Pub Date : 2026-01-30DOI: 10.1016/j.synbio.2026.01.017
Qiao-Qin Zhao , Peng-Cheng Hu , Chuan-Jiang Zhang , Meng-Yao Zhu , Yun Tian , Xiao-Na Yang , Xiao-Jun Ji , Chang-Zhu Li , Xiao-Man Sun , Xiang-Yang Lu , Hu-Hu Liu
Squalene, a multifunctional natural compound with diverse bioactivities, has significant potential in the nutraceutical and health industries. Microbial synthesis using engineered cell factories represents a sustainable alternative to conventional methods of extracting from plants or animals. This study systematically optimized squalene production in Yarrowia lipolytica through integrated metabolic pathway reconstruction and adaptive laboratory evolution. First, overexpression of DGA1 and LRO1 induced lipid droplet expansion and proliferation to achieve efficient steady-state accumulation of intracellular squalene. Second, the catalytic efficiency between ERG20 and SQS was enhanced by fusing ERG20-SQS through an enzyme fusion strategy for increasing the squalene synthesis flux. Then, the expression of ScHMG1, the rate-limiting enzyme of the MVA pathway, was further enhanced to optimize precursor supply. Finally, adaptive laboratory evolution induced by hydrogen peroxide generated the evolved strain SY8–H3, which produced 801.34 mg/L of squalene in shake-flask fermentation and 4.53 g/L via fed-batch fermentation in a 2.4 L bioreactor. This research firstly applies the oxidative stress-driven adaptive evolution to enhance squalene biosynthesis in Y. lipolytica, establishing the reference for synthesizing squalene and its derived compounds in engineered Y. lipolytica.
{"title":"Metabolic engineering and adaptive laboratory evolution enhance squalene production in Yarrowia lipolytica","authors":"Qiao-Qin Zhao , Peng-Cheng Hu , Chuan-Jiang Zhang , Meng-Yao Zhu , Yun Tian , Xiao-Na Yang , Xiao-Jun Ji , Chang-Zhu Li , Xiao-Man Sun , Xiang-Yang Lu , Hu-Hu Liu","doi":"10.1016/j.synbio.2026.01.017","DOIUrl":"10.1016/j.synbio.2026.01.017","url":null,"abstract":"<div><div>Squalene, a multifunctional natural compound with diverse bioactivities, has significant potential in the nutraceutical and health industries. Microbial synthesis using engineered cell factories represents a sustainable alternative to conventional methods of extracting from plants or animals. This study systematically optimized squalene production in <em>Yarrowia lipolytica</em> through integrated metabolic pathway reconstruction and adaptive laboratory evolution. First, overexpression of <em>DGA1</em> and <em>LRO1</em> induced lipid droplet expansion and proliferation to achieve efficient steady-state accumulation of intracellular squalene. Second, the catalytic efficiency between ERG20 and SQS was enhanced by fusing ERG20-SQS through an enzyme fusion strategy for increasing the squalene synthesis flux. Then, the expression of ScHMG1, the rate-limiting enzyme of the MVA pathway, was further enhanced to optimize precursor supply. Finally, adaptive laboratory evolution induced by hydrogen peroxide generated the evolved strain SY8–H3, which produced 801.34 mg/L of squalene in shake-flask fermentation and 4.53 g/L via fed-batch fermentation in a 2.4 L bioreactor. This research firstly applies the oxidative stress-driven adaptive evolution to enhance squalene biosynthesis in <em>Y. lipolytica</em>, establishing the reference for synthesizing squalene and its derived compounds in engineered <em>Y. lipolytica</em>.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"13 ","pages":"Pages 86-97"},"PeriodicalIF":4.4,"publicationDate":"2026-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146080398","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}
Microbial cell factories represent the primary approach for heterologous lycopene synthesis, where gene source selection and pathway regulation have been demonstrated to have a significant impact on lycopene titer. In this study, key lycopene biosynthesis genes (crtE, crtB and crtI) derived from the extremophile Deinococcus wulumuqiensis R12 were introduced into Escherichia coli, generating the chassis strain H0. Fermentation optimization revealed sodium pyruvate significantly enhanced lycopene production and cell growth. Quantitative polymerase chain reaction (qPCR) analysis revealed that sodium pyruvate upregulated the expression of dxr, ispA, crtE, crtB and crtI genes, while downregulating the expression of dxs and idi genes. Consequently, different sources of dxs, dxr, idi and ispA genes were screened and co-expressed to reinforce the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in E. coli. The optimized combination of dxs from E. coli MG1655 with idi from D. wulumuqiensis R12 achieved maximal lycopene titer of 293.70 mg/L (112.49 mg/g DCW), which was 33.88-fold higher than that of the initial strain H0. This study offers genetic resources for heterologous carotenoid synthesis and establishes a reference framework for the synthesis of analogous complex isoprenoid metabolites.
