl</em> system suggested selection pressures for maintaining λ-prophage dormancy (lysogeny). We further enhanced our CRISPR toolkit using yeast for in vivo assembly of donors and expression cassettes, enabling the establishment of polyhydroxybutyrate synthesis from sucrose. Overall, our study highlights the importance of combining ALE with streamlined CRISPR-mediated allele editing to advance microbial production using cost-effective carbon sources.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"403 ","pages":"Pages 126-139"},"PeriodicalIF":4.1,"publicationDate":"2025-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143863562","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 : 2025-04-17DOI: 10.1016/j.jbiotec.2025.04.014
Jiawei Zhou , Haoyu Long , Yan Guo , Jian Lu , Nengfei Wang , Haifeng Liu , Xiangshan Zhou , Menghao Cai
The polar fungus Geomyces sp. WNF-15A produces high-quality red pigment (AGRP), but the cold-dependent characteristic of AGRP synthesis restricts its industrialization. This study employed transcriptome analysis to compare gene expression profiles of the wild-type strain with cold-independent mutants of scaffold1.t692 (Δ1–692) and scaffold2.t704 (Δ2–704). From the analysis, 23 candidate genes were identified and functionally characterized among 22,600 differentially expressed genes. Knockout and recovery of scaffold5.t61, scaffold7.t586, or scaffold7.t712 proved their regulatory functions in AGRP synthesis, among which scaffold5.t61 functioned as a transcription factor, while scaffold7.t586 and scaffold7.t712 were involved in the glutamate-related nitrogen metabolism. Exogenous addition of nitrate, glutamine, and glutamate, combined with transcriptional regulation studies, revealed the importance of glutamate metabolism for cold-adaptive synthesis of AGRP. Scaffold5.t61 responded to the cold environment and regulated the transcription of scaffold2.t704 and scaffold1.t692. It subsequently increased glutamate synthesis by regulating the key nitrogen metabolism genes of scaffold7.t586 and scaffold7.t712, ultimately resulting in cold-dependent synthesis of AGRP in Geomyces sp. WNF-15A. This study offers new insights into the mechanisms of cold adaptation in polar fungi and serves as a reference for the development of psychrophilic fungal resources.
{"title":"Glutamate-related nitrogen metabolism regulates cold-adaptive synthesis of red pigment in polar fungus Geomyces sp. WNF-15A","authors":"Jiawei Zhou , Haoyu Long , Yan Guo , Jian Lu , Nengfei Wang , Haifeng Liu , Xiangshan Zhou , Menghao Cai","doi":"10.1016/j.jbiotec.2025.04.014","DOIUrl":"10.1016/j.jbiotec.2025.04.014","url":null,"abstract":"<div><div>The polar fungus <em>Geomyces</em> sp. WNF-15A produces high-quality red pigment (AGRP), but the cold-dependent characteristic of AGRP synthesis restricts its industrialization. This study employed transcriptome analysis to compare gene expression profiles of the wild-type strain with cold-independent mutants of <em>scaffold1.t692</em> (Δ1–692) and <em>scaffold2.t704</em> (Δ2–704). From the analysis, 23 candidate genes were identified and functionally characterized among 22,600 differentially expressed genes. Knockout and recovery of <em>scaffold5.t61</em>, <em>scaffold7.t586</em>, or <em>scaffold7.t712</em> proved their regulatory functions in AGRP synthesis, among which <em>scaffold5.t61</em> functioned as a transcription factor, while <em>scaffold7.t586</em> and <em>scaffold7.t712</em> were involved in the glutamate-related nitrogen metabolism. Exogenous addition of nitrate, glutamine, and glutamate, combined with transcriptional regulation studies, revealed the importance of glutamate metabolism for cold-adaptive synthesis of AGRP. <em>Scaffold5.t61</em> responded to the cold environment and regulated the transcription of <em>scaffold2.t704</em> and <em>scaffold1.t692</em>. It subsequently increased glutamate synthesis by regulating the key nitrogen metabolism genes of <em>scaffold7.t586</em> and <em>scaffold7.t712</em>, ultimately resulting in cold-dependent synthesis of AGRP in <em>Geomyces</em> sp. WNF-15A. This study offers new insights into the mechanisms of cold adaptation in polar fungi and serves as a reference for the development of psychrophilic fungal resources.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"404 ","pages":"Pages 121-131"},"PeriodicalIF":4.1,"publicationDate":"2025-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143864102","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}
