Pub Date : 2025-12-29DOI: 10.1016/j.synbio.2025.11.016
Yong Feng , Xihua Chen , Zeyang Li , Zhong Ni , Zhengfen Wu , Zhongjian Guo , Fubao Sun , Huiqing Chen , Huayou Chen
Glucose oxidase (GOD) is a widely used enzyme in biotechnology, yet its narrow substrate specificity limits its application in complex bioconversion processes such as agricultural waste valorization. In this study, we employed synthetic biology and protein engineering strategies to engineer a broad-spectrum glucose oxidase from Aureobasidium sp. (AreGOD). Initially, site-directed mutagenesis at N82, a key gatekeeper at the dimer interface, modulated substrate channel geometry, leading to increased catalytic activity towards various sugars, particularly stachyose and xylose. Furthermore, systematic linker engineering between the spore anchor protein CotG and AreGOD revealed that flexible linkers, particularly the (GGGGS)5 repeat (LK3), dramatically expanded the enzyme's substrate spectrum towards various mono-, di-, and oligosaccharides. The optimized spore-displayed AreGOD (CotG-LK3-AreGOD) exhibited strong synergistic effects with cellulase in wheat straw degradation, significantly enhancing the hydrolysis of cellulose, hemicellulose, and lignin. Our work demonstrates an effective and generalizable strategy for engineering substrate-promiscuous oxidases, highlighting the potential of integrative enzyme design for sustainable bioprocessing and agricultural biotechnology.
{"title":"Engineering a broad-spectrum glucose oxidase via substrate channel and linker design for enhanced lignocellulose bioconversion","authors":"Yong Feng , Xihua Chen , Zeyang Li , Zhong Ni , Zhengfen Wu , Zhongjian Guo , Fubao Sun , Huiqing Chen , Huayou Chen","doi":"10.1016/j.synbio.2025.11.016","DOIUrl":"10.1016/j.synbio.2025.11.016","url":null,"abstract":"<div><div>Glucose oxidase (GOD) is a widely used enzyme in biotechnology, yet its narrow substrate specificity limits its application in complex bioconversion processes such as agricultural waste valorization. In this study, we employed synthetic biology and protein engineering strategies to engineer a broad-spectrum glucose oxidase from <em>Aureobasidium</em> sp. (AreGOD). Initially, site-directed mutagenesis at N82, a key gatekeeper at the dimer interface, modulated substrate channel geometry, leading to increased catalytic activity towards various sugars, particularly stachyose and xylose. Furthermore, systematic linker engineering between the spore anchor protein CotG and AreGOD revealed that flexible linkers, particularly the (GGGGS)<sub>5</sub> repeat (LK3), dramatically expanded the enzyme's substrate spectrum towards various mono-, di-, and oligosaccharides. The optimized spore-displayed AreGOD (CotG-LK3-AreGOD) exhibited strong synergistic effects with cellulase in wheat straw degradation, significantly enhancing the hydrolysis of cellulose, hemicellulose, and lignin. Our work demonstrates an effective and generalizable strategy for engineering substrate-promiscuous oxidases, highlighting the potential of integrative enzyme design for sustainable bioprocessing and agricultural biotechnology.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 218-228"},"PeriodicalIF":4.4,"publicationDate":"2025-12-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145883634","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 : 2025-12-22DOI: 10.1016/j.synbio.2025.11.009
Nga Yu Poon , Anthony J. Sinskey , Kang Zhou
1,3-propanediol (1,3-PDO) is used to synthesize plastics used in many consumer products. As the demand and production of such plastics increase, a technology will be needed to utilize 1,3-PDO released from the plastics after their disposal. In our previous study, we developed the strain (BA07Δ) that could use malonate semialdehyde (MSA, an important intermediate in the 1,3-PDO assimilation pathway) as the major carbon source. Here, we present construction of PA16, a strain which could grow to an OD600 of 7 by consuming 6.5 g/L of 1,3-PDO within 72 h in M9-based medium supplemented with 1 g/L of complete supplement mixture (CSM). This was achieved by adaptive laboratory evolution (ALE) after extending the pathway in BA07Δ through the introduction of a 1,3-propanediol dehydrogenase from Klebsiella pneumoniae (KpDhaT), an aldehyde dehydrogenase from E. coli (EcPuuC) and a 3-hydroxypropionate dehydrogenase from Halomonas bluephagenesis (HbDddA). Comparing the transcriptome of PA16 and its ancestor in the ALE (PA1) revealed the upregulation of two genes, threonine dehydrogenase (EcTdh) and 2-amino-3-ketobutyrate CoA ligase (EcKbl) responsible for threonine degradation. The overexpression of these genes in PA1 resulted in a 5-fold increase in the 72-h cell density. This finding helped simplify the growth medium of PA16: the supplement mixture containing more than 10 amino acids/nucleobases was reduced to just having 0.1 g/L threonine. PA16's OD600 reached 3 when it grew in a defined medium containing 10 g/L 1,3-PDO and 0.1 g/L threonine as carbon sources. E. coliPA16 should be a useful strain to the subsequent research on upcycling 1,3-PDO derived from plastic wastes.