微生物细胞工厂代表了外源番茄红素合成的主要途径，其中基因来源选择和途径调控已被证明对番茄红素滴度有显著影响。本研究将嗜极细菌乌鲁穆奇Deinococcus wulumuqiensis R12衍生的关键番茄红素生物合成基因（crtE、crtB和crtI）导入大肠杆菌，生成底盘菌株H0。发酵优化结果表明，丙酮酸钠能显著提高番茄红素产量和细胞生长。定量聚合酶链反应（qPCR）结果显示，丙酮酸钠上调了dxr、ispA、crtE、crtB和crtI基因的表达，下调了dxs和idi基因的表达。因此，筛选不同来源的dxs、dxr、idi和ispA基因，并共同表达以增强大肠杆菌中2- c -甲基-d-赤藓糖醇4-磷酸（MEP）途径。大肠杆菌MG1655的dxs与乌鲁穆歧杆菌R12的idi经优化组合后，番茄红素滴度最高为293.70 mg/L (112.49 mg/g DCW)，比初始菌株H0提高了33.88倍。本研究为异源类胡萝卜素合成提供了遗传资源，并为合成类似的类异戊二烯复合物代谢物建立了参考框架。
{"title":"Metabolic engineering of endogenous MEP pathway for enhanced lycopene production in Escherichia coli","authors":"Xian Xu, Hongyu Xing, Hui Zhi, Chen Qin, Yuyue Deng, Wanqi Wei, Chunyan Huang","doi":"10.1016/j.synbio.2026.01.014","DOIUrl":"10.1016/j.synbio.2026.01.014","url":null,"abstract":"<div><div>Microbial cell factories represent the primary approach for heterologous lycopene synthesis, where gene source selection and pathway regulation have been demonstrated to have a significant impact on lycopene titer. In this study, key lycopene biosynthesis genes (<em>crtE</em>, <em>crtB</em> and <em>crtI</em>) derived from the extremophile <em>Deinococcus wulumuqiensis</em> R12 were introduced into <em>Escherichia coli</em>, generating the chassis strain H0. Fermentation optimization revealed sodium pyruvate significantly enhanced lycopene production and cell growth. Quantitative polymerase chain reaction (qPCR) analysis revealed that sodium pyruvate upregulated the expression of <em>dxr</em>, <em>ispA</em>, <em>crtE</em>, <em>crtB</em> and <em>crtI</em> genes, while downregulating the expression of <em>dxs</em> and <em>idi</em> genes. Consequently, different sources of <em>dxs</em>, <em>dxr</em>, <em>idi</em> and <em>ispA</em> genes were screened and co-expressed to reinforce the 2-C-methyl-<span>d</span>-erythritol 4-phosphate (MEP) pathway in <em>E. coli</em>. The optimized combination of <em>dxs</em> from <em>E. coli</em> MG1655 with <em>idi</em> from <em>D. wulumuqiensis</em> R12 achieved maximal lycopene titer of 293.70 mg/L (112.49 mg/g DCW), which was 33.88-fold higher than that of the initial strain H0. This study offers genetic resources for heterologous carotenoid synthesis and establishes a reference framework for the synthesis of analogous complex isoprenoid metabolites.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"13 ","pages":"Pages 109-119"},"PeriodicalIF":4.4,"publicationDate":"2026-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146080400","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 : 2026-01-30DOI: 10.1016/j.synbio.2026.01.015
Yuxin Zhang , Meiyan Wang , Kaijie Dou , Ruizhi Zhang , Chunfang Wang , Xiaoying Bian , Jun Si , Guoqing Niu
Prodigiosin, a bioactive tripyrrole pigment, exhibits a broad spectrum of biological activities-including antimicrobial, anticancer, and antimalarial properties-thereby holding significant promise for use in pharmaceutical applications and industrial biotechnology. In this study, three isolates of Serratia marcescens were recovered from the cabbage rhizosphere. Genomic analysis revealed a highly conserved prodigiosin biosynthetic gene cluster embedded within the chromosomes of all three isolates. Though prodigiosin production was detected in these three S. marcescens isolates, the relatively low yield severely limits the feasibility of its large-scale production. To address this issue, we employed a stepwise strategy involving heterologous expression, promoter engineering, genome-wide transposon mutagenesis, and optimization of fermentation media with the aim to achieve high-level prodigiosin production. The introduction of an engineered prodigiosin gene cluster into a tailored Pseudomonas putida KT2440 chassis strain yielded a maximum prodigiosin titer of 665 mg/L in shake-flask cultures, significantly outperforming production levels of the native S. marcescens isolates. When cultured in a small-scale stirred-tank bioreactor, the engineered strain further elevated the prodigiosin yield to 1161 mg/L. Our study presents a robust platform for prodigiosin overproduction, which can be adapted to improve the titers of other prodiginine family compounds.