Biorefineries play a crucial role in advancing the circular bioeconomy by integrating the environmental and socio-economic dimensions of the industrial sector. This study investigated the potential of integrated ultrasound (UL)-deep eutectic solvent (DES, choline chloride/glycerol) pretreatment of pineapple leaf (PL) waste for efficient bioethanol production, emphasizing its sustainability and environmental benefits. The pretreatment conditions were optimized using response surface methodology, with variables including ultrasound amplitude (45 %), time (30 min), and solid loading (10 %, w/w). The solid PL biomass was physico-chemically characterized, revealing prominent variations in functional groups, surface morphology, crystallinity, and surface area across samples subjected to individual and integrated pretreatment approaches. A high reducing sugar yield of 324.41 mg/g PL biomass was recovered after enzymatic hydrolysis of integrated UL-ChCl/glycerol pretreated PL samples under optimized conditions. The fermentation of the sugar hydrolysate yielded 121.36 mg/g ethanol with 89.61 % fermentation efficiency. Notably, DES recyclability experiments indicated significant performance up to the third cycle, after which activity marginally declined in correlation with sugar yield. The synergistic UL-ChCl/glycerol pretreatment process supports circular bioeconomy by promoting sustainable biomass conversion and offering a promising approach to reducing environmental impacts by utilizing agricultural waste for renewable energy production.
{"title":"Bioprocessing of pineapple leaf waste biomass using an integrated ultrasound-deep eutectic solvent pretreatment approach for improved bioethanol production","authors":"Vishal Sharma , Jia Xiang Wang , Mei-Ling Tsai , Aditya Yadav , Cheng-Di Dong , Parushi Nargotra , Pei-Pei Sun","doi":"10.1016/j.jbiotec.2025.04.011","DOIUrl":"10.1016/j.jbiotec.2025.04.011","url":null,"abstract":"<div><div>Biorefineries play a crucial role in advancing the circular bioeconomy by integrating the environmental and socio-economic dimensions of the industrial sector. This study investigated the potential of integrated ultrasound (UL)-deep eutectic solvent (DES, choline chloride/glycerol) pretreatment of pineapple leaf (PL) waste for efficient bioethanol production, emphasizing its sustainability and environmental benefits. The pretreatment conditions were optimized using response surface methodology, with variables including ultrasound amplitude (45 %), time (30 min), and solid loading (10 %, w/w). The solid PL biomass was physico-chemically characterized, revealing prominent variations in functional groups, surface morphology, crystallinity, and surface area across samples subjected to individual and integrated pretreatment approaches. A high reducing sugar yield of 324.41 mg/g PL biomass was recovered after enzymatic hydrolysis of integrated UL-ChCl/glycerol pretreated PL samples under optimized conditions. The fermentation of the sugar hydrolysate yielded 121.36 mg/g ethanol with 89.61 % fermentation efficiency. Notably, DES recyclability experiments indicated significant performance up to the third cycle, after which activity marginally declined in correlation with sugar yield. The synergistic UL-ChCl/glycerol pretreatment process supports circular bioeconomy by promoting sustainable biomass conversion and offering a promising approach to reducing environmental impacts by utilizing agricultural waste for renewable energy production.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"404 ","pages":"Pages 83-93"},"PeriodicalIF":4.1,"publicationDate":"2025-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143848250","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 : 2025-04-17DOI: 10.1016/j.jbiotec.2025.04.013
Lan Yao , Zhe Zhang , Guangyu Chen , Zhiyuan Sun , Xiong Chen , Haitao Yang
The enzymatic hydrolysis of lignocellulose continues to be encumbered by elevated production costs and diminished cellulase efficiency. In this work, modified DES recovered lignin was obtained by grafting acrylamide and acryloyl chloride to enhance glucose release. At a cellulase dosage of 5 FPU/g-cellulose and pH of 5.5, modified lignin promoted glucose yield of dilute-acid-pretreated wheat straw by 158 % compared with control. The mechanism by which modified lignin promotes enzymatic hydrolysis was further explored. The binding constant was reduced from (3.3510 ± 0.8361)* 104 to (2.7600 ± 0.6027)* 103 L•mol−1 after modification. Modified lignin could make α-helix content enhancement so that cellulase had a compact and stable spatial structure. Lignin binds within the catalytic tunnel of cellulase and that the modified lignin interacts with cellulase with increased hydrogen bonding, resulting in a more compact cellulase structure. The modified lignin might reduce the unproductive adsorption of cellulase, and increase stability and cellulose accessibility to reduce cellulase cost.
