Pub Date : 2026-01-02DOI: 10.1016/j.synbio.2025.12.003
Xiaochun Zheng , Yajun Li , Zhenhua Liu , Peng Sun , Yaqi Dong , Xue Wang , Yanan Wang , Xiaobing Yang
Patchoulol is a widely used sesquiterpenoid in perfumes, cosmetics, foods and pharmaceuticals. The plant-dependent production is suffering from limited growing area, long seasonal cycle, etc. Microbial production represents a sustainable alternative as it is featured with mild operating conditions and eco-friendliness. Herein, we engineered the oleaginous Rhodotorula toruloides toward patchoulol production. First, the patchoulol biosynthesis baseline was constructed by employing a chimeric enzyme of the Pogostemon cablin originated patchoulol synthase and the native FPPS. Second, the supply of essential intermediates was streamlined by redeploying the mevalonate (MVA) pathway while the recycling of NADPH was enhanced through over-expressing related enzymes. Third, the patchoulol production was further enhanced to 724.8 mg/L, 6.0 mg/L/h and 36.2 mg/g glucose by down-regulating the squalene biosynthesis and tuning the cultivation condition in shake flask. Finally, the production of patchoulol was increased to 1.31 g/L and 13.8 mg/g glucose in the minimal medium in a 3-L bioreactor. Our study demonstrated the potential of R. toruloides in producing patchoulol, and should shed light on the microbial synthesis of other sesquiterpenes.
{"title":"Biosynthesis of patchoulol via metabolic engineering the oleaginous red yeast Rhodotorula toruloides","authors":"Xiaochun Zheng , Yajun Li , Zhenhua Liu , Peng Sun , Yaqi Dong , Xue Wang , Yanan Wang , Xiaobing Yang","doi":"10.1016/j.synbio.2025.12.003","DOIUrl":"10.1016/j.synbio.2025.12.003","url":null,"abstract":"<div><div>Patchoulol is a widely used sesquiterpenoid in perfumes, cosmetics, foods and pharmaceuticals. The plant-dependent production is suffering from limited growing area, long seasonal cycle, etc. Microbial production represents a sustainable alternative as it is featured with mild operating conditions and eco-friendliness. Herein, we engineered the oleaginous <em>Rhodotorula toruloides</em> toward patchoulol production. First, the patchoulol biosynthesis baseline was constructed by employing a chimeric enzyme of the <em>Pogostemon cablin</em> originated patchoulol synthase and the native FPPS. Second, the supply of essential intermediates was streamlined by redeploying the mevalonate (MVA) pathway while the recycling of NADPH was enhanced through over-expressing related enzymes. Third, the patchoulol production was further enhanced to 724.8 mg/L, 6.0 mg/L/h and 36.2 mg/g glucose by down-regulating the squalene biosynthesis and tuning the cultivation condition in shake flask. Finally, the production of patchoulol was increased to 1.31 g/L and 13.8 mg/g glucose in the minimal medium in a 3-L bioreactor. Our study demonstrated the potential of <em>R. toruloides</em> in producing patchoulol, and should shed light on the microbial synthesis of other sesquiterpenes.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 293-300"},"PeriodicalIF":4.4,"publicationDate":"2026-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145883698","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 : 2026-01-02DOI: 10.1016/j.synbio.2025.12.004
Yucheng Hu , Jinde Chen , Shaofang Tian , Yang Zhang , Zhiqian Zhang , Ao Jiang , Yi-Rui Wu , Baoshun Zhang
Poor aqueous solubility of steroid precursors, such as pregnenolone and progesterone, limits microbial biotransformation and high-throughput strain screening, representing a bottleneck for strain improvement and potential industrial applications.
To address this, we developed a growth-coupled progesterone-responsive biosensor in Saccharomyces cerevisiae, integrated with a hydroxypropyl-β-cyclodextrin (HP-β-CD) system to enhance intracellular steroid availability. The biosensor links progesterone formation to cell growth and fluorescence, with selection stringency finely tuned via an IPTG-inducible lac operator and 3-aminotriazole (3-AT) to suppress low-producing cells. Coupled with atmospheric and room temperature plasma (ARTP) mutagenesis, the growth-coupled biosensor–FADS platform identified five yeast variants capable of improved conversion of pregnenolone to progesterone while expressing 3β-hydroxysteroid dehydrogenase (3β-HSD) without altering the enzyme itself. The progesterone production of these selected variants was subsequently validated using 1 mM pregnenolone as the substrate, showing 2.0–3.37-fold higher titers than the wild-type strain, demonstrating proof-of-concept. Microfluidic droplet encapsulation allowed clear separation of high-producers, highlighting the platform's selectivity, robustness, and scalability. This synthetic biology–driven system integration platform provides a practical, modular, and high-throughput strategy for screening poorly water-soluble steroid-producing yeast. It is adaptable to other bioactive molecules, can support future enzyme evolution, and demonstrates potential for broader biotechnological applications.