{"title":"High-level prodigiosin production in Pseudomonas putida enabled by combinatorial metabolic engineering","authors":"Yuxin Zhang , Meiyan Wang , Kaijie Dou , Ruizhi Zhang , Chunfang Wang , Xiaoying Bian , Jun Si , Guoqing Niu","doi":"10.1016/j.synbio.2026.01.015","DOIUrl":"10.1016/j.synbio.2026.01.015","url":null,"abstract":"<div><div>Prodigiosin, a bioactive tripyrrole pigment, exhibits a broad spectrum of biological activities-including antimicrobial, anticancer, and antimalarial properties-thereby holding significant promise for use in pharmaceutical applications and industrial biotechnology. In this study, three isolates of <em>Serratia marcescens</em> were recovered from the cabbage rhizosphere. Genomic analysis revealed a highly conserved prodigiosin biosynthetic gene cluster embedded within the chromosomes of all three isolates. Though prodigiosin production was detected in these three <em>S. marcescens</em> isolates, the relatively low yield severely limits the feasibility of its large-scale production. To address this issue, we employed a stepwise strategy involving heterologous expression, promoter engineering, genome-wide transposon mutagenesis, and optimization of fermentation media with the aim to achieve high-level prodigiosin production. The introduction of an engineered prodigiosin gene cluster into a tailored <em>Pseudomonas putida</em> KT2440 chassis strain yielded a maximum prodigiosin titer of 665 mg/L in shake-flask cultures, significantly outperforming production levels of the native <em>S</em>. <em>marcescens</em> isolates. When cultured in a small-scale stirred-tank bioreactor, the engineered strain further elevated the prodigiosin yield to 1161 mg/L. Our study presents a robust platform for prodigiosin overproduction, which can be adapted to improve the titers of other prodiginine family compounds.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"13 ","pages":"Pages 98-108"},"PeriodicalIF":4.4,"publicationDate":"2026-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146080401","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 : 2026-01-25DOI: 10.1016/j.synbio.2026.01.010
Xing Fan , Xin Zhang , Minguo Tang , Shihao Wei , Ziyue Guo , Fucai Ren , Jiaying Hao , Lingqi Hua , Lin Zhou , Jie Xu , Wei Huang , Qianjin Kang , Linquan Bai
Sesquiterpenes have highly diverse chemical structures and biological activities, resulting in considerable interest in terms of their application and exploration of new analogs. In this study, we identified a new cryptic bacterial terpenoid biosynthetic gene cluster (i.e., pha) in the genome of the soil microorganism Streptomyces phaeochromogenes OSK-123 (strain OSK-123). According to online BiG-FAM and sequence similarity network analyses, pha was revealed to span an approximately 18 kb DNA region with 14 open reading frames, which likely include sequences encoding enzymes catalyzing the production of new sesquiterpenes. To facilitate the identification of target compounds, strong constitutive promoters were incorporated into pha. Additionally, we conducted a comparative analysis of different fermentation extracts for wild-type and promoter-substituted strains as well as RT-qPCR and LC-ESI-MS analyses to efficiently detect and identify target compounds. An examination of spectroscopic data identified four new 6/5-fused bicyclic sesquiterpenoid compounds, designated as phaterpene A–D (compounds 1–4). The terpene synthase PhaA catalyzed the formation of a six-membered ring sesquiterpene skeleton via heterologous expression in Escherichia coli. The complete pha sequence was incorporated into plasmid pLQ1512 and heterologously expressed in Streptomyces albus J1074 to elucidate the phaterpene biosynthetic pathway. Candidate modification genes were disrupted and functionally validated in S. albus J1074. The protoporphyrinogen/coproporphyrinogen oxidase PhaB was identified to catalyze the formation of a 6,5-fused bicyclic sesquiterpenoid scaffold. On the basis of these findings, the new phaterpene biosynthetic pathway was established. This research not only presented a practical approach for discovery of the targeted compounds through integration of multiple pipelines, but also enriched the understanding of chemical diversity and biosynthetic machinery of the new sesquiterpenes.