{"title":"Enhancing biomass enzymatic hydrolysis performance by modified DES lignin","authors":"Lan Yao , Zhe Zhang , Guangyu Chen , Zhiyuan Sun , Xiong Chen , Haitao Yang","doi":"10.1016/j.jbiotec.2025.04.013","DOIUrl":"10.1016/j.jbiotec.2025.04.013","url":null,"abstract":"<div><div>The enzymatic hydrolysis of lignocellulose continues to be encumbered by elevated production costs and diminished cellulase efficiency. In this work, modified DES recovered lignin was obtained by grafting acrylamide and acryloyl chloride to enhance glucose release. At a cellulase dosage of 5 FPU/g-cellulose and pH of 5.5, modified lignin promoted glucose yield of dilute-acid-pretreated wheat straw by 158 % compared with control. The mechanism by which modified lignin promotes enzymatic hydrolysis was further explored. The binding constant was reduced from (3.3510 ± 0.8361)* 10<sup>4</sup> to (2.7600 ± 0.6027)* 10<sup>3</sup> L•mol<sup>−1</sup> after modification. Modified lignin could make α-helix content enhancement so that cellulase had a compact and stable spatial structure. Lignin binds within the catalytic tunnel of cellulase and that the modified lignin interacts with cellulase with increased hydrogen bonding, resulting in a more compact cellulase structure. The modified lignin might reduce the unproductive adsorption of cellulase, and increase stability and cellulose accessibility to reduce cellulase cost.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"403 ","pages":"Pages 115-125"},"PeriodicalIF":4.1,"publicationDate":"2025-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143859398","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}
Current techniques for recombinant protein synthesis mostly depend on traditional expression systems, including bacterial, insect, and mammalian cell cultures. However, these platforms are expensive to build and operate at commercial scales and/or have limited capacities to produce complex proteins. In recent years, plant-based expression systems have become top candidates for the production of recombinant proteins as they are highly scalable, robust, and safe. Lettuce, with its strong foundation in agriculture, is an excellent host for pharmaceutical protein production. Although vaccines, antibodies, and therapeutic proteins have been produced in plants, the technologies required for safe, efficient, scalable manufacture of recombinant proteins have matured to the point where several products have already been tested and will soon be followed by a rich pipeline of recombinant vaccines, and therapeutic proteins, showcasing their role in addressing global health and industrial demands. By synthesizing recent research, this review article aims to provide a comprehensive perspective on the current status, challenges, and future directions of transient overexpression systems in lettuce, paving the way for their broader adoption in biotechnology.
