ACS Synthetic Biology最新文献_第8页

Improving Yield and Assessing Quality of Secreted Proteins Over-Expressed in Pichia Pastoris with Chaperones and Structural Mass Spectrometry 利用伴侣蛋白和结构质谱技术提高毕赤酵母过表达分泌蛋白的产量和质量评估

IF 4.5 2区生物学

ACS Synthetic Biology Pub Date : 2026-09-18 Epub Date: 2026-09-04 DOI: 10.1021/acssynbio.6c00497

Xinzhe Ren, E-Ming Rau, Lauren Coutu, Travis Tran, Thomas J. Begley, Peter C. Dedon, Shane R. Byrne, Stephen D. Fried

{"title":"Improving Yield and Assessing Quality of Secreted Proteins Over-Expressed in Pichia Pastoris with Chaperones and Structural Mass Spectrometry","authors":"Xinzhe Ren, E-Ming Rau, Lauren Coutu, Travis Tran, Thomas J. Begley, Peter C. Dedon, Shane R. Byrne, Stephen D. Fried","doi":"10.1021/acssynbio.6c00497","DOIUrl":"https://doi.org/10.1021/acssynbio.6c00497","url":null,"abstract":"Pichia pastoris is a widely used host for recombinant protein production because it combines the advantages of microbial cultivation with eukaryotic protein folding and secretion. However, secretion efficiency is often limited by the folding capacity of the endoplasmic reticulum (ER), where recombinant proteins must be translocated, folded, and processed prior to export. When ER folding capacity is exceeded, proteins may be retained, degraded, or secreted in non-native conformations, reducing both yield and product quality. Chaperone engineering and codon optimization represent two promising strategies to address these limitations. Here, we generated stable Pichia strains expressing four model secreted proteins (human serum albumin, interleukin-2, thaumatin-I, and thaumatin-II) using either conventional codon optimization or Epi-MAX codon engineering, which adapts transgene codon usage to stress-responsive translational programs. We also engineered strains containing an additional chromosomal copy of either the ER Hsp70 chaperone Kar2 or protein disulfide isomerase (Pdi1). To assess protein quality, we applied limited proteolysis mass spectrometry (LiP-MS), a structural proteomics approach that can detect subtle conformational differences to secreted proteins. Increased Pdi1 levels improved secretion of all four proteins tested, whereas Kar2 overexpression generally reduced yield. For thaumatin-II, Pdi1 enhanced secretion but promoted release of a non-native conformation, which we could correct through codon engineering. Together, these results demonstrate that maximizing recombinant protein production requires optimization of both yield and structural quality and establish complementary strategies for improving secreted protein expression in Pichia.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 9","pages":"3807–3820"},"PeriodicalIF":4.5,"publicationDate":"2026-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149003791","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

Production of Archaeal Inositol Phospholipids in Engineered Saccharomyces cerevisiae 工程酿酒酵母中古细菌肌醇磷脂的生产

IF 4.5 2区生物学

ACS Synthetic Biology Pub Date : 2026-09-18 Epub Date: 2026-08-24 DOI: 10.1021/acssynbio.6c00268

