Journal of biotechnology最新文献_第2页

Impact of liquid and solid-state cultures on hemoglobin production and oxidative state in Saccharomyces cerevisiae

IF 4.1 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-02-08 DOI: 10.1016/j.jbiotec.2025.01.020

Facundo Muñiz-Paredes , Olena P. Ishchuk , Dina Petranovic

Recombinant human hemoglobin gained attention due to its potential use as a blood-free oxygen carrier substitute. To enhance human hemoglobin production in Saccharomyces cerevisiae, various genetic engineering strategies have been employed, including: increasing intracellular heme levels, minimizing heme and protein degradation pathways, and co-expressing the α-hemoglobin stabilizing protein (AHSP). Solid-state culture (SSC) may enhance hemoglobin production by increasing heme biosynthesis, as it relates to intracellular oxygen availability. A comparative analysis of heme and hemoglobin production was conducted between liquid culture (LC) and SSC using the S. cerevisiae AHSP strain. While both systems exhibited comparable heme and hemoglobin yields per cell, a significant 18 % increase in biomass was observed in SSC. The expression of the aerobic master gene HAP1 remained consistent between both systems, however, CYC1 (regulated by HAP1) was two-fold overexpressed in SSC, indicating higher oxygen transference and possibly more efficient electron transport. Several antioxidant genes were downregulated in the SSC, suggesting that LC may be more susceptible to electron leakage during oxidative phosphorylation, potentially due to the lower expression of CYC1. It is proposed that high expression of antioxidant genes in LC inhibits biomass production due to the metabolic burden of maintaining redox homeostasis. These differences between LC and SSC may explain the suitability of SSC as a platform for recombinant protein production.

{"title":"Impact of liquid and solid-state cultures on hemoglobin production and oxidative state in Saccharomyces cerevisiae","authors":"Facundo Muñiz-Paredes , Olena P. Ishchuk , Dina Petranovic","doi":"10.1016/j.jbiotec.2025.01.020","DOIUrl":"10.1016/j.jbiotec.2025.01.020","url":null,"abstract":"<div><div>Recombinant human hemoglobin gained attention due to its potential use as a blood-free oxygen carrier substitute. To enhance human hemoglobin production in <em>Saccharomyces cerevisiae</em>, various genetic engineering strategies have been employed, including: increasing intracellular heme levels, minimizing heme and protein degradation pathways, and co-expressing the α-hemoglobin stabilizing protein (AHSP). Solid-state culture (SSC) may enhance hemoglobin production by increasing heme biosynthesis, as it relates to intracellular oxygen availability. A comparative analysis of heme and hemoglobin production was conducted between liquid culture (LC) and SSC using the <em>S. cerevisiae</em> AHSP strain. While both systems exhibited comparable heme and hemoglobin yields per cell, a significant 18 % increase in biomass was observed in SSC. The expression of the aerobic master gene <em>HAP1</em> remained consistent between both systems, however, <em>CYC1</em> (regulated by <em>HAP1</em>) was two-fold overexpressed in SSC, indicating higher oxygen transference and possibly more efficient electron transport. Several antioxidant genes were downregulated in the SSC, suggesting that LC may be more susceptible to electron leakage during oxidative phosphorylation, potentially due to the lower expression of <em>CYC1</em>. It is proposed that high expression of antioxidant genes in LC inhibits biomass production due to the metabolic burden of maintaining redox homeostasis. These differences between LC and SSC may explain the suitability of SSC as a platform for recombinant protein production.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"400 ","pages":"Pages 1-7"},"PeriodicalIF":4.1,"publicationDate":"2025-02-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143391005","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}

引用次数: 0

Progress on production of malic acid and succinic acid by industrially-important engineered microorganisms

IF 4.1 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-02-07 DOI: 10.1016/j.jbiotec.2025.02.001

Na Wu , Wenxin Wang , Jianguo Zhu

Organic acids are widely used as additives in the food, pharmaceutical, chemical, and plastic industries. Currently, the industrial production methods of organic acids mainly include plant extraction and chemical synthesis. The latter mainly uses petroleum-based compounds as raw materials to synthesize organic acids through a series of chemical reactions. All of these methods have problems such as environmental pollution, high cost, and unsustainability. By contrast, microbial fermentation can effectively utilize a variety of carbon sources. Due to its low production cost, environmental friendliness, and high product purity, microbial fermentation has received increasing attention in recent years. However, the low yield and long fermentation cycle of microbial fermentation limits its industrial application. With the development of genomics, transcriptomics, and other omics technologies, the metabolic pathways of various strains producing organic acids have gradually been elucidated. Based on this, new technologies such as synthetic biology and high-throughput screening have also been extensively studied. This review summarizes the latest research progress in improving organic acid biosynthesis through metabolic engineering, focusing on L-malic acid (L-MA) and succinic acid (SA). Finally, we also discuss the challenges and future prospects of this field. This review has important reference value in the fields of food, pharmaceuticals, and chemicals, providing a theoretical basis for the study of organic acid biosynthesis.