{"title":"Metabolic engineering enables Escherichia coli to grow on 1,3-propanediol","authors":"Nga Yu Poon , Anthony J. Sinskey , Kang Zhou","doi":"10.1016/j.synbio.2025.11.009","DOIUrl":"10.1016/j.synbio.2025.11.009","url":null,"abstract":"<div><div>1,3-propanediol (1,3-PDO) is used to synthesize plastics used in many consumer products. As the demand and production of such plastics increase, a technology will be needed to utilize 1,3-PDO released from the plastics after their disposal. In our previous study, we developed the strain (<strong>BA07Δ</strong>) that could use malonate semialdehyde (MSA, an important intermediate in the 1,3-PDO assimilation pathway) as the major carbon source. Here, we present construction of <strong>PA16</strong>, a strain which could grow to an OD<sub>600</sub> of 7 by consuming 6.5 g/L of 1,3-PDO within 72 h in M9-based medium supplemented with 1 g/L of complete supplement mixture (CSM). This was achieved by adaptive laboratory evolution (ALE) after extending the pathway in <strong>BA07Δ</strong> through the introduction of a 1,3-propanediol dehydrogenase from <em>Klebsiella pneumoniae</em> (KpDhaT), an aldehyde dehydrogenase from <em>E. coli</em> (EcPuuC) and a 3-hydroxypropionate dehydrogenase from <em>Halomonas bluephagenesis</em> (HbDddA). Comparing the transcriptome of <strong>PA16</strong> and its ancestor in the ALE (<strong>PA1</strong>) revealed the upregulation of two genes, threonine dehydrogenase (EcTdh) and 2-amino-3-ketobutyrate CoA ligase (EcKbl) responsible for threonine degradation. The overexpression of these genes in <strong>PA1</strong> resulted in a 5-fold increase in the 72-h cell density. This finding helped simplify the growth medium of <strong>PA16</strong>: the supplement mixture containing more than 10 amino acids/nucleobases was reduced to just having 0.1 g/L threonine. <strong>PA16</strong>'s OD<sub>600</sub> reached 3 when it grew in a defined medium containing 10 g/L 1,3-PDO and 0.1 g/L threonine as carbon sources. <em>E. coli</em> <strong>PA16</strong> should be a useful strain to the subsequent research on upcycling 1,3-PDO derived from plastic wastes.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 209-217"},"PeriodicalIF":4.4,"publicationDate":"2025-12-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145839459","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 : 2025-12-18DOI: 10.1016/j.synbio.2025.12.001
Qi Zhao , Hui-Jie Zhang , Ming-Ming Han , Jumai Abiti , Yan-Ju Dong , Jia-Ning Wang , Jiang-Tao Lu , Wen Wang , Xi Zhang , Shao-Lei Geng , Le-Le Qiu , Xiao-Yin Wang , Zi-Chun Hua , Tian-Yun Wang , Yan-Long Jia
Chinese hamster ovary (CHO) cells undergo endoplasmic reticulum stress (ERS) during intensive recombinant protein production, triggering the unfolded protein response (UPR) to balance cell survival and protein output. Nevertheless, key regulatory components of this process remain incompletely characterized. In this study, we demonstrate that Annexin A1 (ANXA1) functions as a UPR suppressor in CHO cells. Employing the PiggyBac transposon system, we generated a stable ANXA1-knockdown cell line exhibiting a 4.5-fold increase in recombinant antibody expression and a 4.2-fold increase in specific productivity. Pharmacological inhibition using AC2-26 similarly enhanced recombinant protein expression in low-productivity cell populations. Mechanistically, ANXA1 depletion remodeled the UPR by activating the PERK-eIF2α-ATF4 and IRE1-XBP1 branches. This activation upregulaed ATF4, Bip, and XBP1s; suppressed CHOP; reduced apoptosis; and enhanced autophagic flux. Metabolic profiling revealed increased glucose and lactate utilization, while glutamine consumption and ammonia flux remained unchanged. Collectively, these findings establish that ANXA1 depletion enhances recombinant protein biosynthesis through coordinated pro-survival mechanisms. Targeting ANXA1 thus represents an innovative cell engineering strategy for optimizing CHO cell platforms in industrial biopharmaceutical manufacturing.