{"title":"Advancements in transient overexpression systems for enhanced recombinant protein production in lettuce","authors":"Meryem Zekraoui, Rehman Sarwar, Yong Wang, Wei Zhang, Li-Na Ding, Zheng Wang, Yuanxue Liang, Xiao-Li Tan","doi":"10.1016/j.jbiotec.2025.04.015","DOIUrl":"10.1016/j.jbiotec.2025.04.015","url":null,"abstract":"<div><div>Current techniques for recombinant protein synthesis mostly depend on traditional expression systems, including bacterial, insect, and mammalian cell cultures. However, these platforms are expensive to build and operate at commercial scales and/or have limited capacities to produce complex proteins. In recent years, plant-based expression systems have become top candidates for the production of recombinant proteins as they are highly scalable, robust, and safe. Lettuce<em>,</em> with its strong foundation in agriculture, is an excellent host for pharmaceutical protein production. Although vaccines, antibodies, and therapeutic proteins have been produced in plants, the technologies required for safe, efficient, scalable manufacture of recombinant proteins have matured to the point where several products have already been tested and will soon be followed by a rich pipeline of recombinant vaccines, and therapeutic proteins, showcasing their role in addressing global health and industrial demands. By synthesizing recent research, this review article aims to provide a comprehensive perspective on the current status, challenges, and future directions of transient overexpression systems in lettuce, paving the way for their broader adoption in biotechnology.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"404 ","pages":"Pages 132-143"},"PeriodicalIF":4.1,"publicationDate":"2025-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143870772","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 : 2025-04-15DOI: 10.1016/j.jbiotec.2025.04.009
Qing Li , Min Jiang , Huirong Yang , Xuyan Zong , Teodora Emilia Coldea , Chao Cheng , Haifeng Zhao
The effects of wheat gluten hydrolysates (WGH) preculture on yeast acetic acid tolerance and fermentation performances were investigated. Results showed that WGH preculture significantly increased yeast growth and viability under acetic acid stress. Particularly, the WGH fraction precipitated with 90 % (v/v) gradient ethanol (WGH-C) preculture significantly improved yeast cell membrane integrity and H+-ATPase activity, thereby decreasing the intracellular accumulation of ROS and acetic acid. Meanwhile, WGH-C preculture promoted the ethanol production efficiency, shortening the fermentation lag time by 12 h and increasing the ethanol yield by 37.46 %. These improvements were attributed to that WGH-C preculture regulated intracellular amino acid composition and transport protein related gene expression of yeast. Transcriptome profiling demonstrated that the cell wall and plasma membrane structures were remodeled, reducing the oxidative stress induced by acetic acid. Furthermore, regulation of energy metabolism and transporter activity are prime mechanisms in improving acetic acid tolerance and fermentation efficiency of yeast.
{"title":"Transcriptome profiling unravels improved ethanol production and acetic acid tolerance in yeast by preculture of wheat gluten hydrolysates","authors":"Qing Li , Min Jiang , Huirong Yang , Xuyan Zong , Teodora Emilia Coldea , Chao Cheng , Haifeng Zhao","doi":"10.1016/j.jbiotec.2025.04.009","DOIUrl":"10.1016/j.jbiotec.2025.04.009","url":null,"abstract":"<div><div>The effects of wheat gluten hydrolysates (WGH) preculture on yeast acetic acid tolerance and fermentation performances were investigated. Results showed that WGH preculture significantly increased yeast growth and viability under acetic acid stress. Particularly, the WGH fraction precipitated with 90 % (v/v) gradient ethanol (WGH-C) preculture significantly improved yeast cell membrane integrity and H<sup>+</sup>-ATPase activity, thereby decreasing the intracellular accumulation of ROS and acetic acid. Meanwhile, WGH-C preculture promoted the ethanol production efficiency, shortening the fermentation lag time by 12 h and increasing the ethanol yield by 37.46 %. These improvements were attributed to that WGH-C preculture regulated intracellular amino acid composition and transport protein related gene expression of yeast. Transcriptome profiling demonstrated that the cell wall and plasma membrane structures were remodeled, reducing the oxidative stress induced by acetic acid. Furthermore, regulation of energy metabolism and transporter activity are prime mechanisms in improving acetic acid tolerance and fermentation efficiency of yeast.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"403 ","pages":"Pages 103-114"},"PeriodicalIF":4.1,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143847907","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 : 2025-04-15DOI: 10.1016/j.jbiotec.2025.04.012
Yaping Liu , Zhiqiang Xiong , Wenhao Liu , Zibo Song , Xin Song , Lianzhong Ai
Bifidobacterium animalis, one of the most prevalent bacteria in the digestive tracts of humans and other mammals, is a typical addition to dairy products. The previous study reported an oxygen tolerant B. animalis AR668-R1 domesticated by adaptive laboratory evolution, which is different from most strictly anaerobic Bifidobacterium strains. However, the studies at molecular level of strain AR668-R1 were hindered due to the low electroporation efficiency. This work aims to achieve a high level of reproducibility in the electroporation-mediated transformation efficiency of AR668-R1. When the optimal parameters were the seed inoculum (OD600 = 0.6), inoculation size (2 %), sucrose concentration (0.5 mol/L), sodium chloride concentration (0.25 mol/L), growth stage (OD600 = 0.3), plasmid concentration (500 ng/μL), electric field intensity (15 kV/cm), and resuscitation time (3 h), the electroporation efficiency reached 3.97 × 105 CFU/μg DNA, which was 79-fold higher than that of the unoptimized condition. Moreover, transcriptional analysis revealed that a series of putative competence genes (ssb, gene0596, comEC, and gene1115) in AR668-R1 were significantly upregulated after optimization. It suggested that improving transformation efficiency is attributable to the enhancement of competence gene expression. Overexpression of the above four competence genes further enhanced the transformation efficiency in AR668-R1. Specifically, comEC overexpression resulted in 2.5 times (9.78 ×105 CFU/μg DNA) improvement. Furthermore, knockout of comEC resulted in a transformation efficiency 74.9-fold (5.32 ×103 CFU/μg DNA) lower than the control, which demonstrated that the key competence gene is closely related to transformation efficiency. Together, the transformation efficiency was successfully improved in AR668-R1, which could promote extensive genetic manipulation and functional analysis in B. animalis.