Jinze Li, Shizhe Zhang, Erpeng Guo, Chenli Liu, Zhilai Hong, Tong Si

{"title":"Production of Archaeal Inositol Phospholipids in Engineered Saccharomyces cerevisiae","authors":"Jinze Li, Shizhe Zhang, Erpeng Guo, Chenli Liu, Zhilai Hong, Tong Si","doi":"10.1021/acssynbio.6c00268","DOIUrl":"https://doi.org/10.1021/acssynbio.6c00268","url":null,"abstract":"Despite phospholipid backbone divergence between Archaea (ether-isoprenoid/glycerol-1-phosphate membranes) and Bacteria/Eukarya (ester-fatty acid/glycerol-3-phosphate membranes), known as the “lipid divide”, inositol headgroups are conserved across all domains. Inositol lipids from thermophilic archaea form exceptionally stable liposomes but are difficult to obtain at scale. Here, we engineered eukaryotic Saccharomyces cerevisiae for heterologous biosynthesis of unsaturated archaetidylinositol, revealing unexpected promiscuity of endogenous yeast enzymes toward archaeal substrates. Metabolic engineering achieved production levels of 45.6 ± 6.2 mg/g DCW, substantially exceeding endogenous phosphatidylinositol level measured in the same strain (4.8 ± 1.0 mg/g DCW) by >8-fold, which enabled detailed structural, physiological, and biophysical characterization of unsaturated archaetidylinositol for the first time. Production triggered pleiotropic lipid metabolism changes and enhanced thermotolerance, with engineered yeast retaining 57% viability after 40 min at 50 °C, a condition that eliminates chassis strain viability. Purified archaeal inositol lipid formed stable liposomes, which maintained integrity upon heat shock treatment at 95 °C or during 4 week storage at 4 °C, while eukaryotic lipid-derived vesicles exhibited substantial cargo leakage under identical conditions. This yeast platform will enable access to versatile archaetidylinositol derivatives with tailored bioactivity and material properties for both evolutionary studies and biotechnology applications.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 9","pages":"3664–3675"},"PeriodicalIF":4.5,"publicationDate":"2026-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149003782","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

Structure-Guided Terminal Modifications Enhance the Efficiency of PET Degradation 结构导向末端修饰提高PET降解效率

IF 4.5 2区生物学

ACS Synthetic Biology Pub Date : 2026-09-18 Epub Date: 2026-07-31 DOI: 10.1021/acssynbio.5c00682

Lin Zhang, Keyan Chen, Zhiwen Xi, Zhuo Tian, Xuyang wei, Wan Syaidatul Aqma, Zhiguo Guo, Qi Wang, Qiuhong Niu

{"title":"Structure-Guided Terminal Modifications Enhance the Efficiency of PET Degradation","authors":"Lin Zhang, Keyan Chen, Zhiwen Xi, Zhuo Tian, Xuyang wei, Wan Syaidatul Aqma, Zhiguo Guo, Qi Wang, Qiuhong Niu","doi":"10.1021/acssynbio.5c00682","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00682","url":null,"abstract":"Poly(ethylene terephthalate) (PET) is a widely used plastic whose persistence and improper disposal pose serious environmental and health risks. In this study, three novel PET hydrolases TbPETase, AbPETase, and AfPETase were identified from Thermoanaerobacterales, Acidimicrobiales, and Actinokineospora fastidiosa, respectively. Among these, TbPETase exhibited the highest enzymatic activity and thermostability. Based on structural analysis, we performed semirational truncations targeting the intrinsically disordered N- and C-terminal regions of TbPETase, generating two improved variants ΔN36 and ΔC4. The double mutant, TbPETaseΔN36/ΔC4, demonstrated a 2.3-fold increase in overall enzymatic activity and a 2.6-fold improvement in catalytic efficiency (kcat/Km) compared to the wild-type enzyme, along with significantly enhanced thermal stability. Molecular dynamics simulations revealed that the removal of flexible terminal regions increased the overall structural rigidity of TbPETaseΔN36/ΔC4. This structural stabilization was associated with the formation of a hydrogen bond at T215 and a π−π stacking interaction at W193. In a 100 mL one-pot reaction system, the combination of TbPETaseΔN36/ΔC4 with an engineered BMHETase variant, BMHETase6M, achieved 81.2% degradation of semicrystalline PET powder at 60 °C over 60 h, yielding terephthalic acid as the major product. These findings demonstrate the potential of TbPETaseΔN36/ΔC4 as a highly efficient and industrially applicable biocatalyst for PET degradation.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 9","pages":"3553–3565"},"PeriodicalIF":4.5,"publicationDate":"2026-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149003788","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

Plasmid- and Inducer-Free Biosynthesis of Difucosyllactose through Rational Fusion Enzyme Engineering and Systematic Pathway Optimization 通过合理的融合酶工程和系统途径优化的无质粒和无诱导剂生物合成二焦酰基乳糖。

IF 4.5 2区生物学

ACS Synthetic Biology Pub Date : 2026-08-21 Epub Date: 2026-07-18 DOI: 10.1021/acssynbio.6c00229

Zhiqiang Liu, Binglin Li, Jieying Deng, Yanfeng Liu, Jianghua Li, Guocheng Du, Jian Chen, Xueqin Lv, Xianhao Xu, Long Liu