{"title":"Leveraging ANXA1 to enhance recombinant protein yields in CHO cells: A UPR-Mediated bioprocessing approach","authors":"Qi Zhao , Hui-Jie Zhang , Ming-Ming Han , Jumai Abiti , Yan-Ju Dong , Jia-Ning Wang , Jiang-Tao Lu , Wen Wang , Xi Zhang , Shao-Lei Geng , Le-Le Qiu , Xiao-Yin Wang , Zi-Chun Hua , Tian-Yun Wang , Yan-Long Jia","doi":"10.1016/j.synbio.2025.12.001","DOIUrl":"10.1016/j.synbio.2025.12.001","url":null,"abstract":"<div><div>Chinese hamster ovary (CHO) cells undergo endoplasmic reticulum stress (ERS) during intensive recombinant protein production, triggering the unfolded protein response (UPR) to balance cell survival and protein output. Nevertheless, key regulatory components of this process remain incompletely characterized. In this study, we demonstrate that Annexin A1 (ANXA1) functions as a UPR suppressor in CHO cells. Employing the PiggyBac transposon system, we generated a stable ANXA1-knockdown cell line exhibiting a 4.5-fold increase in recombinant antibody expression and a 4.2-fold increase in specific productivity. Pharmacological inhibition using AC2-26 similarly enhanced recombinant protein expression in low-productivity cell populations. Mechanistically, ANXA1 depletion remodeled the UPR by activating the PERK-eIF2α-ATF4 and IRE1-XBP1 branches. This activation upregulaed ATF4, Bip, and XBP1s; suppressed CHOP; reduced apoptosis; and enhanced autophagic flux. Metabolic profiling revealed increased glucose and lactate utilization, while glutamine consumption and ammonia flux remained unchanged. Collectively, these findings establish that ANXA1 depletion enhances recombinant protein biosynthesis through coordinated pro-survival mechanisms. Targeting ANXA1 thus represents an innovative cell engineering strategy for optimizing CHO cell platforms in industrial biopharmaceutical manufacturing.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 197-208"},"PeriodicalIF":4.4,"publicationDate":"2025-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145797028","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 : 2025-12-18DOI: 10.1016/j.synbio.2025.11.006
Shuo Liu , Fei Xiao , Lanxin Lv , Meiyan Wang , Wenli Li , Guoqing Niu
{"title":"Corrigendum to “Morphology-engineered alleviation of mycelial aggregation in Streptomyces chassis for potentiated production of secondary metabolites” [Synth Syst Biotechnol 10 (3) (2025) 1059–1069]","authors":"Shuo Liu , Fei Xiao , Lanxin Lv , Meiyan Wang , Wenli Li , Guoqing Niu","doi":"10.1016/j.synbio.2025.11.006","DOIUrl":"10.1016/j.synbio.2025.11.006","url":null,"abstract":"","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 183-184"},"PeriodicalIF":4.4,"publicationDate":"2025-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145797029","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}
Formate utilization as a sustainable carbon source for microbial production of high-value chemicals and heterologous proteins presents considerable safety and environmental benefits over conventional feedstocks. As a low-cost, CO2-derived compound, formate serves as a non-flammable and non-toxic alternative to methanol for induction of recombinant expression in Komagataella phaffii. However, since native K. phaffii only utilizes formate as an energy source rather than a carbon substrate for biomass synthesis, we engineered a synthetic peroxisomal formate assimilation pathway by introducing heterologous acetyl-CoA synthetase (ACS) and acetaldehyde dehydrogenase (ACDH), thereby enabling formatotrophic growth with formate as the sole carbon source. This chassis was further optimized through (i) co-expression of the transcriptional activator Mit1 to enhance the coupling efficiency of the ACS-ACDH module, and (ii) reinforcement of the Xu5P pathway by overexpressing dihydroxyacetone synthase (DAS1) and ribulose-5-phosphate-3-epimerase (RPE) to redirect metabolic flux. The resulting formatotrophic K. phaffi strain achieved a specific growth rate of 0.012 h−1 in basal salt medium with formate as the sole carbon source, and produced 30.9 U/(mL·OD600) of xylanase from Aspergillus niger ATCC 1015 as a model heterologous protein. Furthermore, 13C isotopic tracing confirmed the incorporation of formate-derived carbon into central metabolism for the biosynthesis of amino acids, nucleotides, and structural carbohydrates, validating active formate assimilation. This study establishes a microbial platform for formate-based production of heterologous proteins and underscores the potential of metabolic engineering to advance sustainable biomanufacturing from one-carbon feedstocks.