{"title":"High-efficiency transformation of Bifidobacterium animalis AR668-R1 using electroporation","authors":"Yaping Liu , Zhiqiang Xiong , Wenhao Liu , Zibo Song , Xin Song , Lianzhong Ai","doi":"10.1016/j.jbiotec.2025.04.012","DOIUrl":"10.1016/j.jbiotec.2025.04.012","url":null,"abstract":"<div><div><em>Bifidobacterium animalis</em>, one of the most prevalent bacteria in the digestive tracts of humans and other mammals, is a typical addition to dairy products. The previous study reported an oxygen tolerant <em>B. animalis</em> AR668-R1 domesticated by adaptive laboratory evolution, which is different from most strictly anaerobic <em>Bifidobacterium</em> strains. However, the studies at molecular level of strain AR668-R1 were hindered due to the low electroporation efficiency. This work aims to achieve a high level of reproducibility in the electroporation-mediated transformation efficiency of AR668-R1<em>.</em> When the optimal parameters were the seed inoculum (OD<sub>600</sub> = 0.6), inoculation size (2 %), sucrose concentration (0.5 mol/L), sodium chloride concentration (0.25 mol/L), growth stage (OD<sub>600</sub> = 0.3), plasmid concentration (500 ng/μL), electric field intensity (15 kV/cm), and resuscitation time (3 h), the electroporation efficiency reached 3.97 × 10<sup>5</sup> CFU/μg DNA, which was 79-fold higher than that of the unoptimized condition. Moreover, transcriptional analysis revealed that a series of putative competence genes (<em>ssb</em>, <em>gene0596</em>, <em>comEC</em>, and <em>gene1115</em>) in AR668-R1 were significantly upregulated after optimization. It suggested that improving transformation efficiency is attributable to the enhancement of competence gene expression. Overexpression of the above four competence genes further enhanced the transformation efficiency in AR668-R1. Specifically, <em>comEC</em> overexpression resulted in 2.5 times (9.78 ×10<sup>5</sup> CFU/μg DNA) improvement. Furthermore, knockout of <em>comEC</em> resulted in a transformation efficiency 74.9-fold (5.32 ×10<sup>3</sup> CFU/μg DNA) lower than the control, which demonstrated that the key competence gene is closely related to transformation efficiency. Together, the transformation efficiency was successfully improved in AR668-R1, which could promote extensive genetic manipulation and functional analysis in <em>B. animalis</em>.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"404 ","pages":"Pages 94-101"},"PeriodicalIF":4.1,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143848253","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 : 2025-04-12DOI: 10.1016/j.jbiotec.2025.04.008
Sami Kazaz , Jaya Tripathi , Yang Tian , Halbay Turumtay , Dylan Chin , İrem Pamukçu , Monikaben Nimavat , Emine Akyuz Turumtay , Edward E.K. Baidoo , Corinne D. Scown , Aymerick Eudes
Beta-ketoadipate (βKA) is an intermediate of the βKA pathway involved in the degradation of aromatic compounds in several bacteria and fungi. Beta-ketoadipate also represents a promising chemical for the manufacturing of performance-advantaged nylons. We established a strategy for the in planta synthesis of βKA via manipulation of the shikimate pathway and the expression of bacterial enzymes from the βKA pathway. Using Nicotiana benthamiana as a transient expression system, we demonstrated the efficient conversion of protocatechuate (PCA) to βKA when plastid-targeted bacterial-derived PCA 3,4-dioxygenase (PcaHG) and 3-carboxy-cis,cis-muconate cycloisomerase (PcaB) were co-expressed with 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (AroG) and 3-dehydroshikimate dehydratase (QsuB). This metabolic pathway was reconstituted in Arabidopsis by introducing a construct (pAtβKA) with stacked pcaG, pcaH, and pcaB genes into a PCA-overproducing genetic background that expresses AroG and QsuB (referred as QsuB-2). The resulting QsuB-2 x pAtβKA stable lines displayed βKA titers as high as 0.25 % on a dry weight basis in stems, along with a drastic