{"title":"Progress on production of malic acid and succinic acid by industrially-important engineered microorganisms","authors":"Na Wu , Wenxin Wang , Jianguo Zhu","doi":"10.1016/j.jbiotec.2025.02.001","DOIUrl":"10.1016/j.jbiotec.2025.02.001","url":null,"abstract":"<div><div>Organic acids are widely used as additives in the food, pharmaceutical, chemical, and plastic industries. Currently, the industrial production methods of organic acids mainly include plant extraction and chemical synthesis. The latter mainly uses petroleum-based compounds as raw materials to synthesize organic acids through a series of chemical reactions. All of these methods have problems such as environmental pollution, high cost, and unsustainability. By contrast, microbial fermentation can effectively utilize a variety of carbon sources. Due to its low production cost, environmental friendliness, and high product purity, microbial fermentation has received increasing attention in recent years. However, the low yield and long fermentation cycle of microbial fermentation limits its industrial application. With the development of genomics, transcriptomics, and other omics technologies, the metabolic pathways of various strains producing organic acids have gradually been elucidated. Based on this, new technologies such as synthetic biology and high-throughput screening have also been extensively studied. This review summarizes the latest research progress in improving organic acid biosynthesis through metabolic engineering, focusing on L-malic acid (L-MA) and succinic acid (SA). Finally, we also discuss the challenges and future prospects of this field. This review has important reference value in the fields of food, pharmaceuticals, and chemicals, providing a theoretical basis for the study of organic acid biosynthesis.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"400 ","pages":"Pages 8-19"},"PeriodicalIF":4.1,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143382432","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}

引用次数: 0

Experimental study on the mechanism of biological hydrogen sulfide generation from organic sulfur-rich coal

IF 4.1 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-02-06 DOI: 10.1016/j.jbiotec.2025.02.002

Wenjie Zhao , Xianbo Su , Weizhong Zhao , Peipei Yan , Yixuan Zhou

Whether of primary or secondary origin, the presence of hydrogen sulfide (H₂S) in coalbed methane (CBM) is commonly attributed to sulfate reduction facilitated by sulfate-reducing bacteria (SRB). However, the sulfate content in high-sulfur coal is exceptionally low, insufficient to function as a substrate for sulfate-reducing bacteria (SRB). In this study, an anaerobic digestion experiment was conducted with high-organic-sulfur coal collected from the Late Permian Longtan Formation in Guangxi Province as both the carbon and sulfur sources. The formation mechanism of H₂S is revealed from the evolution rules of gas components, liquid organic matter, and microbial communities during the anaerobic digestion process. The findings indicate three distinct mechanisms contributing to the biological formation of H₂S in coal seams: firstly, the degradation of readily degradable organic sulfur in coal by microorganisms possessing denitrification capabilities, primarily attributed to the activity of the Wolinella; secondly, The synergistic consortium involving SRB, Pseudomonas spp., and denitrifying Thiobacillus species mediates SO₄^2- reduction and H₂S biogenesis through cross-metabolic interactions; thirdly, Methylotrophic methanogens employ the methyl groups of organic sulfides to produce CH₄ and H₂S simultaneously. Therefore, biological H₂S can be generated under the presence of a sulfur source, appropriate temperature, and conducive environmental conditions. This comprehension will contribute valuable insights to the discourse on the generation and enrichment patterns of H₂S in natural coalbed methane. Additionally, it can offer practical avenues for the prevention and control of H₂S through technological approaches.

{"title":"Experimental study on the mechanism of biological hydrogen sulfide generation from organic sulfur-rich coal","authors":"Wenjie Zhao , Xianbo Su , Weizhong Zhao , Peipei Yan , Yixuan Zhou","doi":"10.1016/j.jbiotec.2025.02.002","DOIUrl":"10.1016/j.jbiotec.2025.02.002","url":null,"abstract":"<div><div>Whether of primary or secondary origin, the presence of hydrogen sulfide (H<sub>2</sub>S) in coalbed methane (CBM) is commonly attributed to sulfate reduction facilitated by sulfate-reducing bacteria (SRB). However, the sulfate content in high-sulfur coal is exceptionally low, insufficient to function as a substrate for sulfate-reducing bacteria (SRB). In this study, an anaerobic digestion experiment was conducted with high-organic-sulfur coal collected from the Late Permian Longtan Formation in Guangxi Province as both the carbon and sulfur sources. The formation mechanism of H<sub>2</sub>S is revealed from the evolution rules of gas components, liquid organic matter, and microbial communities during the anaerobic digestion process. The findings indicate three distinct mechanisms contributing to the biological formation of H<sub>2</sub>S in coal seams: firstly, the degradation of readily degradable organic sulfur in coal by microorganisms possessing denitrification capabilities, primarily attributed to the activity of the <em>Wolinella</em>; secondly, The synergistic consortium involving SRB, Pseudomonas spp., and denitrifying Thiobacillus species mediates SO<sub>4</sub><sup>2-</sup> reduction and H₂S biogenesis through cross-metabolic interactions; thirdly, Methylotrophic methanogens employ the methyl groups of organic sulfides to produce CH<sub>4</sub> and H<sub>2</sub>S simultaneously. Therefore, biological H<sub>2</sub>S can be generated under the presence of a sulfur source, appropriate temperature, and conducive environmental conditions. This comprehension will contribute valuable insights to the discourse on the generation and enrichment patterns of H<sub>2</sub>S in natural coalbed methane. Additionally, it can offer practical avenues for the prevention and control of H<sub>2</sub>S through technological approaches.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"400 ","pages":"Pages 20-28"},"PeriodicalIF":4.1,"publicationDate":"2025-02-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143374141","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}