{"title":"Formatotrophic Komagataella phaffii expressing recombinant xylanase via metabolic engineering","authors":"Ziwei Zhou , Bing Liu , Wenjie Cong , Hualan Zhou , Yu Zheng , Jianguo Zhang","doi":"10.1016/j.synbio.2025.11.015","DOIUrl":"10.1016/j.synbio.2025.11.015","url":null,"abstract":"<div><div>Formate utilization as a sustainable carbon source for microbial production of high-value chemicals and heterologous proteins presents considerable safety and environmental benefits over conventional feedstocks. As a low-cost, CO<sub>2</sub>-derived compound, formate serves as a non-flammable and non-toxic alternative to methanol for induction of recombinant expression in <em>Komagataella phaffii</em>. However, since native <em>K. phaffii</em> only utilizes formate as an energy source rather than a carbon substrate for biomass synthesis, we engineered a synthetic peroxisomal formate assimilation pathway by introducing heterologous acetyl-CoA synthetase (ACS) and acetaldehyde dehydrogenase (ACDH), thereby enabling formatotrophic growth with formate as the sole carbon source. This chassis was further optimized through (i) co-expression of the transcriptional activator Mit1 to enhance the coupling efficiency of the ACS-ACDH module, and (ii) reinforcement of the Xu5P pathway by overexpressing dihydroxyacetone synthase (DAS1) and ribulose-5-phosphate-3-epimerase (RPE) to redirect metabolic flux. The resulting formatotrophic <em>K. phaffi</em> strain achieved a specific growth rate of 0.012 h<sup>−1</sup> in basal salt medium with formate as the sole carbon source, and produced 30.9 U/(mL·OD<sub>600</sub>) of xylanase from <em>Aspergillus niger</em> ATCC 1015 as a model heterologous protein. Furthermore, <sup>13</sup>C isotopic tracing confirmed the incorporation of formate-derived carbon into central metabolism for the biosynthesis of amino acids, nucleotides, and structural carbohydrates, validating active formate assimilation. This study establishes a microbial platform for formate-based production of heterologous proteins and underscores the potential of metabolic engineering to advance sustainable biomanufacturing from one-carbon feedstocks.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 185-196"},"PeriodicalIF":4.4,"publicationDate":"2025-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145797027","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 : 2025-12-11DOI: 10.1016/j.synbio.2025.11.013
Ziming Liu , Min Tang , Wanze Zhang , Yanjie Tian , Jianjun Qiao , Mingzhang Wen , Weiguo Li , Qinggele Caiyin
Delta (δ)-tocotrienol is a member of the vitamin E family and exhibits bioactivities such as antioxidant, anti-inflammatory, and neuroprotective activities. As a nutrient with protective effects on human health, δ-tocotrienol has broad application prospects in food, cosmetic, and pharmaceutical industries. The construction of efficient microbial cell factories capable of δ-tocotrienol production using synthetic biology approaches is an effective strategy for supplementing or even replacing the vitamin E supply chain in the future. The current study successfully enhanced the biosynthesis of δ-tocotrienol in Saccharomyces cerevisiae by combining metabolic engineering and enzyme engineering strategies. Specifically, the substrate channel constructed by the sequential fusion of the enzymes PaCrtE and SyHPT successfully increased the supply of the key precursor MGGBQ, resulting in a significant increase in the production of δ-tocotrienol. In situ extraction and optimization of the expression of transporter protein PDR1 increased the efflux of δ-tocotrienol, directing the metabolic flux toward the product δ-tocotrienol. To enhance the catalytic activity of the key rate-limiting enzyme tocopherol cyclase from Arabidopsis thaliana (AtTC), semirational protein design was conducted herein. The mutant AtTCT87S was found to increase the production of δ-tocotrienol by 2.3 times compared to that obtained with the wild-type enzyme. AtTCT87S can thus be universally used for synthetic biology strategies in future studies to enhance the microbial heterologous production of δ-tocotrienol. The strain T08 was finally obtained herein; the numerous metabolic engineering strategies discussed in this study were integrated into this strain, allowing the production of 4337.3 μg/L of δ-tocotrienol in a shake-flask fermentation, which is 8.9 times that of the yield obtained with the initial strain T03. Scaling up to a 5-L fermentation tank resulted in a δ-tocotrienol yield of 16.9 mg/L.