{"title":"Targeted identification of new phaterpenes and elucidation of the relevant biosynthetic pathway in Streptomyces phaeochromogenes OSK-123","authors":"Xing Fan , Xin Zhang , Minguo Tang , Shihao Wei , Ziyue Guo , Fucai Ren , Jiaying Hao , Lingqi Hua , Lin Zhou , Jie Xu , Wei Huang , Qianjin Kang , Linquan Bai","doi":"10.1016/j.synbio.2026.01.010","DOIUrl":"10.1016/j.synbio.2026.01.010","url":null,"abstract":"<div><div>Sesquiterpenes have highly diverse chemical structures and biological activities, resulting in considerable interest in terms of their application and exploration of new analogs. In this study, we identified a new cryptic bacterial terpenoid biosynthetic gene cluster (i.e., <em>pha</em>) in the genome of the soil microorganism <em>Streptomyces phaeochromogenes</em> OSK-123 (strain OSK-123). According to online BiG-FAM and sequence similarity network analyses, <em>pha</em> was revealed to span an approximately 18 kb DNA region with 14 open reading frames, which likely include sequences encoding enzymes catalyzing the production of new sesquiterpenes. To facilitate the identification of target compounds, strong constitutive promoters were incorporated into <em>pha</em>. Additionally, we conducted a comparative analysis of different fermentation extracts for wild-type and promoter-substituted strains as well as RT-qPCR and LC-ESI-MS analyses to efficiently detect and identify target compounds. An examination of spectroscopic data identified four new 6/5-fused bicyclic sesquiterpenoid compounds, designated as phaterpene A–D (compounds <strong>1</strong>–<strong>4</strong>). The terpene synthase PhaA catalyzed the formation of a six-membered ring sesquiterpene skeleton via heterologous expression in <em>Escherichia coli</em>. The complete <em>pha</em> sequence was incorporated into plasmid pLQ1512 and heterologously expressed in <em>Streptomyces albus</em> J1074 to elucidate the phaterpene biosynthetic pathway. Candidate modification genes were disrupted and functionally validated in <em>S. albus</em> J1074. The protoporphyrinogen/coproporphyrinogen oxidase PhaB was identified to catalyze the formation of a 6,5-fused bicyclic sesquiterpenoid scaffold. On the basis of these findings, the new phaterpene biosynthetic pathway was established. This research not only presented a practical approach for discovery of the targeted compounds through integration of multiple pipelines, but also enriched the understanding of chemical diversity and biosynthetic machinery of the new sesquiterpenes.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"13 ","pages":"Pages 73-85"},"PeriodicalIF":4.4,"publicationDate":"2026-01-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146080399","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 : 2026-01-24DOI: 10.1016/j.synbio.2026.01.011
Wenxia Song, Sheng Yang, Rong Tan, Qingsheng Qi, Xuemei Lu
Robust surface display systems are crucial for engineering Gram-negative bacteria as whole-cell biocatalysts. In the efficient cellulose degrader Cytophaga hutchinsonii, cellulases are secreted by the Type IX Secretion System (T9SS), yet their mechanism for outer membrane anchoring remained unknown. Here, we report a novel lipopolysaccharide (LPS)-dependent anchoring mechanism for T9SS substrates. We initially found that the anchoring of T9SS substrates to the outer membrane involves a modification, evident as a characteristic ladder-like pattern on PVDF membrane. By expressing heterologous proteins fused to the CTDs of specific cellulases, we demonstrated that this modification and anchoring are strictly CTD-dependent. Using bioinformatic analysis and gene deletion, we identified WaaL, which encodes a key enzyme involved in LPS biosynthesis. LC-MS/MS proteomics demonstrated that the ΔwaaL mutant fails to modify and anchor T9SS substrates. Therefore, we conclude that outer membrane anchoring depends on the combined action of LPS and the substrate CTDs. Leveraging this mechanism, we developed a novel surface display platform by fusing heterologous enzymes to T9SS substrate CTDs. As a proof-of-concept, we successfully displayed a functional polyethylene terephthalate hydrolase (PETase) on the surface of C. hutchinsonii, enabling the degradation of PET. Our work not only uncovers a fundamental mechanism for protein anchoring in C. hutchinsonii but also establishes an LPS-CTD-based platform for programmable surface display in Gram-negative bacteria, significantly expanding the toolbox for synthetic biology and biotechnological applications.