引用次数: 0

Efficient production of spermidine from Bacillus amyloliquefaciens by enhancing synthesis pathway, blocking degradation pathway and increasing precursor supply 解淀粉芽孢杆菌通过增强合成途径、阻断降解途径和增加前体供应来高效生产亚精胺。

IF 4.1 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-02-01 DOI: 10.1016/j.jbiotec.2024.12.001

Ziyue Zhao, Ailing Guo, Dian Zou, Zhou Li, Xuetuan Wei

Spermidine has broad application potential in food, medicine and other fields. In this study, a novel Bacillus amyloliquefaciens cell factory was constructed for production of spermidine from renewable biomass resources. Firstly, the speB gene was found to be optimal for synthesis of spermidine, and the function of SpeB was explained by amino acid sequence analysis and molecular docking. By replacing the native promoter of the speEB operon with the P43, the synthesis of spermidine was significantly enhanced in B. amyloliquefaciens HSPM1-P43speEB. After knockout of the genes yobN and bltD associated with spermidine degradation, the spermidine titer of the strain HSPM2 was further improved to 115.96 mg/L, increased by 108 % compared to HSPM1-P43speEB. Subsequently, the titer of spermidine was further increased to 277.47 mg/L through enhancing the supply of the precursor methionine by overexpression of speD. Finally, the renewable biomass resources, xylose and feather meal were optimized to produce spermidine, and the maximum titer is up to 588.10 mg/L after optimization. In conclusion, an efficient spermidine producing B. amyloliquefaciens was constructed through combinatorial metabolic engineering strategies, and the sustainable production of spermidine was achieved using the biomass resources of xylose and feather meal.

亚精胺在食品、医药等领域具有广阔的应用潜力。本研究建立了一种新型解淀粉芽孢杆菌细胞工厂，用于从可再生生物质资源中生产亚精胺。首先，发现speB基因最适合合成亚精胺，并通过氨基酸序列分析和分子对接对speB基因的功能进行了解释。通过用P43取代speEB操纵子的天然启动子，解淀粉杆菌HSPM1-P43speEB的亚精胺合成显著增强。敲除亚精胺降解相关基因yobN和bltD后，菌株HSPM2的亚精胺滴度进一步提高至115.96mg/L，较HSPM1-P43speEB提高108%。随后，通过过表达speD增加前体蛋氨酸的供应，亚精胺滴度进一步提高到277.47mg/L。最后，以可再生生物质资源、木糖和羽毛粉为原料进行优化生产亚精胺，优化后的最大滴度可达588.10mg/L。综上所述，通过组合代谢工程策略构建了高效产亚精胺解淀粉芽孢杆菌，实现了利用木糖和羽毛粕生物量资源持续生产亚精胺。

{"title":"Efficient production of spermidine from Bacillus amyloliquefaciens by enhancing synthesis pathway, blocking degradation pathway and increasing precursor supply","authors":"Ziyue Zhao, Ailing Guo, Dian Zou, Zhou Li, Xuetuan Wei","doi":"10.1016/j.jbiotec.2024.12.001","DOIUrl":"10.1016/j.jbiotec.2024.12.001","url":null,"abstract":"<div><div>Spermidine has broad application potential in food, medicine and other fields. In this study, a novel <em>Bacillus amyloliquefaciens</em> cell factory was constructed for production of spermidine from renewablebiomass resources. Firstly, the <em>speB</em> gene was found to be optimal for synthesis of spermidine, and the function of SpeB was explained by amino acid sequence analysis and molecular docking. By replacing the native promoter of the <em>speEB</em> operon with the P43, the synthesis of spermidine was significantly enhanced in <em>B. amyloliquefaciens</em> HSPM1-P43<em>speEB</em>. After knockout of the genes <em>yobN</em> and <em>bltD</em> associated with spermidine degradation, the spermidine titer of the strain HSPM2 was further improved to 115.96 mg/L, increased by 108 % compared to HSPM1-P43<em>speEB</em>. Subsequently, the titer of spermidine was further increased to 277.47 mg/L through enhancing the supply of the precursor methionine by overexpression of <em>speD</em>. Finally, the renewable biomass resources, xylose and feather meal were optimized to produce spermidine, and the maximum titer is up to 588.10 mg/L after optimization. In conclusion, an efficient spermidine producing <em>B. amyloliquefaciens</em> was constructed through combinatorial metabolic engineering strategies, and the sustainable production of spermidine was achieved using the biomass resources of xylose and feather meal.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"398 ","pages":"Pages 87-96"},"PeriodicalIF":4.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142794715","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}