{"title":"A growth-coupled progesterone-responsive biosensor for high-throughput microfluidic screening in Saccharomyces cerevisiae","authors":"Yucheng Hu , Jinde Chen , Shaofang Tian , Yang Zhang , Zhiqian Zhang , Ao Jiang , Yi-Rui Wu , Baoshun Zhang","doi":"10.1016/j.synbio.2025.12.004","DOIUrl":"10.1016/j.synbio.2025.12.004","url":null,"abstract":"<div><div>Poor aqueous solubility of steroid precursors, such as pregnenolone and progesterone, limits microbial biotransformation and high-throughput strain screening, representing a bottleneck for strain improvement and potential industrial applications.</div><div>To address this, we developed a growth-coupled progesterone-responsive biosensor in <em>Saccharomyces cerevisiae</em>, integrated with a hydroxypropyl-β-cyclodextrin (HP-β-CD) system to enhance intracellular steroid availability. The biosensor links progesterone formation to cell growth and fluorescence, with selection stringency finely tuned via an IPTG-inducible lac operator and 3-aminotriazole (3-AT) to suppress low-producing cells. Coupled with atmospheric and room temperature plasma (ARTP) mutagenesis, the growth-coupled biosensor–FADS platform identified five yeast variants capable of improved conversion of pregnenolone to progesterone while expressing 3β-hydroxysteroid dehydrogenase (3β-HSD) without altering the enzyme itself. The progesterone production of these selected variants was subsequently validated using 1 mM pregnenolone as the substrate, showing 2.0–3.37-fold higher titers than the wild-type strain, demonstrating proof-of-concept. Microfluidic droplet encapsulation allowed clear separation of high-producers, highlighting the platform's selectivity, robustness, and scalability. This synthetic biology–driven system integration platform provides a practical, modular, and high-throughput strategy for screening poorly water-soluble steroid-producing yeast. It is adaptable to other bioactive molecules, can support future enzyme evolution, and demonstrates potential for broader biotechnological applications.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 301-311"},"PeriodicalIF":4.4,"publicationDate":"2026-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145883696","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 : 2025-12-31DOI: 10.1016/j.synbio.2025.12.012
Chenglong Zhang , Jia Wang , Longfei Zhao , Nan Wu , Yi Shi , Xia Li , Changqiang Ke , Jia Liu , Yang Ye , Ying Wang , Bingzhi Li , Wenhai Xiao , Mingdong Yao , Yingjin Yuan
Utilizing synthetic biology techniques to construct recombinant engineered cells for the synthesis of paclitaxel and its key precursors has emerged as an effective method to address the supply–demand imbalance and protect rare plant resources. Taxadiene, a critical precursor of paclitaxel, exhibits limited yield in eukaryotic systems, constraining its biosynthetic potential. Previous research has demonstrated that glycosylation modifications in Saccharomyces cerevisiae substantially impact the regulation of exogenous protein expression. In this study, we found that knocking out endogenous protein glycosylation genes in the chassis significantly improved the expression of heterologous proteins, especially the key taxadiene synthase (TS), and thereby increased the yield of taxadiene. In particular, we identified that the deletion of the glycosyltransferase gene mnn11 increased taxadiene production levels by 65.2 %. The subsequent multi-copy integration of the key enzyme taxadiene synthase further elevated taxadiene production levels in shake flasks by more than 3-fold, reaching 113.5 mg/L. Moreover, the enhancement of geranylgeranyl diphosphate synthesis-related expression modules resulted in a 2.69-fold increase in taxadiene yield, to 420.4 mg/L. Following the optimization of fed-batch fermentation conditions, taxadiene production levels of up to 1.26 g/L were achieved, representing a 63-fold improvement over that obtained with the initial strain. Our findings provide valuable insights into enhancing heterologous taxadiene production efficiency by blocking endogenous protein glycosylation modifications in S. cerevisiae, establishing a critical precedent for improving compatibility between natural product synthesis and microbial cell factories.