{"title":"Plasmid- and Inducer-Free Biosynthesis of Difucosyllactose through Rational Fusion Enzyme Engineering and Systematic Pathway Optimization","authors":"Zhiqiang Liu, Binglin Li, Jieying Deng, Yanfeng Liu, Jianghua Li, Guocheng Du, Jian Chen, Xueqin Lv, Xianhao Xu, Long Liu","doi":"10.1021/acssynbio.6c00229","DOIUrl":"10.1021/acssynbio.6c00229","url":null,"abstract":"Difucosyllactose (DFL), a fucosylatedhuman milk oligosaccharide,exhibits significant application potential in the food and pharmaceuticalindustries. However, the microbial production of DFL faces challengessuch as the accumulation of the intermediate 2′-fucosyllactose(2′-FL) and the high costs associated with the use of antibioticsand inducers. In this study, we constructed a plasmid- and inducer-free Escherichia coli MG1655 strain to enhance the biosynthesisof DFL while minimizing 2′-FL accumulation. Initially, the de novo DFL biosynthetic pathway was established by introducingdifferent α-1,3-fucosyltransferases (α-1,3-FucT) intoa 2′-FL-producing strain. Combinatorial metabolic engineeringstrategies were then employed to improve DFL accumulation. Subsequently,guided by the in silico multienzyme assembling byreshaping space (iMARS) framework, a fusion enzyme, FucTaY218K-L60-FutC, was rationally designed to effectively alleviate2′-FL accumulation. Furthermore, modulation of the hydrophobicmicroenvironment in the substrate-binding pocket of FucTaY218K yielded the beneficial mutant FucTaY218K/W31R. Finally,modifications to the lactose operon significantly improved straingrowth while concurrently enhancing DFL production. The best-performingstrain achieved a DFL titer of 74 g/L in a 5-L bioreactor, correspondingto a productivity of 0.96 g/L/h, representing the highest productivityreported to date. This study establishes a robust and environmentallyfriendly platform for the industrial-scale production of DFL and providesa strategy for the microbial synthesis of other high-value human milkoligosaccharides.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 8","pages":"3357–3368"},"PeriodicalIF":4.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148496837","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

Incoherent Input as a Key Strategy for Robust Time-Dependent Biphasic Dynamics 非相干输入是鲁棒时变双相动力学的关键策略。

IF 4.5 2区生物学

ACS Synthetic Biology Pub Date : 2026-08-21 Epub Date: 2026-07-09 DOI: 10.1021/acssynbio.6c00099

Qinlin Li, Yuzhi Hu, Hong Qi, Tingting Wang, Meng Jin, Shoufang Huang, Maosheng Wang, Jiqian Zhang, Xiang Li, Xin Chen, Jianwei Shuai, Fei Xu

{"title":"Incoherent Input as a Key Strategy for Robust Time-Dependent Biphasic Dynamics","authors":"Qinlin Li, Yuzhi Hu, Hong Qi, Tingting Wang, Meng Jin, Shoufang Huang, Maosheng Wang, Jiqian Zhang, Xiang Li, Xin Chen, Jianwei Shuai, Fei Xu","doi":"10.1021/acssynbio.6c00099","DOIUrl":"10.1021/acssynbio.6c00099","url":null,"abstract":"Biological systems maintain functional order throughprecise responses,with time-dependent biphasic dynamics (TBD) serving as a key regulatorymechanism. However, the network topologies supporting TBD and theprinciples underlying their robustness remain poorly understood. Here,we systematically investigate all possible one-, two-, and three-nodenetwork motifs using extensive random parameter sampling to evaluatetheir capacity to generate TBDs. Statistical analysis of the resultingdynamics reveals that incoherent feedforward loops and negative feedbackloops constitute core topological modules underlying TBD generation.We identify 13 minimal core three-node motifs and construct a comprehensivetopological landscape linking network structure to functional robustness.Extending this framework, we propose a motif-to-hypermotif designparadigm and demonstrate that coupling motifs through node sharingor edge connectivity to form feedback-enriched hypermotifs significantlyenhances TBD robustness. Our findings reveal that incoherent inputtargeting the output node, particularly via inhibitory regulation,is a key design strategy for robust TBDs, providing new insights intothe modular principles governing biological network architecture.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 8","pages":"3215–3226"},"PeriodicalIF":4.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148409736","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