引用次数: 0

A review towards sustainable analyte detection: Biomimetic inspiration in biosensor technology 可持续分析物检测综述：生物传感器技术的仿生启示。

IF 4.1 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-02-01 DOI: 10.1016/j.jbiotec.2024.11.015

Pratistha Bhagat , Lata Sheo Bachan Upadhyay

The branch of biomimetics has witnessed a profound impact on the field of biosensor technology, reflected in sustainable analyte detection. A vast array of biosensor platforms with improved/upgraded performance have been developed and reported. No wonder the motivation from the field of biomimetics has a huge impact on generating detection systems with escalated degrees of manipulation and tunability at different levels. More recently, biomimetic biosensor technology has found potential in constructing bio-inspired materials such as aptamers, MIPs, nanozymes, DNAzymes, Synzymes, etc. to be integrated with biosensor fabrication. The establishment of a sensing setup is not limited to the bioreceptor fabrication; the construction of transducing element using biomimetic material have been reported too. Moreover, to serve a biosensing of target analyte from a fatal diseased sample different biomimetic architectures can be designed that mimic in-vivo microenvironmental surroundings to get an exact microenvironment equivalent to natural conditions leading towards designing of a precise treatment strategy. This research area is ever-evolving as there is a scope for upgradation and refinement due to advancing technologies including nanotechnology, biomimetic nanomaterials, microfluidics, optical sensors, etc. This review is an attempt to comprehend and juxtapose the very primary innovations in the field of biomimetic biosensor technology to realize its comprehensive and wide-range scope and possibilities.

仿生学分支对生物传感器技术领域产生了深远的影响，体现在可持续分析物检测上。大量具有改进/升级性能的生物传感器平台已经被开发和报道。难怪来自仿生学领域的动机对产生具有不同级别操作和可调性升级程度的检测系统产生巨大影响。最近，仿生生物传感器技术已经发现了构建生物启发材料的潜力，如适配体、mip、纳米酶、DNAzymes、Synzymes等，与生物传感器制造相结合。传感装置的建立不限于生物受体的制造；利用仿生材料构建转导元件也有报道。此外，为了从致命的患病样本中对目标分析物进行生物传感，可以设计不同的仿生结构，模拟体内微环境，以获得相当于自然条件的精确微环境，从而设计精确的治疗策略。由于纳米技术、仿生纳米材料、微流体、光学传感器等先进技术的发展，这一研究领域不断发展，升级和完善的范围不断扩大。本文试图对仿生生物传感器技术领域的主要创新进行理解和并列，以实现其全面和广泛的范围和可能性。

{"title":"A review towards sustainable analyte detection: Biomimetic inspiration in biosensor technology","authors":"Pratistha Bhagat , Lata Sheo Bachan Upadhyay","doi":"10.1016/j.jbiotec.2024.11.015","DOIUrl":"10.1016/j.jbiotec.2024.11.015","url":null,"abstract":"<div><div>The branch of biomimetics has witnessed a profound impact on the field of biosensor technology, reflected in sustainable analyte detection. A vast array of biosensor platforms with improved/upgraded performance have been developed and reported. No wonder the motivation from the field of biomimetics has a huge impact on generating detection systems with escalated degrees of manipulation and tunability at different levels. More recently, biomimetic biosensor technology has found potential in constructing bio-inspired materials such as aptamers, MIPs, nanozymes, DNAzymes, Synzymes, etc. to be integrated with biosensor fabrication. The establishment of a sensing setup is not limited to the bioreceptor fabrication; the construction of transducing element using biomimetic material have been reported too. Moreover, to serve a biosensing of target analyte from a fatal diseased sample different biomimetic architectures can be designed that mimic in-vivo microenvironmental surroundings to get an exact microenvironment equivalent to natural conditions leading towards designing of a precise treatment strategy. This research area is ever-evolving as there is a scope for upgradation and refinement due to advancing technologies including nanotechnology, biomimetic nanomaterials, microfluidics, optical sensors, etc. This review is an attempt to comprehend and juxtapose the very primary innovations in the field of biomimetic biosensor technology to realize its comprehensive and wide-range scope and possibilities.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"398 ","pages":"Pages 51-65"},"PeriodicalIF":4.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142769258","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}