{"title":"Regulating protein glycosylation modification enhances the synthesis of taxadiene in Saccharomyces cerevisiae","authors":"Chenglong Zhang , Jia Wang , Longfei Zhao , Nan Wu , Yi Shi , Xia Li , Changqiang Ke , Jia Liu , Yang Ye , Ying Wang , Bingzhi Li , Wenhai Xiao , Mingdong Yao , Yingjin Yuan","doi":"10.1016/j.synbio.2025.12.012","DOIUrl":"10.1016/j.synbio.2025.12.012","url":null,"abstract":"<div><div>Utilizing synthetic biology techniques to construct recombinant engineered cells for the synthesis of paclitaxel and its key precursors has emerged as an effective method to address the supply–demand imbalance and protect rare plant resources. Taxadiene, a critical precursor of paclitaxel, exhibits limited yield in eukaryotic systems, constraining its biosynthetic potential. Previous research has demonstrated that glycosylation modifications in <em>Saccharomyces cerevisiae</em> substantially impact the regulation of exogenous protein expression. In this study, we found that knocking out endogenous protein glycosylation genes in the chassis significantly improved the expression of heterologous proteins, especially the key taxadiene synthase (TS), and thereby increased the yield of taxadiene. In particular, we identified that the deletion of the glycosyltransferase gene <em>mnn11</em> increased taxadiene production levels by 65.2 %. The subsequent multi-copy integration of the key enzyme taxadiene synthase further elevated taxadiene production levels in shake flasks by more than 3-fold, reaching 113.5 mg/L. Moreover, the enhancement of geranylgeranyl diphosphate synthesis-related expression modules resulted in a 2.69-fold increase in taxadiene yield, to 420.4 mg/L. Following the optimization of fed-batch fermentation conditions, taxadiene production levels of up to 1.26 g/L were achieved, representing a 63-fold improvement over that obtained with the initial strain. Our findings provide valuable insights into enhancing heterologous taxadiene production efficiency by blocking endogenous protein glycosylation modifications in <em>S. cerevisiae</em>, establishing a critical precedent for improving compatibility between natural product synthesis and microbial cell factories.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 284-292"},"PeriodicalIF":4.4,"publicationDate":"2025-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145883633","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 : 2025-12-31DOI: 10.1016/j.synbio.2025.12.013
Huixue Chen , Yan Gao , Shixue Jin , Qian Yun , Xinchen Ruan , Xudong Qu , Chun Lei
The azinothricin family of hybrid hexadepsipeptide-polyketide natural products exhibit remarkable bioactivities, including potent antibacterial, antitumor, antimalarial and anti-inflammatory activities. However, only a few azinothricin-type natural products are currently known, and the biosynthetic potential of microbes remains underexplored. In this work, 137 candidate biosynthetic gene clusters (BGCs) were identified using a genome-mining strategy based on cblaster homology screening. Furthermore, Streptomyces durmitorensis DSM 41863 was prioritized for in-depth experiment due to its unique PKS module expansion, leading to the discovery of kettapeptin, the azinothricin-type metabolite isolated from this species for the first time. Additionally, a putative biosynthetic pathway for kettapeptin was proposed. This work expands the azinothricin-type BGC landscape and establishes S. durmitorensis DSM 41863 as a genetically tractable platform for bioengineering novel derivatives.