Quantitative Measurement of Synthetic Repression Curves Reveals Design Challenges for Genetic Circuit Engineering under Growth Arrest 合成抑制曲线的定量测量揭示了生长抑制下基因电路工程设计的挑战。

IF 4.5 2区生物学

ACS Synthetic Biology Pub Date : 2026-08-21 Epub Date: 2026-05-13 DOI: 10.1021/acssynbio.6c00123

John P. Marken, Mark L. Prator, Bruce A. Hay, Richard M. Murray

{"title":"Quantitative Measurement of Synthetic Repression Curves Reveals Design Challenges for Genetic Circuit Engineering under Growth Arrest","authors":"John P. Marken, Mark L. Prator, Bruce A. Hay, Richard M. Murray","doi":"10.1021/acssynbio.6c00123","DOIUrl":"10.1021/acssynbio.6c00123","url":null,"abstract":"Despite the factthat microbes in natural environments spend mostof their time in growth arrest, we understand little about how thisphysiological state affects the performance of engineered geneticcircuits. Here, we measure repression curves from a library of geneticNOT gates at single-cell resolution in Escherichia coli under both active growth and growth arrest tosystematically investigate how growth arrest affects circuit behavior.We find that the impact of growth arrest on circuit performance isalmost entirely dominated by a >100-fold reduction in unrepressedexpression levels. Growth arrest caused gene expression noise to increaseonly moderately and had minimal impact on the sensitivity and sharpnessof the repression curves. Our work shows both that conventional geneticcircuit design paradigms are currently insufficient to develop circuitsthat can function properly under growth arrest, and that addressingthe reduction in just a single performance parameter would be sufficientto resolve this problem. This work expands our understanding of bacterialgene regulation under growth arrest and lays the groundwork for newdesign paradigms that will be essential to ensure the safe and reliableperformance of synthetic biology systems in real-world environments.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 8","pages":"3235–3248"},"PeriodicalIF":4.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147924471","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 Framework for the In Vivo Production of Extensively Engineered Thiopeptides 广泛工程硫肽的体内生产框架。

IF 4.5 2区生物学

ACS Synthetic Biology Pub Date : 2026-08-21 Epub Date: 2026-07-03 DOI: 10.1021/acssynbio.6c00311

Shinta Ijichi, Shotaro Hoshino, Emiko Nagai, Shumpei Asamizu, Hiroyasu Onaka

{"title":"A Framework for the In Vivo Production of Extensively Engineered Thiopeptides","authors":"Shinta Ijichi, Shotaro Hoshino, Emiko Nagai, Shumpei Asamizu, Hiroyasu Onaka","doi":"10.1021/acssynbio.6c00311","DOIUrl":"10.1021/acssynbio.6c00311","url":null,"abstract":"Thiopeptidesare a family of macrocycle-containing ribosomallysynthesized and post-translationally modified peptides. Their elaboratescaffolds offer considerable potential for bioengineering toward thiopeptide-basedpharmaceuticals and other practical applications. Among thiopeptides,lactazoles possess uniquely promiscuous biosynthetic machinery thatenables the creation of diverse macrocyclic peptides. Previous bioengineeringefforts have exploited this machinery to achieve the de novo designof bioactive lactazole-based thiopeptides in vitro. However, it remainsunclear whether microbial systems can produce lactazole-based thiopeptideswith dramatically engineered macrocycles, particularly those thatare expanded and contain more than 50% amino acid divergence relativeto native lactazole macrocycles. Here, we established a frameworkfor the in vivo production of lactazole-based thiopeptides by tuningexpression cassettes and selecting suitable heterologous hosts andculture conditions. Initially, we focused on transcriptional terminatorsin the 3′-untranslated region of the precursor gene and identifiedthe lazA terminator as a critical determinant forlactazole production. We then evaluated several heterologous Streptomyces hosts and selected Streptomyces sp. TP-A0584 ΔgodA for the production oflactazole-based thiopeptides. Under optimized conditions, includinglow-temperature cultivation, more than 90% of the tested lactazole-basedthiopeptides were successfully produced, and a large part of themreached mg-scale production, with a maximum titer of 46.4 mg/L. Notably,although their macrocycles showed up to 73% amino acid divergencefrom those of native lactazoles, most of these lactazole-based thiopeptideswere successfully produced in vivo. Our framework represents an initialstep toward enabling the large-scale supply of lactazole-based thiopeptidesand should facilitate the development of thiopeptide-based bioactivemolecules.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 8","pages":"3426–3437"},"PeriodicalIF":4.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148373664","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