reduction in lignin content and improvement of biomass saccharification efficiency compared to wild-type controls, and without any significant reduction in biomass yields. Using biomass sorghum as a potential crop for large-scale βKA production, techno-economic analysis indicated that βKA accumulated at titers of 0.25 % and 4 % on a dry weight basis could be competitively priced in the range of $2.04–34.49/kg and $0.47–2.12/kg, respectively, depending on the selling price of the residual biomass recovered after βKA extraction. This study lays the foundation for a more environmentally-friendly synthesis of βKA using plants as production hosts.
{"title":"In planta production of the nylon precursor beta-ketoadipate","authors":"Sami Kazaz , Jaya Tripathi , Yang Tian , Halbay Turumtay , Dylan Chin , İrem Pamukçu , Monikaben Nimavat , Emine Akyuz Turumtay , Edward E.K. Baidoo , Corinne D. Scown , Aymerick Eudes","doi":"10.1016/j.jbiotec.2025.04.008","DOIUrl":"10.1016/j.jbiotec.2025.04.008","url":null,"abstract":"<div><div>Beta-ketoadipate (βKA) is an intermediate of the βKA pathway involved in the degradation of aromatic compounds in several bacteria and fungi. Beta-ketoadipate also represents a promising chemical for the manufacturing of performance-advantaged nylons. We established a strategy for the <em>in planta</em> synthesis of βKA via manipulation of the shikimate pathway and the expression of bacterial enzymes from the βKA pathway. Using <em>Nicotiana benthamiana</em> as a transient expression system, we demonstrated the efficient conversion of protocatechuate (PCA) to βKA when plastid-targeted bacterial-derived PCA 3,4-dioxygenase (PcaHG) and 3-carboxy-<em>cis</em>,<em>cis</em>-muconate cycloisomerase (PcaB) were co-expressed with 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (AroG) and 3-dehydroshikimate dehydratase (QsuB). This metabolic pathway was reconstituted in Arabidopsis by introducing a construct (<em>pAtβKA</em>) with stacked <em>pcaG</em>, <em>pcaH</em>, and <em>pcaB</em> genes into a PCA-overproducing genetic background that expresses AroG and QsuB (referred as <em>QsuB-2</em>). The resulting <em>QsuB-2 x pAtβKA</em> stable lines displayed βKA titers as high as 0.25 % on a dry weight basis in stems, along with a drastic reduction in lignin content and improvement of biomass saccharification efficiency compared to wild-type controls, and without any significant reduction in biomass yields. Using biomass sorghum as a potential crop for large-scale βKA production, techno-economic analysis indicated that βKA accumulated at titers of 0.25 % and 4 % on a dry weight basis could be competitively priced in the range of $2.04–34.49/kg and $0.47–2.12/kg, respectively, depending on the selling price of the residual biomass recovered after βKA extraction. This study lays the foundation for a more environmentally-friendly synthesis of βKA using plants as production hosts.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"404 ","pages":"Pages 102-111"},"PeriodicalIF":4.1,"publicationDate":"2025-04-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143848254","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 : 2025-04-11DOI: 10.1016/j.jbiotec.2025.04.007
Sumedha Saha , Delrin Shaina Xaxa , Sudip K. Ghosh , Mrinal K. Maiti
Microalgae with rapid growth rate, ability to be cultivated in non-agricultural land, and producing numerous bioactive compounds have attracted attention for biofuel production and extraction of valuable co-products. This work focuses on the development of an industrially viable Chlorella vulgaris strain more suitable for biorefinery by downregulating the ADP-glucose pyrophosphorylase (AGPase) enzyme involved in starch biosynthesis. Transgenic lines of C. vulgaris generated through RNA interference (RNAi) demonstrated diminished starch content with an average of 6.7 % (dry cell weight, DCW) from 10 % (DCW) in the untransformed control, after 5 days of shake flask culture in