{"title":"Molecular landscape analysis of azinothricin-type natural products enables the identification of kettapeptin from Streptomyces durmitorensis","authors":"Huixue Chen , Yan Gao , Shixue Jin , Qian Yun , Xinchen Ruan , Xudong Qu , Chun Lei","doi":"10.1016/j.synbio.2025.12.013","DOIUrl":"10.1016/j.synbio.2025.12.013","url":null,"abstract":"<div><div>The azinothricin family of hybrid hexadepsipeptide-polyketide natural products exhibit remarkable bioactivities, including potent antibacterial, antitumor, antimalarial and anti-inflammatory activities. However, only a few azinothricin-type natural products are currently known, and the biosynthetic potential of microbes remains underexplored. In this work, 137 candidate biosynthetic gene clusters (BGCs) were identified using a genome-mining strategy based on cblaster homology screening. Furthermore, <em>Streptomyces durmitorensis</em> DSM 41863 was prioritized for in-depth experiment due to its unique PKS module expansion, leading to the discovery of kettapeptin, the azinothricin-type metabolite isolated from this species for the first time. Additionally, a putative biosynthetic pathway for kettapeptin was proposed. This work expands the azinothricin-type BGC landscape and establishes <em>S</em>. <em>durmitorensis</em> DSM 41863 as a genetically tractable platform for bioengineering novel derivatives.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 265-273"},"PeriodicalIF":4.4,"publicationDate":"2025-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145883631","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 : 2025-12-31DOI: 10.1016/j.synbio.2025.12.006
Yunyue Chen , Siyifei Wang , Leiying Xie , Luhao Zhang , Min Zhu , Yingke Xu
Precise regulation of protein abundance is essential for understanding dynamic cellular processes and for advancing therapeutic development. However, existing approaches lack the spatiotemporal resolution required to these cellular processes. Recent advances in optogenetics have enabled the design of optogenetic targeted protein degradation systems (Opto-TPD) allowing reversible and non-invasive control of protein stability with high spatiotemporal precision. In this review, we systematically summarize the design principles of Opto-TPD tools, including those based on light-oxygen-voltage (LOV)-domain conformational systems, light-inducible dimerization systems, and light-controlled degradation tool expression systems. We further highlight their applications in probing protein function, modulating signaling pathways, and therapeutic translations. By comparing the mechanistic features, performance, and limitations of each platform, we aim to provide a comprehensive resource for guiding future tool optimization. Altogether, these Opto-TPD tools represent a powerful and versatile complement to existing protein manipulation technologies, expanding the toolbox for precise control of protein homeostasis in living systems.
{"title":"Design principles for optogenetic-based targeted protein degradation","authors":"Yunyue Chen , Siyifei Wang , Leiying Xie , Luhao Zhang , Min Zhu , Yingke Xu","doi":"10.1016/j.synbio.2025.12.006","DOIUrl":"10.1016/j.synbio.2025.12.006","url":null,"abstract":"<div><div>Precise regulation of protein abundance is essential for understanding dynamic cellular processes and for advancing therapeutic development. However, existing approaches lack the spatiotemporal resolution required to these cellular processes. Recent advances in optogenetics have enabled the design of optogenetic targeted protein degradation systems (Opto-TPD) allowing reversible and non-invasive control of protein stability with high spatiotemporal precision. In this review, we systematically summarize the design principles of Opto-TPD tools, including those based on light-oxygen-voltage (LOV)-domain conformational systems, light-inducible dimerization systems, and light-controlled degradation tool expression systems. We further highlight their applications in probing protein function, modulating signaling pathways, and therapeutic translations. By comparing the mechanistic features, performance, and limitations of each platform, we aim to provide a comprehensive resource for guiding future tool optimization. Altogether, these Opto-TPD tools represent a powerful and versatile complement to existing protein manipulation technologies, expanding the toolbox for precise control of protein homeostasis in living systems.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 255-264"},"PeriodicalIF":4.4,"publicationDate":"2025-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145883629","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 : 2025-12-31DOI: 10.1016/j.synbio.2025.12.002
Xiao-Nan Hou , Dan Shu , Tian-Fu Li , Qi Yang , Zhe-Min Li , Di Luo , Jie Yang , Zhi-Ying Yan , Hong Tan
Compared with conventional microbial hosts, filamentous fungi have distinct advantages for the industrial-scale biosynthesis of high-value chemical compounds. However, current research on strain engineering and fermentation optimization strategies for synthetic biology applications is limited in filamentous fungi, especially in industrial production strains. In this study, we established a CRISPR/Cas9-based gene editing system in Botrytis cinerea strain TB-31, an important filamentous fungal platform for the study of the biosynthesis and regulation of the sesquiterpenoid abscisic acid (ABA). This system enables efficient single- and multigene knockout, large-fragment deletion, and heterologous protein expression. Among the engineered mutant strains, the △bcaba1234 strain with complete ablation of the ABA biosynthetic gene cluster (BGC) demonstrated significant metabolic flux rewiring, redirecting cellular resources toward terpenoid precursor biosynthesis; this metabolic reprogramming proves pivotal for high-value terpenoid biosynthesis. This study not only establishes an efficient genome editing tool for the ABA-producing strain B. cinerea TB-31 but also provides a foundation for its development as a new potential terpenoid-producing chassis strain.