{"title":"Enhancing delta-tocotrienol production in Saccharomyces cerevisiae via metabolic engineering strategies in conjunction with the mutagenesis of tocopherol cyclase","authors":"Ziming Liu , Min Tang , Wanze Zhang , Yanjie Tian , Jianjun Qiao , Mingzhang Wen , Weiguo Li , Qinggele Caiyin","doi":"10.1016/j.synbio.2025.11.013","DOIUrl":"10.1016/j.synbio.2025.11.013","url":null,"abstract":"<div><div>Delta (δ)-tocotrienol is a member of the vitamin E family and exhibits bioactivities such as antioxidant, anti-inflammatory, and neuroprotective activities. As a nutrient with protective effects on human health, δ-tocotrienol has broad application prospects in food, cosmetic, and pharmaceutical industries. The construction of efficient microbial cell factories capable of δ-tocotrienol production using synthetic biology approaches is an effective strategy for supplementing or even replacing the vitamin E supply chain in the future. The current study successfully enhanced the biosynthesis of δ-tocotrienol in <em>Saccharomyces cerevisiae</em> by combining metabolic engineering and enzyme engineering strategies. Specifically, the substrate channel constructed by the sequential fusion of the enzymes PaCrtE and SyHPT successfully increased the supply of the key precursor MGGBQ, resulting in a significant increase in the production of δ-tocotrienol. <em>In situ</em> extraction and optimization of the expression of transporter protein PDR1 increased the efflux of δ-tocotrienol, directing the metabolic flux toward the product δ-tocotrienol. To enhance the catalytic activity of the key rate-limiting enzyme tocopherol cyclase from <em>Arabidopsis thaliana</em> (AtTC), semirational protein design was conducted herein. The mutant AtTC<sup>T87S</sup> was found to increase the production of δ-tocotrienol by 2.3 times compared to that obtained with the wild-type enzyme. AtTC<sup>T87S</sup> can thus be universally used for synthetic biology strategies in future studies to enhance the microbial heterologous production of δ-tocotrienol. The strain T08 was finally obtained herein; the numerous metabolic engineering strategies discussed in this study were integrated into this strain, allowing the production of 4337.3 μg/L of δ-tocotrienol in a shake-flask fermentation, which is 8.9 times that of the yield obtained with the initial strain T03. Scaling up to a 5-L fermentation tank resulted in a δ-tocotrienol yield of 16.9 mg/L.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 172-182"},"PeriodicalIF":4.4,"publicationDate":"2025-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145747943","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 : 2025-12-08DOI: 10.1016/j.synbio.2025.11.011
Jin-Peng Zhang , Zi-Lun Mei , Jia-Wei Ren , Xiao-Mei Zhang , Jin-Song Gong , Guo-Qiang Xu , Hui Li , Xiao-Juan Zhang , Zheng-Hong Xu
The Shine-Dalgarno (SD) sequence and its adjacent flanking regions, including the translation standby site (TSS) and the N-terminal coding sequence (NCS), play critical roles in regulating ribosome recruitment, translation efficiency, and mRNA stability against RNase degradation. However, structure-activity relationships governing these regions remain poorly characterized, and their functional interplay introduces substantial complexity. In this study, we employed one-pot technology to build a post-transcriptional regulatory component (PTRC) library of 576 variants to clarify the relationship between sequence variants and both protein expression and mRNA levels via high-throughput sequencing. Our results show that although unstructured TSSs do not enhance mRNA stability, they markedly increase translation efficiency, causing a 16 %–100 % rise in protein expression. In contrast, structured TSSs increase mRNA levels by 43 %–90 %. Additionally, highly conserved SD sequences boost translation efficiency by up to 10 % and mRNA abundance by up to 12 %. Moreover, it was found that optimized linear N-terminal coding sequence (NCS) positively affects protein expression and mRNA levels. The effects of these optimized regulatory components were verified in the expression control of the nrk and sam2 genes, resulting in enhanced production. These findings underscore the crucial role of structural optimization, guiding the rational design of synthetic post-transcriptional regulatory elements.