{"title":"A novel LPS-dependent outer membrane-anchoring mechanism for T9SS substrates enables engineered enzyme display and whole-cell PET degradation in Cytophaga hutchinsonii","authors":"Wenxia Song, Sheng Yang, Rong Tan, Qingsheng Qi, Xuemei Lu","doi":"10.1016/j.synbio.2026.01.011","DOIUrl":"10.1016/j.synbio.2026.01.011","url":null,"abstract":"<div><div>Robust surface display systems are crucial for engineering Gram-negative bacteria as whole-cell biocatalysts. In the efficient cellulose degrader <em>Cytophaga hutchinsonii</em>, cellulases are secreted by the Type IX Secretion System (T9SS), yet their mechanism for outer membrane anchoring remained unknown. Here, we report a novel lipopolysaccharide (LPS)-dependent anchoring mechanism for T9SS substrates. We initially found that the anchoring of T9SS substrates to the outer membrane involves a modification, evident as a characteristic ladder-like pattern on PVDF membrane. By expressing heterologous proteins fused to the CTDs of specific cellulases, we demonstrated that this modification and anchoring are strictly CTD-dependent. Using bioinformatic analysis and gene deletion, we identified WaaL, which encodes a key enzyme involved in LPS biosynthesis. LC-MS/MS proteomics demonstrated that the Δ<em>waaL</em> mutant fails to modify and anchor T9SS substrates. Therefore, we conclude that outer membrane anchoring depends on the combined action of LPS and the substrate CTDs. Leveraging this mechanism, we developed a novel surface display platform by fusing heterologous enzymes to T9SS substrate CTDs. As a proof-of-concept, we successfully displayed a functional polyethylene terephthalate hydrolase (PETase) on the surface of <em>C. hutchinsonii</em>, enabling the degradation of PET. Our work not only uncovers a fundamental mechanism for protein anchoring in <em>C. hutchinsonii</em> but also establishes an LPS-CTD-based platform for programmable surface display in Gram-negative bacteria, significantly expanding the toolbox for synthetic biology and biotechnological applications.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"13 ","pages":"Pages 61-72"},"PeriodicalIF":4.4,"publicationDate":"2026-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146039778","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 : 2026-01-23DOI: 10.1016/j.synbio.2026.01.003
Jie Liu , Dan Mei , Xuan-Jun Zhang , Wei-Guo Zhang , Long-Bao Zhu
Glutaric acid is a significant C5 dicarboxylic acid, extensively utilized in the chemical industry, medicine, and biomaterials. In recent years, the advancement of synthetic biology and metabolic engineering has rendered microbial production of glutaric acid a sustainable alternative to conventional chemical synthesis. This study reviews recent advancements in glutaric acid biosynthesis, primarily concentrating on the design of biosynthetic pathways and metabolic engineering strategies for the development of engineered strains. The utilization of systems biology technologies in the development of the glutaric acid biosynthetic pathway is examined. This study outlines the issues associated with glutaric acid biosynthesis and its prospective developmental trajectory, intending to offer theoretical insights and technological guidance for the sustainable production of glutaric acid and related fine chemicals.
{"title":"Sustainable production of glutaric acid in microbial cell factories: Current advances and future prospects","authors":"Jie Liu , Dan Mei , Xuan-Jun Zhang , Wei-Guo Zhang , Long-Bao Zhu","doi":"10.1016/j.synbio.2026.01.003","DOIUrl":"10.1016/j.synbio.2026.01.003","url":null,"abstract":"<div><div>Glutaric acid is a significant C5 dicarboxylic acid, extensively utilized in the chemical industry, medicine, and biomaterials. In recent years, the advancement of synthetic biology and metabolic engineering has rendered microbial production of glutaric acid a sustainable alternative to conventional chemical synthesis. This study reviews recent advancements in glutaric acid biosynthesis, primarily concentrating on the design of biosynthetic pathways and metabolic engineering strategies for the development of engineered strains. The utilization of systems biology technologies in the development of the glutaric acid biosynthetic pathway is examined. This study outlines the issues associated with glutaric acid biosynthesis and its prospective developmental trajectory, intending to offer theoretical insights and technological guidance for the sustainable production of glutaric acid and related fine chemicals.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"13 ","pages":"Pages 50-60"},"PeriodicalIF":4.4,"publicationDate":"2026-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146039777","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 : 2026-01-22DOI: 10.1016/j.synbio.2026.01.007
Manman Sun , Alex Xiong Gao , Bin Ye , Yimeng Zhao , Rodrigo Ledesma-Amaro , Jin Gao , Peng Wang
Membraneless organelles (MLOs) formed through liquid-liquid phase separation (LLPS) constitute crucial dynamic microenvironments within cells, capable of selectively concentrating specific molecules and regulating biochemical reactions. Based on the working mechanisms of natural MLOs, researchers have designed and constructed various synthetic MLOs. These MLOs have been applied in regulating enzyme activity, optimizing metabolic pathways, regulating gene expression, producing recombinant proteins, and developing functional biomaterials. Here, we systematically summarized the design strategies, characterization techniques, and client protein recruitment methods for synthetic MLOs, and categorically reviewed their application progress in the biotechnology field. We also discussed current challenges faced in the practical applications of synthetic MLOs and future research directions. This review aims to provide theoretical guidance and practical reference for the design and application of LLPS-driven synthetic MLOs, thereby promoting their innovative development in synthetic biology and biotechnology.