引用次数: 0

Bioinformatics-assisted mining and design of novel pullulanase suitable for starch cold hydrolysis 生物信息学辅助下适用于淀粉冷水解的新型普鲁兰酶的挖掘与设计。

IF 4.1 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-02-01 DOI: 10.1016/j.jbiotec.2024.12.005

Xin Wang , Zixing Wang , Xueting Zhang , Yanli Zhang , Wenxia Zhang , Yu Zhang , Xuecheng Zhang , Yazhong Xiao , Yinliang Zhang , Wei Fang

Cold-active pullulanases with good catalytic performance possess promising applications in cold hydrolysis of starch. Adopting bioinformatics-assisted mining strategies, 7 candidate cold-active pullulanases were initially screened out from IMG/MER database. Among the candidates, PulBs exhibited good thermostability and the highest specific activity of 147.4 U/mg. The half-life of PulBs was about 200 h at 35 °C. Employing PulBs as the initial enzyme, the active-site design of FuncLib was implemented to enhance the activity. The design PulBs-20 exhibited an enhanced specific activity of 209.9 U/mg, which was 1.4 times that of PulBs. Furthermore, the thermostability of PulBs-20 was augmented, with a half-life of 250 h at 35 °C. When applied in the cold hydrolysis of starch, PulBs-20 can effectively enhance the hydrolysis effect of raw starch. Supplemented with the raw starch-hydrolyzing α-amylase AmyZ1 and PulBs-20, the hydrolysis rate of raw corn starch increased to 53.5 %, which was 1.3 times that of using AmyZ1 alone. Due to its high hydrolysis activity and good thermostability, PulBs-20 can serve as an efficient accessory enzyme in starch cold hydrolysis.

冷活性普鲁兰酶具有良好的催化性能，在淀粉的冷水解方面具有广阔的应用前景。采用生物信息学辅助挖掘策略，从IMG/MER数据库中初步筛选出7个候选冷活性普鲁兰酶。其中，PulBs具有良好的热稳定性，比活性最高，达147.4 U/mg。在35℃时，PulBs的半衰期约为200h。以PulBs为起始酶，对FuncLib进行活性位点设计，增强活性。设计的PulBs-20比活性为209.9 U/mg，是PulBs的1.4倍。此外，PulBs-20的热稳定性得到了提高，在35℃下的半衰期为250h。PulBs-20应用于淀粉冷水解时，可有效提高原料淀粉的水解效果。添加原淀粉水解α-淀粉酶AmyZ1和PulBs-20后，原玉米淀粉的水解率达到53.5%，是单独使用AmyZ1的1.3倍。PulBs-20具有较高的水解活性和良好的热稳定性，可作为淀粉冷水解的有效辅助酶。

{"title":"Bioinformatics-assisted mining and design of novel pullulanase suitable for starch cold hydrolysis","authors":"Xin Wang , Zixing Wang , Xueting Zhang , Yanli Zhang , Wenxia Zhang , Yu Zhang , Xuecheng Zhang , Yazhong Xiao , Yinliang Zhang , Wei Fang","doi":"10.1016/j.jbiotec.2024.12.005","DOIUrl":"10.1016/j.jbiotec.2024.12.005","url":null,"abstract":"<div><div>Cold-active pullulanases with good catalytic performance possess promising applications in cold hydrolysis of starch. Adopting bioinformatics-assisted mining strategies, 7 candidate cold-active pullulanases were initially screened out from IMG/MER database. Among the candidates, PulBs exhibited good thermostability and the highest specific activity of 147.4 U/mg. The half-life of PulBs was about 200 h at 35 °C. Employing PulBs as the initial enzyme, the active-site design of FuncLib was implemented to enhance the activity. The design PulBs-20 exhibited an enhanced specific activity of 209.9 U/mg, which was 1.4 times that of PulBs. Furthermore, the thermostability of PulBs-20 was augmented, with a half-life of 250 h at 35 °C. When applied in the cold hydrolysis of starch, PulBs-20 can effectively enhance the hydrolysis effect of raw starch. Supplemented with the raw starch-hydrolyzing α-amylase AmyZ1 and PulBs-20, the hydrolysis rate of raw corn starch increased to 53.5 %, which was 1.3 times that of using AmyZ1 alone. Due to its high hydrolysis activity and good thermostability, PulBs-20 can serve as an efficient accessory enzyme in starch cold hydrolysis.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"398 ","pages":"Pages 106-116"},"PeriodicalIF":4.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142835780","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}