sine - dalgarno （SD）序列及其相邻的侧翼区域，包括翻译备用位点（TSS）和n端编码序列（NCS），在调节核糖体招募、翻译效率和mRNA抗rna酶降解的稳定性方面发挥着关键作用。然而，控制这些区域的结构-活性关系仍然很不清楚，它们的功能相互作用引入了大量的复杂性。在本研究中，我们采用一锅技术构建了576个变异的转录后调控成分（PTRC）文库，通过高通量测序来阐明序列变异与蛋白表达和mRNA水平的关系。我们的研究结果表明，尽管非结构化的tss不提高mRNA的稳定性，但它们显著提高了翻译效率，导致蛋白质表达增加16% - 100%。相反，结构化的tss使mRNA水平增加43% - 90%。此外，高度保守的SD序列可使翻译效率提高10%，mRNA丰度提高12%。此外，优化后的线性n端编码序列（NCS）对蛋白质表达和mRNA水平有积极影响。这些优化后的调控成分在调控nrk和sam2基因的表达中得到了验证，从而提高了产量。这些发现强调了结构优化的关键作用，指导合理设计合成的转录后调控元件。
{"title":"Evaluation of synthetic post-transcription regulatory sequences reveals design principle to enhance mRNA stability and translation efficiency","authors":"Jin-Peng Zhang , Zi-Lun Mei , Jia-Wei Ren , Xiao-Mei Zhang , Jin-Song Gong , Guo-Qiang Xu , Hui Li , Xiao-Juan Zhang , Zheng-Hong Xu","doi":"10.1016/j.synbio.2025.11.011","DOIUrl":"10.1016/j.synbio.2025.11.011","url":null,"abstract":"<div><div>The Shine-Dalgarno (SD) sequence and its adjacent flanking regions, including the translation standby site (TSS) and the N-terminal coding sequence (NCS), play critical roles in regulating ribosome recruitment, translation efficiency, and mRNA stability against RNase degradation. However, structure-activity relationships governing these regions remain poorly characterized, and their functional interplay introduces substantial complexity. In this study, we employed one-pot technology to build a post-transcriptional regulatory component (PTRC) library of 576 variants to clarify the relationship between sequence variants and both protein expression and mRNA levels via high-throughput sequencing. Our results show that although unstructured TSSs do not enhance mRNA stability, they markedly increase translation efficiency, causing a 16 %–100 % rise in protein expression. In contrast, structured TSSs increase mRNA levels by 43 %–90 %. Additionally, highly conserved SD sequences boost translation efficiency by up to 10 % and mRNA abundance by up to 12 %. Moreover, it was found that optimized linear N-terminal coding sequence (NCS) positively affects protein expression and mRNA levels. The effects of these optimized regulatory components were verified in the expression control of the <em>nrk</em> and <em>sam2</em> genes, resulting in enhanced production. These findings underscore the crucial role of structural optimization, guiding the rational design of synthetic post-transcriptional regulatory elements.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 160-171"},"PeriodicalIF":4.4,"publicationDate":"2025-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145747942","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 : 2025-12-05DOI: 10.1016/j.synbio.2025.11.014
Dandan Feng , Tianshu Zhang , Guan Zhou , Yanli Cao , Quan Luo , Huifang Xu , Xuefeng Lu
2′-Deoxy-2′-fluoroadenosine (2′-F-dA) is a nucleoside analogue used as a key building block for oligonucleotide drugs. It can be biosynthesized from a low-cost 2′-deoxy-2′-fluorouridine via one-pot transglycosylation catalyzed by a thymidine phosphorylase (TP) and a purine nucleoside phosphorylase (PNP). However, reliance on purified enzymes and low space-time yields present challenges for industrial application of the process. Here, we develop a whole-cell-based biocatalytic system employing TP and PNP from Escherichia coli, which demonstrates high catalytic efficiency and operational simplicity in scaled-up reaction. In particular, a thermal pretreatment of TP- and PNP-expressing whole cells, determined as 50 °C for 3 h, effectively suppressed endogenous deamination side reaction while enhancing 2′-F-dA yield. Subsequent optimization of enzyme and substrate loadings and their relative ratios achieved an unprecedented space-time yield of 1.22 g/L/h with 88.1 g/L product titer in a 500 mL scaled-up reaction, manifesting a highest total conversion of 68.2 %. An integrated purification process yielded gram-scale solid powder of 2′-F-dA with 98.0 % chemical purity and 85.0 % recovery. This novel whole-cell biocatalytic process demonstrates significant industrial potential for the production of 2′-F-dA.