{"title":"Advances in engineering and applications of synthetic phase-separated membraneless organelles in biotechnology","authors":"Manman Sun , Alex Xiong Gao , Bin Ye , Yimeng Zhao , Rodrigo Ledesma-Amaro , Jin Gao , Peng Wang","doi":"10.1016/j.synbio.2026.01.007","DOIUrl":"10.1016/j.synbio.2026.01.007","url":null,"abstract":"<div><div>Membraneless organelles (MLOs) formed through liquid-liquid phase separation (LLPS) constitute crucial dynamic microenvironments within cells, capable of selectively concentrating specific molecules and regulating biochemical reactions. Based on the working mechanisms of natural MLOs, researchers have designed and constructed various synthetic MLOs. These MLOs have been applied in regulating enzyme activity, optimizing metabolic pathways, regulating gene expression, producing recombinant proteins, and developing functional biomaterials. Here, we systematically summarized the design strategies, characterization techniques, and client protein recruitment methods for synthetic MLOs, and categorically reviewed their application progress in the biotechnology field. We also discussed current challenges faced in the practical applications of synthetic MLOs and future research directions. This review aims to provide theoretical guidance and practical reference for the design and application of LLPS-driven synthetic MLOs, thereby promoting their innovative development in synthetic biology and biotechnology.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"13 ","pages":"Pages 37-49"},"PeriodicalIF":4.4,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146039779","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 : 2026-01-21DOI: 10.1016/j.synbio.2026.01.005
Xiaojing Li , Siyuan Zhu , Xiting Huang , Zhenxiang Xu , Tingting Guo , Jian Kong , Wentao Kong
Lacticaseibacillus paracasei (L. paracasei), a probiotic bacterium commonly found in the intestinal tract and fermented products, has been utilized as a tool for generating various bioactive components. However, its potential as a host cell for protein secretion is constrained by the limited availability of secretion signal peptides. This study explores the potential of endogenous signal peptides from L. paracasei for improving heterologous protein secretion. Endogenous secretory proteins from L. paracasei BL23 were analyzed to characterize their signal peptides. Using reporter genes such as nuc (nuclease) and amy (amylase) under the control of the nisin-inducible promoter PnisA, signal peptide SP230 was shown to be the most potent endogenous signal peptide for heterologous protein secretion in L. paracasei BL23. Additionally, a tandem linkage strategy employing identical signal peptides increased the secretion levels of heterologous proteins. The secretion of heterologous proteins was found to be dependent on the utilization of their optimal signal peptides. Tandem linkage of proteins with preferred signal peptides proved to be critical for efficient secretion of two distinct heterologous proteins simultaneously. The identification of novel signal peptides and the development of tandem linkage strategies in this study offer valuable insights for improving heterologous protein expression and secretion in L. paracasei. These findings enhance the potential of L. paracasei as a host for biotechnological applications, facilitating the secretion of diverse proteins.