引用次数: 0

Corrigendum to “Cell-free reduction of carboxylic acids with secreted carboxylic acid reductase” [J. Biotechnol. 382 (2024) 44–50] 用分泌的羧酸还原酶进行无细胞羧酸还原的勘误表[J]。生物工程学报，2016,44(2):444 - 451。

IF 4.1 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-02-01 DOI: 10.1016/j.jbiotec.2024.12.002

Dominic Goj , Stella Ebner , Melissa Horvat , Simon Arhar , Ludmila Martínková , Margit Winkler

引用次数: 0

Discovery and characterization of NADH oxidases for selective sustainable synthesis of 5-hydroxymethylfuran carboxylic acid 发现并鉴定用于选择性可持续合成 5- 羟甲基呋喃羧酸的 NADH 氧化酶。

IF 4.1 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-02-01 DOI: 10.1016/j.jbiotec.2024.11.009

Karishma Shah , Daniel Kracher , Peter Macheroux , Silvia Wallner , André Pick , Robert Kourist

Efficient regeneration of NAD⁺ remains a significant challenge for oxidative biotransformations. In order to identify enzymes with higher activity and stability, a panel of NADH oxidases (Nox) was investigated in the regeneration of nicotinamide cofactors for the oxidation of hydroxymethyl furfural (HMF) to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). We present novel Nox that exhibit remarkable catalytic activities, elevated thermal and pH stabilities, and higher intrinsic flavin loadings, thus eliminating the need for external flavin addition. The kinetic analysis of the NADH oxidases indicates that AdNox, GdNox, CmNox, and LvNox exhibit V_max values of 86 U/mg, 50 U/mg, 4.3 U/mg, and 23 U/mg, respectively. When these NADH oxidases were applied in a HMF oxidation reaction, LvNox demonstrated the highest HMFCA yield of 97 % in the presence of 0.1 mM NAD and 10 mM HMF. In contrast to previously reported NADH oxidases from the same family, these NADH oxidases naturally accept NADPH as a substrate. Rapid kinetics experiments identified the oxidative reaction as the rate-limiting step of the reaction. NADH oxidases achieved high atom economy, a high reaction mass efficiency and a low process mass intensity. The findings contribute significantly to the field of biocatalysis and offer potential avenues for more environmentally friendly cofactor regeneration in chemical synthesis.

NAD+ 的高效再生仍然是氧化生物转化过程中的一项重大挑战。为了鉴定具有更高活性和稳定性的酶，我们研究了一组 NADH 氧化酶（Nox），它们在将羟甲基糠醛（HMF）氧化为 5-hydroxymethyl-2-furancarboxylic acid（HMFCA）过程中用于烟酰胺辅助因子的再生。我们展示了新型 Nox，它们具有显著的催化活性、较高的热稳定性和 pH 稳定性，以及较高的固有黄素负载，因此无需添加外部黄素。NADH 氧化酶的动力学分析表明，AdNox、GdNox、CmNox 和 LvNox 的 Vmax 值分别为 86 U/mg、50 U/mg、4.3 U/mg 和 23 U/mg。将这些 NADH 氧化酶用于 HMF 氧化反应时，在 0.1mM NAD 和 10mM HMF 存在下，LvNox 的 HMFCA 产率最高，达到 97%。与之前报道的同族 NADH 氧化酶不同，这些 NADH 氧化酶天然接受 NADPH 作为底物。快速动力学实验确定氧化反应是该反应的限速步骤。NADH 氧化酶实现了高原子经济性、高反应质量效率和低过程质量强度。这些发现为生物催化领域做出了重要贡献，并为化学合成中更环保的辅助因子再生提供了潜在途径。

{"title":"Discovery and characterization of NADH oxidases for selective sustainable synthesis of 5-hydroxymethylfuran carboxylic acid","authors":"Karishma Shah , Daniel Kracher , Peter Macheroux , Silvia Wallner , André Pick , Robert Kourist","doi":"10.1016/j.jbiotec.2024.11.009","DOIUrl":"10.1016/j.jbiotec.2024.11.009","url":null,"abstract":"<div><div>Efficient regeneration of NAD<sup>+</sup> remains a significant challenge for oxidative biotransformations. In order to identify enzymes with higher activity and stability, a panel of NADH oxidases (Nox) was investigated in the regeneration of nicotinamide cofactors for the oxidation of hydroxymethyl furfural (HMF) to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). We present novel Nox that exhibit remarkable catalytic activities, elevated thermal and pH stabilities, and higher intrinsic flavin loadings, thus eliminating the need for external flavin addition. The kinetic analysis of the NADH oxidases indicates that <em>Ad</em>Nox, <em>Gd</em>Nox, <em>Cm</em>Nox, and <em>Lv</em>Nox exhibit V<sub>max</sub> values of 86 U/mg, 50 U/mg, 4.3 U/mg, and 23 U/mg, respectively. When these NADH oxidases were applied in a HMF oxidation reaction, LvNox demonstrated the highest HMFCA yield of 97 % in the presence of 0.1 mM NAD and 10 mM HMF. In contrast to previously reported NADH oxidases from the same family, these NADH oxidases naturally accept NADPH as a substrate. Rapid kinetics experiments identified the oxidative reaction as the rate-limiting step of the reaction. NADH oxidases achieved high atom economy, a high reaction mass efficiency and a low process mass intensity. The findings contribute significantly to the field of biocatalysis and offer potential avenues for more environmentally friendly cofactor regeneration in chemical synthesis.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"398 ","pages":"Pages 18-28"},"PeriodicalIF":4.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142739743","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}