{"title":"Metabolic reprogramming of abscisic acid-producing strain Botrytis cinerea TB-31 toward terpenoid biosynthesis using a CRISPR/Cas9 ribonucleoprotein system","authors":"Xiao-Nan Hou , Dan Shu , Tian-Fu Li , Qi Yang , Zhe-Min Li , Di Luo , Jie Yang , Zhi-Ying Yan , Hong Tan","doi":"10.1016/j.synbio.2025.12.002","DOIUrl":"10.1016/j.synbio.2025.12.002","url":null,"abstract":"<div><div>Compared with conventional microbial hosts, filamentous fungi have distinct advantages for the industrial-scale biosynthesis of high-value chemical compounds. However, current research on strain engineering and fermentation optimization strategies for synthetic biology applications is limited in filamentous fungi, especially in industrial production strains. In this study, we established a CRISPR/Cas9-based gene editing system in <em>Botrytis cinerea</em> strain TB-31, an important filamentous fungal platform for the study of the biosynthesis and regulation of the sesquiterpenoid abscisic acid (ABA). This system enables efficient single- and multigene knockout, large-fragment deletion, and heterologous protein expression. Among the engineered mutant strains, the △<em>bcaba1234</em> strain with complete ablation of the ABA biosynthetic gene cluster (BGC) demonstrated significant metabolic flux rewiring, redirecting cellular resources toward terpenoid precursor biosynthesis; this metabolic reprogramming proves pivotal for high-value terpenoid biosynthesis. This study not only establishes an efficient genome editing tool for the ABA-producing strain <em>B. cinerea</em> TB-31 but also provides a foundation for its development as a new potential terpenoid-producing chassis strain.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 238-254"},"PeriodicalIF":4.4,"publicationDate":"2025-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145883630","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 : 2025-12-31DOI: 10.1016/j.synbio.2025.12.011
Wei Li , Di Liu , Linbo Gou , Shengfang Wu , Xiuwen Zhou , Tai-Ping Fan , Long Wang , Yujie Cai
(-)-α-Bisabolol, a valuable monocyclic sesquiterpene alcohol, has garnered significant attention in the pharmaceutical and cosmetic industries due to its remarkable anti-inflammatory, antibacterial, and skin-care properties. In this study, Serratia marcescens HBQA7 (S. marcescens HBQA7), a non-model strain resistant to terpenoid toxicity, was used as the production host, and the expression intensities of different integration sites were screened. The complete (-)-α-bisabolol synthesis pathway was integrated into these sites, achieving a production titer of 3.5 g L−1. On this basis, by knocking out competitive pathway genes (such as slaB and adhE) and global regulatory factors (arcA and iclR), and introducing efficient glucose transport and activation (glf and glk), the shake flask fermentation titer was increased to 7.21 g L−1. Through optimization of fermentation culture by orthogonal experiments and others, the titer was further increased to 9.90 g L−1. Finally, through the fed-batch fermentation process conducted in a 50 L bioreactor, a titer of 102.3 g L−1 was achieved after 110 h of cultivation. The productivity reached 0.93 g L−1 h−1. This study not only establishes the most efficient microbial production system for (-)-α-bisabolol reported to date, but also demonstrates the outstanding potential of S. marcescens as a chassis for terpenoid biosynthesis. It provides a novel strategy for the industrial production of high-value terpenoids.
（-）-α-比abolol是一种有价值的单环倍半萜醇，由于其显著的抗炎、抗菌和护肤特性，在制药和化妆品行业引起了极大的关注。本研究以抗萜类毒性的非模式菌株粘质Serratia marcescens HBQA7 （S. marcescens HBQA7）为生产宿主，筛选不同整合位点的表达强度。完整的(-)-α-双abolol合成途径被整合到这些位点，生产滴度达到3.5 g L−1。在此基础上，通过敲除竞争通路基因（如slaB和adhE）和全局调控因子（arcA和iclR），引入高效葡萄糖运输和激活（glf和glk），摇瓶发酵滴度提高到7.21 g L−1。通过正交试验等优化发酵培养，进一步将滴度提高到9.90 g L−1。最后，在50 L的生物反应器中进行补料分批发酵，培养110 h后滴度达到102.3 g L−1。产率达到0.93 g L−1 h−1。本研究不仅建立了迄今为止报道的最有效的(-)-α-双abolol微生物生产系统，而且还证明了S. marcescens作为萜类生物合成基质的巨大潜力。为高值萜类化合物的工业化生产提供了新的思路。