{"title":"Exploration of endogenous signal peptides and tandem linkage strategies for enhanced heterologous protein secretion in Lacticaseibacillus paracasei","authors":"Xiaojing Li , Siyuan Zhu , Xiting Huang , Zhenxiang Xu , Tingting Guo , Jian Kong , Wentao Kong","doi":"10.1016/j.synbio.2026.01.005","DOIUrl":"10.1016/j.synbio.2026.01.005","url":null,"abstract":"<div><div><em>Lacticaseibacillus paracasei</em> (<em>L. paracasei</em>), a probiotic bacterium commonly found in the intestinal tract and fermented products, has been utilized as a tool for generating various bioactive components. However, its potential as a host cell for protein secretion is constrained by the limited availability of secretion signal peptides. This study explores the potential of endogenous signal peptides from <em>L. paracasei</em> for improving heterologous protein secretion. Endogenous secretory proteins from <em>L. paracasei</em> BL23 were analyzed to characterize their signal peptides. Using reporter genes such as <em>nuc</em> (nuclease) and <em>amy</em> (amylase) under the control of the nisin-inducible promoter PnisA, signal peptide SP230 was shown to be the most potent endogenous signal peptide for heterologous protein secretion in <em>L. paracasei</em> BL23. Additionally, a tandem linkage strategy employing identical signal peptides increased the secretion levels of heterologous proteins. The secretion of heterologous proteins was found to be dependent on the utilization of their optimal signal peptides. Tandem linkage of proteins with preferred signal peptides proved to be critical for efficient secretion of two distinct heterologous proteins simultaneously. The identification of novel signal peptides and the development of tandem linkage strategies in this study offer valuable insights for improving heterologous protein expression and secretion in <em>L. paracasei</em>. These findings enhance the potential of <em>L. paracasei</em> as a host for biotechnological applications, facilitating the secretion of diverse proteins.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"13 ","pages":"Pages 25-36"},"PeriodicalIF":4.4,"publicationDate":"2026-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146039776","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 : 2026-01-20DOI: 10.1016/j.synbio.2026.01.006
Jiugong Lv , Jiuquan An , Zhenyu Sun , Guofu Zhao , Xinyao Ding , Xi Deng , Huiping Tan , Jiajia Cai , Liya Liang , Rongming Liu
Succinate is a biobased platform chemical with wide applications in food, pharmaceuticals, and biodegradable polymers such as polybutylene succinate. Despite advances in microbial fermentation, cost-effective production remains limited by inefficient utilization of lignocellulosic hydrolysates, where glucose and xylose are the predominant sugars. In this study, we systematically engineered Escherichia coli C600 to enhance succinate biosynthesis from mixed sugars and hydrolysates. Competing by-product pathways were eliminated, the phosphotransferase system was modified to relieve carbon catabolite repression, and the pck gene from Bacillus subtilis was introduced to alleviate the ATP burden in xylose metabolism. To further improve xylose utilization, heterologous oxidative pathways (Weimberg and Dahms) from Caulobacter crescentus were integrated and fine-tuned using ribosome binding site libraries. The optimized strain exhibited flexible glucose–xylose co-utilization across varying sugar ratios, maintaining high succinate yields. A global transcriptional regulator library was then applied, and a crp mutant ESC6crp-W68+ was identified and enabled efficient growth and succinate production using inorganic nitrogen as the sole nitrogen source. Scale-up fermentation in a 5-L bioreactor confirmed the industrial relevance of the engineered strain: ESC6crp-W68+ produced 87.7 g/L succinate from synthetic mixed sugars with a yield of 1.15 mol/mol, and 77.3 g/L from corn stover hydrolysate with a yield of 1.02 mol/mol. This multi-layered engineering framework established a metabolically robust and cost-efficient E. coli platform, enabling high-titer succinate production directly from lignocellulosic hydrolysates.
{"title":"Engineering Escherichia coli for robust Co-utilization of glucose and xylose enables high-titer succinate production from lignocellulosic hydrolysates","authors":"Jiugong Lv , Jiuquan An , Zhenyu Sun , Guofu Zhao , Xinyao Ding , Xi Deng , Huiping Tan , Jiajia Cai , Liya Liang , Rongming Liu","doi":"10.1016/j.synbio.2026.01.006","DOIUrl":"10.1016/j.synbio.2026.01.006","url":null,"abstract":"<div><div>Succinate is a biobased platform chemical with wide applications in food, pharmaceuticals, and biodegradable polymers such as polybutylene succinate. Despite advances in microbial fermentation, cost-effective production remains limited by inefficient utilization of lignocellulosic hydrolysates, where glucose and xylose are the predominant sugars. In this study, we systematically engineered <em>Escherichia coli</em> C600 to enhance succinate biosynthesis from mixed sugars and hydrolysates. Competing by-product pathways were eliminated, the phosphotransferase system was modified to relieve carbon catabolite repression, and the <em>pck</em> gene from <em>Bacillus subtilis</em> was introduced to alleviate the ATP burden in xylose metabolism. To further improve xylose utilization, heterologous oxidative