tris acetate phosphate medium with 16 h:8 h light:dark cycle. Under the same growth condition, the total carbohydrate content decreased by an average of 23.5 %, while the lipid content and carotenoid level improved by an average of 19.3 % and 23 %, respectively, in the RNAi lines compared to the control. RNAi lines showed a higher yield of other important co-products, like protein and exopolysaccharide by an average of 30.6 % and 19.6 %, respectively, compared to the control. Interestingly, when polyethylene glycol (PEG) 6000 (0.5 % w/v) was supplemented in the culture medium of C. vulgaris grown till mid-log phase, RNAi lines along with the untransformed control exhibited enhanced level of valuable metabolites after 7 days of PEG treatment. The average carotenoid content of 25.5 μg/mg was recorded in PEG-treated RNAi lines compared to 15.34 μg/mg in PEG-untreated untransformed control alga. Under the similar growth condition, the average lipid content increased to 22.5 % (DCW) in PEG-treated RNAi lines compared to 16.28 % (DCW) in PEG-untreated untransformed control. Overall, the study encompasses use of genetic engineering tool in combination with the application of biochemical modulator PEG to divert the carbon flux from starch biosynthesis towards improved production of important metabolites in microalga.
{"title":"Enhanced accumulation of important bioproducts in Chlorella vulgaris through AGPase gene silencing coupled with polyethylene glycol treatment","authors":"Sumedha Saha , Delrin Shaina Xaxa , Sudip K. Ghosh , Mrinal K. Maiti","doi":"10.1016/j.jbiotec.2025.04.007","DOIUrl":"10.1016/j.jbiotec.2025.04.007","url":null,"abstract":"<div><div>Microalgae with rapid growth rate, ability to be cultivated in non-agricultural land, and producing numerous bioactive compounds have attracted attention for biofuel production and extraction of valuable co-products. This work focuses on the development of an industrially viable <em>Chlorella vulgaris</em> strain more suitable for biorefinery by downregulating the ADP-glucose pyrophosphorylase (AGPase) enzyme involved in starch biosynthesis. Transgenic lines of <em>C. vulgaris</em> generated through RNA interference (RNAi) demonstrated diminished starch content with an average of 6.7 % (dry cell weight, DCW) from 10 % (DCW) in the untransformed control, after 5 days of shake flask culture in tris acetate phosphate medium with 16 h:8 h light:dark cycle. Under the same growth condition, the total carbohydrate content decreased by an average of 23.5 %, while the lipid content and carotenoid level improved by an average of 19.3 % and 23 %, respectively, in the RNAi lines compared to the control. RNAi lines showed a higher yield of other important co-products, like protein and exopolysaccharide by an average of 30.6 % and 19.6 %, respectively, compared to the control. Interestingly, when polyethylene glycol (PEG) 6000 (0.5 % w/v) was supplemented in the culture medium of <em>C. vulgaris</em> grown till mid-log phase, RNAi lines along with the untransformed control exhibited enhanced level of valuable metabolites after 7 days of PEG treatment. The average carotenoid content of 25.5 μg/mg was recorded in PEG-treated RNAi lines compared to 15.34 μg/mg in PEG-untreated untransformed control alga. Under the similar growth condition, the average lipid content increased to 22.5 % (DCW) in PEG-treated RNAi lines compared to 16.28 % (DCW) in PEG-untreated untransformed control. Overall, the study encompasses use of genetic engineering tool in combination with the application of biochemical modulator PEG to divert the carbon flux from starch biosynthesis towards improved production of important metabolites in microalga.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"403 ","pages":"Pages 81-92"},"PeriodicalIF":4.1,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143828386","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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