{"title":"Efficient whole-cell biocatalytic synthesis of 2′-deoxy-2′-fluoroadenosine, a key building block for nucleic acid drugs","authors":"Dandan Feng , Tianshu Zhang , Guan Zhou , Yanli Cao , Quan Luo , Huifang Xu , Xuefeng Lu","doi":"10.1016/j.synbio.2025.11.014","DOIUrl":"10.1016/j.synbio.2025.11.014","url":null,"abstract":"<div><div>2′-Deoxy-2′-fluoroadenosine (2′-F-dA) is a nucleoside analogue used as a key building block for oligonucleotide drugs. It can be biosynthesized from a low-cost 2′-deoxy-2′-fluorouridine via one-pot transglycosylation catalyzed by a thymidine phosphorylase (TP) and a purine nucleoside phosphorylase (PNP). However, reliance on purified enzymes and low space-time yields present challenges for industrial application of the process. Here, we develop a whole-cell-based biocatalytic system employing TP and PNP from <em>Escherichia coli</em>, which demonstrates high catalytic efficiency and operational simplicity in scaled-up reaction. In particular, a thermal pretreatment of TP- and PNP-expressing whole cells, determined as 50 °C for 3 h, effectively suppressed endogenous deamination side reaction while enhancing 2′-F-dA yield. Subsequent optimization of enzyme and substrate loadings and their relative ratios achieved an unprecedented space-time yield of 1.22 g/L/h with 88.1 g/L product titer in a 500 mL scaled-up reaction, manifesting a highest total conversion of 68.2 %. An integrated purification process yielded gram-scale solid powder of 2′-F-dA with 98.0 % chemical purity and 85.0 % recovery. This novel whole-cell biocatalytic process demonstrates significant industrial potential for the production of 2′-F-dA.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 152-159"},"PeriodicalIF":4.4,"publicationDate":"2025-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145690537","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 : 2025-12-04DOI: 10.1016/j.synbio.2025.11.005
Yu Qin , Jiaxiang Hu , Kezhen Su
Synthetic biology, as an emerging field that integrates life sciences and engineering technology, is driving profound transformations in global science, ethics, and legal systems. In international legal framework, the Biological Weapons Convention (BWC) and the Convention on Biological Diversity (CBD) have established initial hard law governance systems. However, these frameworks still face structural limitations in terms of technical adaptability, the scope of provisions, and institutional coordination. Soft law, with its flexibility, non-binding nature, and ability to build consensus, is increasingly becoming an essential supplement to the international response to the ethical risks of synthetic biology. International organizations, industry alliances, and non-governmental actors are constructing a multi-layered soft law governance network through ethical guidelines, policy recommendations, and codes of conduct, providing institutional support for risk identification, technology classification, and behavioral guidance. Soft law is well-suited to perform the roles of guiding and providing feedback in governance, while hard law should focus on the construction of systems of rights and responsibilities and the establishment of obligations. There is a collaborative governance model that integrates both soft and hard law. This model, characterized by “soft law guidance, hard law consolidation, and soft law feedback,” aims to create a flexible and enforceable governance framework. This approach ensures that soft law provides a timely and adaptive starting point, hard law offers a uniform and accountable foundation, and a feedback loop allows for continuous adjustment based on practical experience.