Tandem-sgRNA Provides an Effective Reverse Genetic Approach for Suppression of Streptomyces Biosynthetic Gene Clusters and Secondary Metabolism Tandem-sgRNA为抑制链霉菌生物合成基因簇和次生代谢提供了有效的反向遗传途径。

IF 4.5 2区生物学

ACS Synthetic Biology Pub Date : 2026-08-21 Epub Date: 2026-07-11 DOI: 10.1021/acssynbio.6c00274

Chengxi Zhang, Nitya Kalyani Josyula, Ivette Cornejo-Corona, Rebecca Watson, José D. Cediel-Becerra, Marc G. Chevrette, Timothy P. Devarenne, Paul D. Straight

{"title":"Tandem-sgRNA Provides an Effective Reverse Genetic Approach for Suppression of Streptomyces Biosynthetic Gene Clusters and Secondary Metabolism","authors":"Chengxi Zhang, Nitya Kalyani Josyula, Ivette Cornejo-Corona, Rebecca Watson, José D. Cediel-Becerra, Marc G. Chevrette, Timothy P. Devarenne, Paul D. Straight","doi":"10.1021/acssynbio.6c00274","DOIUrl":"10.1021/acssynbio.6c00274","url":null,"abstract":"Bacterial biosynthetic gene clusters (BGCs) encode secondarymetaboliteswith diverse biological activities; however, most BGC products remainuncharacterized. One approach to identifying products and their metabolismis to use reverse genetics to identify metabolite-associated phenotypes.CRISPR interference (CRISPRi) offers a promising approach to disruptBGC functions in high-GC genomes, typical of Streptomyces species. In many of these organisms, single-guide RNA (sgRNA)-mediatedCRISPRi often results in incomplete product suppression, resultingin partial phenotypes that are unsuitable for functional studies.Using Streptomyces sp. Mg1, we found that a tandem-sgRNAconfiguration for CRISPRi improved the efficiency of target metabolitesuppression. We engineered strains to express two sgRNAs to targetthe same promoter region within a BGC, resulting in greater than 80%metabolite suppression across diverse secondary metabolite classes.We used tandem-sgRNA CRISPRi to identify phenotypes associated withthe loss of polyketide linearmycins, the siderophore desferrioxamine,the terpene β-carotene, and an uncharacterized nonribosomalpeptide synthetase (NRPS). This approach revealed that β-carotenedepletion substantially reduced intrinsic cellular autofluorescence.Targeting the unknown NRPS produced developmental phenotypes and enabledthe identification of the biosynthetic genes for the antibiotic lavendomycin,revealing a noncollinear organization of genes in the BGC. We suggestthat tandem-sgRNA CRISPRi provides an efficient reverse genetics platformfor the functional characterization of Streptomyces BGCs, enabling the correlation of metabolites with the gene function,identification of associated phenotypes, and prioritization of crypticBGCs for natural product discovery.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 8","pages":"3382–3395"},"PeriodicalIF":4.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148416975","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

Synthetic Intrinsically Disordered Proteins Enable Soluble Expression of Disulfide-Rich Therapeutics in E. coli 合成内在无序蛋白使富含二硫化物的治疗药物在大肠杆菌中可溶表达。

IF 4.5 2区生物学

ACS Synthetic Biology Pub Date : 2026-08-21 Epub Date: 2026-07-16 DOI: 10.1021/acssynbio.6c00115

Yulia Shmidov, Parul Sirohi, Jonathan C. Su, Joy (Shijia). Tong, Rohit Suresh, Joshua J. Milligan, Daniel M. Shapiro, Ashutosh Chilkoti