引用次数: 0

Potency by design: Novel insights in transfection and purification for manufacturing of rAAV gene therapy vectors 效价设计：rAAV基因治疗载体转染和纯化的新见解。

IF 4.1 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-02-01 DOI: 10.1016/j.jbiotec.2024.12.007

Andreas Maccani , Robert Pachlinger , Peter Eisenhut, Sabine Unterthurner, Susanne Heider, Christoph Repik, Stefan Reuberger, Peter Andorfer, Johannes Lengler, Bernhard Kinastberger, Dieter Seczer, Petra Gruber, Alexander Apschner, Lucia Micutkova

This study investigates the crucial role of transfection methods in the manufacturability and potency of recombinant adeno-associated virus (rAAV) gene therapies. By employing a novel analytical approach, multiplex digital PCR (dPCR), we evaluated the impact of different transfection reagents and conditions on the scalability and quality of rAAV. Our research demonstrates that the selection of transfection approach significantly influences not only the yield and ease of scale-up but also the potency of the final product. Importantly, later changes to transfection parameters established in the early stages of development can be challenging, potentially compromising product quality and leading to comparability issues. Leveraging multiplex dPCR has proven instrumental in guiding these early-stage decisions, ensuring a reliable manufacturing process that consistently delivers high-quality therapeutic products. Our findings highlight the importance of optimizing transfection strategies early in development to guarantee the successful production of potent and cost-effective gene therapies, ultimately impacting patient accessibility.

本研究探讨转染方法在重组腺相关病毒（rAAV）基因治疗的可制造性和效力中的关键作用。通过采用一种新的分析方法，多重数字PCR (dPCR)，我们评估了不同转染试剂和条件对rAAV可扩展性和质量的影响。我们的研究表明，转染方法的选择不仅显著影响产量和扩大规模的便利性，而且影响最终产品的效力。重要的是，后期对早期开发阶段建立的转染参数的更改可能具有挑战性，可能会损害产品质量并导致可比性问题。利用多重dPCR已被证明有助于指导这些早期决策，确保可靠的制造过程始终如一地提供高质量的治疗产品。我们的研究结果强调了在开发早期优化转染策略的重要性，以确保成功生产有效且具有成本效益的基因疗法，最终影响患者的可及性。

{"title":"Potency by design: Novel insights in transfection and purification for manufacturing of rAAV gene therapy vectors","authors":"Andreas Maccani , Robert Pachlinger , Peter Eisenhut, Sabine Unterthurner, Susanne Heider, Christoph Repik, Stefan Reuberger, Peter Andorfer, Johannes Lengler, Bernhard Kinastberger, Dieter Seczer, Petra Gruber, Alexander Apschner, Lucia Micutkova","doi":"10.1016/j.jbiotec.2024.12.007","DOIUrl":"10.1016/j.jbiotec.2024.12.007","url":null,"abstract":"<div><div>This study investigates the crucial role of transfection methods in the manufacturability and potency of recombinant adeno-associated virus (rAAV) gene therapies. By employing a novel analytical approach, multiplex digital PCR (dPCR), we evaluated the impact of different transfection reagents and conditions on the scalability and quality of rAAV. Our research demonstrates that the selection of transfection approach significantly influences not only the yield and ease of scale-up but also the potency of the final product. Importantly, later changes to transfection parameters established in the early stages of development can be challenging, potentially compromising product quality and leading to comparability issues. Leveraging multiplex dPCR has proven instrumental in guiding these early-stage decisions, ensuring a reliable manufacturing process that consistently delivers high-quality therapeutic products. Our findings highlight the importance of optimizing transfection strategies early in development to guarantee the successful production of potent and cost-effective gene therapies, ultimately impacting patient accessibility.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"398 ","pages":"Pages 127-132"},"PeriodicalIF":4.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142864376","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}

引用次数: 0

Thermal degradation kinetics and purification of C-phycocyanin from thermophilic and mesophilic cyanobacteria 嗜热和中温蓝藻热降解动力学及c -藻蓝蛋白的纯化。