{"title":"Functions and optimization of soft law in the international governance of synthetic biology: The predicament of hard law vs. the rise of soft law","authors":"Yu Qin , Jiaxiang Hu , Kezhen Su","doi":"10.1016/j.synbio.2025.11.005","DOIUrl":"10.1016/j.synbio.2025.11.005","url":null,"abstract":"<div><div>Synthetic biology, as an emerging field that integrates life sciences and engineering technology, is driving profound transformations in global science, ethics, and legal systems. In international legal framework, the Biological Weapons Convention (BWC) and the Convention on Biological Diversity (CBD) have established initial hard law governance systems. However, these frameworks still face structural limitations in terms of technical adaptability, the scope of provisions, and institutional coordination. Soft law, with its flexibility, non-binding nature, and ability to build consensus, is increasingly becoming an essential supplement to the international response to the ethical risks of synthetic biology. International organizations, industry alliances, and non-governmental actors are constructing a multi-layered soft law governance network through ethical guidelines, policy recommendations, and codes of conduct, providing institutional support for risk identification, technology classification, and behavioral guidance. Soft law is well-suited to perform the roles of guiding and providing feedback in governance, while hard law should focus on the construction of systems of rights and responsibilities and the establishment of obligations. There is a collaborative governance model that integrates both soft and hard law. This model, characterized by “soft law guidance, hard law consolidation, and soft law feedback,” aims to create a flexible and enforceable governance framework. This approach ensures that soft law provides a timely and adaptive starting point, hard law offers a uniform and accountable foundation, and a feedback loop allows for continuous adjustment based on practical experience.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 134-151"},"PeriodicalIF":4.4,"publicationDate":"2025-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145690538","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 : 2025-12-04DOI: 10.1016/j.synbio.2025.11.010
Yue Chen , Linbo Gou , Di Liu , Shengfang Wu , Xiuwen Zhou , Tai-Ping Fan , Long Wang , Yujie Cai
(S)-(+)-linalool, a valuable acyclic monoterpenol secondary metabolite of plants, finds extensive applications in the food, flavor and fragrance, pharmaceutical, and daily chemical industries. Microbial synthesis offers two pathways for its production, among which the mevalonate (MVA) pathway derived from halophilic archaea is more widely employed. However, the traditional Haloarchaea-type MVA pathway relies on bifunctional enzyme catalysis, consuming 3 molecules of ATP. Moreover, the catalytic activity of natural linalool synthase (LIS) is generally low, failing to meet industrial requirements. Additionally, linalool exhibits significant toxicity to microbial hosts, thereby limiting the production capacity of conventional chassis microorganisms. To address these bottlenecks, this study implemented systematic optimizations: Firstly, the Archaeal mevalonate pathway was reconstructed by replacing the original bifunctional enzyme with two monofunctional enzymes, successfully reducing ATP consumption to 2 molecules. Secondly, through directed screening and rational design, a high-activity linalool synthase mutant, CsMLISI331V/I444L, derived from Coriandrum sativum, was obtained. Furthermore, an enzyme fusion strategy was adopted, involving the introduction of a long flexible linker between key genes, which significantly enhanced catalytic efficiency. Finally, S. marcescens HBQA7ΔsIaAB-pyc, a strain previously screened in our laboratory with broad-spectrum tolerance to terpenoids, was selected as the novel chassis cell. Collectively, these efforts resulted in the construction of a microbial cell factory for the efficient synthesis of (S)-(+)-linalool, laying a solid foundation for industrial-scale production.
{"title":"Engineering an ATP-saving mevalonate pathway for high-efficiency S-(+)-linalool production in Serratia marcescens","authors":"Yue Chen , Linbo Gou , Di Liu , Shengfang Wu , Xiuwen Zhou , Tai-Ping Fan , Long Wang , Yujie Cai","doi":"10.1016/j.synbio.2025.11.010","DOIUrl":"10.1016/j.synbio.2025.11.010","url":null,"abstract":"<div><div>(S)-(+)-linalool, a valuable acyclic monoterpenol secondary metabolite of plants, finds extensive applications in the food, flavor and fragrance, pharmaceutical, and daily chemical industries. Microbial synthesis offers two pathways for its production, among which the mevalonate (MVA) pathway derived from halophilic archaea is more widely employed. However, the traditional Haloarchaea-type MVA pathway relies on bifunctional enzyme catalysis, consuming 3 molecules of ATP. Moreover, the catalytic activity of natural linalool synthase (LIS) is generally low, failing to meet industrial requirements. Additionally, linalool exhibits significant toxicity to microbial hosts, thereby limiting the production capacity of conventional chassis microorganisms. To address these bottlenecks, this study implemented systematic optimizations: Firstly, the Archaeal mevalonate pathway was reconstructed by replacing the original bifunctional enzyme with two monofunctional enzymes, successfully reducing ATP consumption to 2 molecules. Secondly, through directed screening and rational design, a high-activity linalool synthase mutant, CsMLIS<sup>I331V/I444L</sup>, derived from <em>Coriandrum sativum</em>, was obtained. Furthermore, an enzyme fusion strategy was adopted, involving the introduction of a long flexible linker between key genes, which significantly enhanced catalytic efficiency. Finally, <em>S</em>. <em>marcescens</em> HBQA7Δ<em>sIaAB-pyc</em>, a strain previously screened in our laboratory with broad-spectrum tolerance to terpenoids, was selected as the novel chassis cell. Collectively, these efforts resulted in the construction of a microbial cell factory for the efficient synthesis of (S)-(+)-linalool, laying a solid foundation for industrial-scale production.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 124-133"},"PeriodicalIF":4.4,"publicationDate":"2025-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145690539","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}
扫码关注我们
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号