pathways (Weimberg and Dahms) from <em>Caulobacter crescentus</em> were integrated and fine-tuned using ribosome binding site libraries. The optimized strain exhibited flexible glucose–xylose co-utilization across varying sugar ratios, maintaining high succinate yields. A global transcriptional regulator library was then applied, and a <em>crp</em> mutant ESC6<sub>crp</sub>-W68<sup>+</sup> was identified and enabled efficient growth and succinate production using inorganic nitrogen as the sole nitrogen source. Scale-up fermentation in a 5-L bioreactor confirmed the industrial relevance of the engineered strain: ESC6<sub>crp</sub>-W68<sup>+</sup> produced 87.7 g/L succinate from synthetic mixed sugars with a yield of 1.15 mol/mol, and 77.3 g/L from corn stover hydrolysate with a yield of 1.02 mol/mol. This multi-layered engineering framework established a metabolically robust and cost-efficient <em>E</em>. <em>coli</em> platform, enabling high-titer succinate production directly from lignocellulosic hydrolysates.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"13 ","pages":"Pages 14-24"},"PeriodicalIF":4.4,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146001776","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 : 2026-01-20DOI: 10.1016/j.synbio.2026.01.008
Jiacheng Li , Zhongmei Hu , Yanjie Li , Hao Zha , Yujie Xie , Mingtao Zhao , Lili Ren , Biao Zhang
This study engineered the thermotolerant yeast Kluyveromyces marxianus to produce 3-hydroxypropionic acid (3-HP), a key precursor for biodegradable plastics, via the malonyl-CoA pathway using non-food feedstocks. The 3-HP titer was further increased through deleting Adh2A and Ach1, which prevents the synthesis of byproducts ethanol and acetic acid. Using Jerusalem artichoke tuber powder, engineered strain produced 27.32 and 32.31 g/L of 3-HP at 37 °C and 42 °C through fed-batch fermentation. Metabolic reconstruction replaced the native FADH2-dependent glycerol pathway (GUT1/GUT2) with an NADH-generating GDH1/DAK1 pathway, significantly enhancing glycerol utilization and increasing intracellular NADH supply by 62 %. Overexpression of Utr1 can further enhance the NADPH supply. Combined with heterologous expression of a codon-optimized, high-activity malonyl-CoA reductase (MCR) mutant (MCRN940V/K1106W/S1114R), the engineered strain achieved 3-HP titers of 33.15 g/L in fed-batch fermentation using pure glycerol at 42 °C. Crucially, it also produced 26.57 g/L 3-HP directly from crude glycerol at 42 °C. The thermotolerant fermentation at 42 °C, unprecedented for yeast-based 3-HP synthesis, reduces cooling water consumption by approximately 60 %, translating to an estimated annual CO2 reduction of 27.1 tons per 1000-ton fermenter. This work establishes a cost-effective, industrially scalable bioprocess for valorizing Jerusalem artichoke tubers and crude glycerol into a key platform chemical for biodegradable plastics and green chemicals, leveraging the strain's substrate flexibility, process robustness, and significant environmental advantages.
{"title":"Engineering Kluyveromyces marxianus for 3-hydroxypropionic acid production at elevated temperature from Jerusalem artichoke tubers and crude glycerol","authors":"Jiacheng Li , Zhongmei Hu , Yanjie Li , Hao Zha , Yujie Xie , Mingtao Zhao , Lili Ren , Biao Zhang","doi":"10.1016/j.synbio.2026.01.008","DOIUrl":"10.1016/j.synbio.2026.01.008","url":null,"abstract":"<div><div>This study engineered the thermotolerant yeast <em>Kluyveromyces marxianus</em> to produce 3-hydroxypropionic acid (3-HP), a key precursor for biodegradable plastics, via the malonyl-CoA pathway using non-food feedstocks. The 3-HP titer was further increased through deleting <em>Adh2A</em> and <em>Ach1</em>, which prevents the synthesis of byproducts ethanol and acetic acid. Using Jerusalem artichoke tuber powder, engineered strain produced 27.32 and 32.31 g/L of 3-HP at 37 °C and 42 °C through fed-batch fermentation. Metabolic reconstruction replaced the native FADH<sub>2</sub>-dependent glycerol pathway (GUT1/GUT2) with an NADH-generating GDH1/DAK1 pathway, significantly enhancing glycerol utilization and increasing intracellular NADH supply by 62 %. Overexpression of <em>Utr1</em> can further enhance the NADPH supply. Combined with heterologous expression of a codon-optimized, high-activity malonyl-CoA reductase (MCR) mutant (MCR<sup>N940V/K1106W/S1114R</sup>), the engineered strain achieved 3-HP titers of 33.15 g/L in fed-batch fermentation using pure glycerol at 42 °C. Crucially, it also produced 26.57 g/L 3-HP directly from crude glycerol at 42 °C. The thermotolerant fermentation at 42 °C, unprecedented for yeast-based 3-HP synthesis, reduces cooling water consumption by approximately 60 %, translating to an estimated annual CO<sub>2</sub> reduction of 27.1 tons per 1000-ton fermenter. This work establishes a cost-effective, industrially scalable bioprocess for valorizing Jerusalem artichoke tubers and crude glycerol into a key platform chemical for biodegradable plastics and green chemicals, leveraging the strain's substrate flexibility, process robustness, and significant environmental advantages.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"13 ","pages":"Pages 1-13"},"PeriodicalIF":4.4,"publicationDate":"2026-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146001775","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