IF 4.1 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-02-01 DOI: 10.1016/j.jbiotec.2024.11.018

Supenya Chittapun , Kattiya Suwanmanee , Chatchol Kongsinkaew , Soisuda Pornpukdeewattana , Yusuf Chisti , Theppanya Charoenrat

The natural blue colorant C-phycocyanin (C-PC) has many potential applications but its poor heat stability limits its commercial use. This study compares the production and thermal stability of C-PC from two cyanobacteria: the thermophilic Thermosynechococcus sp. TUBT-T01 and the mesophilic Synechococcus cedrorum TISTR8589. Thermosynechococcus sp. produced nearly 1.9-fold more C-PC than S. cedrorum. Batch adsorption using a chromatographic cationic ion exchange resin (Streamline Direct HST1) was used to effectively purify the C-PC. The equilibrium adsorption capacity (Q_eq) of the resin for C-PC was the highest at pH 5. At this pH, the Q_eq for the thermophilic C-PC was 5.5 ± 0.1 mg mL⁻¹ , whereas for the mesophilic C-PC it was 1.5 ± 0.2 mg mL⁻¹ . Purification increased the concentration of the thermophilic C-PC by 5.9-fold, and that of mesophilic C-PC by 4.2-fold. The purity ratios of the final products from the two cyanobacteria were similar at ∼2.2. At 60 °C and pH 7, the C-PC of Thermosynechococcus sp. had ∼12-times longer half-life than the mesophilic C-PC; however, the productivity of the thermophilic C-PC was comparatively low because of a low biomass productivity of Thermosynechococcus sp.

天然蓝色着色剂c -藻蓝蛋白（C-PC）有许多潜在的应用，但其热稳定性差限制了其商业应用。本研究比较了两种蓝藻：嗜热的热聚球菌TUBT-T01和嗜热的蜡状聚球菌TISTR8589的C-PC产量和热稳定性。热聚球菌产生的C-PC几乎是塞德勒姆球菌的1.9倍。采用色谱阳离子交换树脂（Streamline Direct HST1）批量吸附法对C-PC进行了有效的纯化。树脂对C-PC的平衡吸附量（Qeq）在pH为5时最高。在这个pH下，嗜热的C-PC的Qeq是5.5±0.1mg - 1，而嗜温的C-PC的Qeq是1.5±0.2mg - 1。纯化后，嗜热C-PC浓度提高5.9倍，中温C-PC浓度提高4.2倍。两种蓝藻的最终产物纯度比相似，为~2.2。在60℃和pH 7条件下，热聚球菌C- pc的半衰期比中温性C- pc长约12倍；然而，由于热聚球菌的生物量生产力较低，嗜热性C-PC的生产力相对较低。

{"title":"Thermal degradation kinetics and purification of C-phycocyanin from thermophilic and mesophilic cyanobacteria","authors":"Supenya Chittapun , Kattiya Suwanmanee , Chatchol Kongsinkaew , Soisuda Pornpukdeewattana , Yusuf Chisti , Theppanya Charoenrat","doi":"10.1016/j.jbiotec.2024.11.018","DOIUrl":"10.1016/j.jbiotec.2024.11.018","url":null,"abstract":"<div><div>The natural blue colorant C-phycocyanin (C-PC) has many potential applications but its poor heat stability limits its commercial use. This study compares the production and thermal stability of C-PC from two cyanobacteria: the thermophilic <em>Thermosynechococcus</em> sp. TUBT-T01 and the mesophilic <em>Synechococcus cedrorum</em> TISTR8589. <em>Thermosynechococcus</em> sp. produced nearly 1.9-fold more C-PC than <em>S. cedrorum</em>. Batch adsorption using a chromatographic cationic ion exchange resin (Streamline Direct HST1) was used to effectively purify the C-PC. The equilibrium adsorption capacity (<em>Q</em><sub><em>eq</em></sub>) of the resin for C-PC was the highest at pH 5. At this pH, the <em>Q</em><sub><em>eq</em></sub> for the thermophilic C-PC was 5.5 ± 0.1 mg mL⁻¹ , whereas for the mesophilic C-PC it was 1.5 ± 0.2 mg mL⁻¹ . Purification increased the concentration of the thermophilic C-PC by 5.9-fold, and that of mesophilic C-PC by 4.2-fold. The purity ratios of the final products from the two cyanobacteria were similar at ∼2.2. At 60 °C and pH 7, the C-PC of <em>Thermosynechococcus</em> sp. had ∼12-times longer half-life than the mesophilic C-PC; however, the productivity of the thermophilic C-PC was comparatively low because of a low biomass productivity of <em>Thermosynechococcus</em> sp.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"398 ","pages":"Pages 76-86"},"PeriodicalIF":4.1,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142769264","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}

引用次数: 0