{"title":"Synthetic Intrinsically Disordered Proteins Enable Soluble Expression of Disulfide-Rich Therapeutics in E. coli","authors":"Yulia Shmidov, Parul Sirohi, Jonathan C. Su, Joy (Shijia). Tong, Rohit Suresh, Joshua J. Milligan, Daniel M. Shapiro, Ashutosh Chilkoti","doi":"10.1021/acssynbio.6c00115","DOIUrl":"10.1021/acssynbio.6c00115","url":null,"abstract":"We previously reported the de novodesign of three small (<20kDa), highly soluble synthetic intrinsically disordered proteins (SynIDPs)and demonstrated their utility as solubility tags for proteins andantifouling agents. Building on this work, we now show that thesehypersoluble SynIDPs significantly enhance the soluble expressionof disulfide-rich proteins (DRPs) of therapeutic relevance, includingfibroblast growth factor 21 (FGF-21), interleukin-15 (IL-15), andbovine pancreatic trypsin inhibitor (BPTI). Through SynIDP fusions,we achieve soluble recombinant production of functionally active DRPscontaining a single disulfide bond in the E. coli strain BL21(DE3) and up to three nonconsecutive disulfide bondsin the E. coli SHuffle T7 Express strain, withoutthe need for refolding. The resulting SynIDP-DRP fusion proteins retainbiological activity, confirming correct folding and disulfide-bondformation with minimal interference from the SynIDP tag, which obviatesthe need for tag removal. Collectively, these findings highlight theversatility and utility of SynIDPs as molecular tools to advance theproduction and development of protein therapeutics.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 8","pages":"3227–3234"},"PeriodicalIF":4.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148467804","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

Rational Engineering of a Sucrose Phosphorylase for Switchable Regioselective Synthesis of Antioxidant Gallic Acid α-Glucosides 可切换区域选择性合成抗氧化没食子酸α-糖苷的蔗糖磷酸化酶的合理工程。

IF 4.5 2区生物学

ACS Synthetic Biology Pub Date : 2026-08-21 Epub Date: 2026-07-14 DOI: 10.1021/acssynbio.6c00370

Zhiwei Li, Zijian Wang, Jingwei Guo, Xueqin Hu, Hongbin Zhang, Jingwen Yang

{"title":"Rational Engineering of a Sucrose Phosphorylase for Switchable Regioselective Synthesis of Antioxidant Gallic Acid α-Glucosides","authors":"Zhiwei Li, Zijian Wang, Jingwei Guo, Xueqin Hu, Hongbin Zhang, Jingwen Yang","doi":"10.1021/acssynbio.6c00370","DOIUrl":"10.1021/acssynbio.6c00370","url":null,"abstract":"Gallic acid is a bioactive polyphenol with poor solubilityandstability. Enzymatic α-glucosylation can address these limitations,but regioselective synthesis among multiple phenolic hydroxyls remainschallenging. Here, we engineer a sucrose phosphorylase from Bifidobacterium adolescentis (BaSP) for the switchable synthesisof gallic acid 4-O- and 3-O-α-d-glucopyranosides. Structure-guidedrational design identifies four key regions controlling substrateorientation. The double mutant I231A/D400E achieves >90% selectivityfor the 4-O isomer with 82% yield in 2 h. Strikingly, the mutant L341V/T157G completely reverses regioselectivity, producing the 3-O isomerwith >98% yield and >99% selectivity. Molecular docking revealsthatthese mutations remodel the active pocket and substrate channel, positioningthe target hydroxyl group adjacent to the catalytic center. The resultingα-glucosides show up to 12-fold improved water solubility andsuperior cytoprotective, anti-inflammatory (IL-1β), and antioxidant(HO-1/NQO1) activities compared to gallic acid. This work providestwo high-performance, regiocomplementary biocatalysts for green synthesisof pharmaceutically relevant gallic acid glycosides and offers mechanisticinsights into regioselectivity control in sucrose phosphorylases.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"15 8","pages":"3474–3484"},"PeriodicalIF":4.5,"publicationDate":"2026-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148443519","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