{"title":"Antibiotic-free high-yield (-)-α-bisabolol production in Serratia marcescens via metabolic engineering and genomic integration of mevalonate pathway genes","authors":"Wei Li , Di Liu , Linbo Gou , Shengfang Wu , Xiuwen Zhou , Tai-Ping Fan , Long Wang , Yujie Cai","doi":"10.1016/j.synbio.2025.12.011","DOIUrl":"10.1016/j.synbio.2025.12.011","url":null,"abstract":"<div><div>(-)-α-Bisabolol, a valuable monocyclic sesquiterpene alcohol, has garnered significant attention in the pharmaceutical and cosmetic industries due to its remarkable anti-inflammatory, antibacterial, and skin-care properties. In this study, <em>Serratia marcescens</em> HBQA7 (<em>S. marcescens</em> HBQA7), a non-model strain resistant to terpenoid toxicity, was used as the production host, and the expression intensities of different integration sites were screened. The complete (-)-α-bisabolol synthesis pathway was integrated into these sites, achieving a production titer of 3.5 g L<sup>−1</sup>. On this basis, by knocking out competitive pathway genes (such as <em>slaB</em> and <em>adhE</em>) and global regulatory factors (<em>arcA</em> and <em>iclR</em>), and introducing efficient glucose transport and activation (<em>glf</em> and <em>glk</em>), the shake flask fermentation titer was increased to 7.21 g L<sup>−1</sup>. Through optimization of fermentation culture by orthogonal experiments and others, the titer was further increased to 9.90 g L<sup>−1</sup>. Finally, through the fed-batch fermentation process conducted in a 50 L bioreactor, a titer of 102.3 g L<sup>−1</sup> was achieved after 110 h of cultivation. The productivity reached 0.93 g L<sup>−1</sup> h<sup>−1</sup>. This study not only establishes the most efficient microbial production system for (-)-α-bisabolol reported to date, but also demonstrates the outstanding potential of <em>S. marcescens</em> as a chassis for terpenoid biosynthesis. It provides a novel strategy for the industrial production of high-value terpenoids.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 274-283"},"PeriodicalIF":4.4,"publicationDate":"2025-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145883632","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 : 2025-12-30DOI: 10.1016/j.synbio.2025.12.010
Ruizhou Tang , Jiahuan Li , Xiaole Yang , Xia Tian , Zehui Wang , Xuning Zhang , Tingting Li
Aminotransferases are promising green biocatalysts for the synthesis of chiral amines, yet their limited catalytic efficiencies restrict broader industrial applications. In this study, a novel (R)-amine transaminase, FalAT, was identified from Fusarium albosuccineum through genome mining. FalAT exhibited optimal activity at 30 °C and pH 7.0 and catalyzed the conversion of 1-Boc-3-piperidone to (R)-1-Boc-3-aminopiperidine with >99 % enantiomeric excess. To efficiently enhance its catalytic performance, a Substrate–Protein Interaction Network (SPIN) strategy was implemented, integrating structure-guided analysis, molecular docking, virtual saturation mutagenesis, and dual energy–distance filtering. SPIN first screened and constructed a mutational library covering 70 amino acid positions, which was subsequently narrowed to 9 key residues, ultimately yielding 15 candidate mutants for experimental validation. Experimental results showed that five mutants exhibited higher catalytic activity than the wild-type enzyme, among which R126A was the most effective, displaying approximately a 4-fold increase in catalytic activity and a 13-fold enhancement in catalytic efficiency (kcat/Km = 2.05 s−1 mM−1). Molecular dynamics simulations revealed that the R126A mutation expanded the active-site cavity, alleviated steric hindrance, and strengthened hydrophobic interactions, thereby improving substrate binding and catalytic turnover. Furthermore, substrate profiling demonstrated that FalAT possesses moderate substrate promiscuity. Overall, the SPIN strategy significantly improved the catalytic performance of FalAT while markedly reducing experimental workload, providing an efficient and generalizable approach for the directed evolution of (R)-amine transaminases for the green synthesis of chiral amines.
{"title":"SPIN-guided engineering of a novel (R)-amine transaminase from Fusarium albosuccineum for enantioselective synthesis of chiral piperidyl amines","authors":"Ruizhou Tang , Jiahuan Li , Xiaole Yang , Xia Tian , Zehui Wang , Xuning Zhang , Tingting Li","doi":"10.1016/j.synbio.2025.12.010","DOIUrl":"10.1016/j.synbio.2025.12.010","url":null,"abstract":"<div><div>Aminotransferases are promising green biocatalysts for the synthesis of chiral amines, yet their limited catalytic efficiencies restrict broader industrial applications. In this study, a novel (<em>R</em>)-amine transaminase, FalAT, was identified from <em>Fusarium albosuccineum</em> through genome mining. FalAT exhibited optimal activity at 30 °C and pH 7.0 and catalyzed the conversion of 1-Boc-3-piperidone to (<em>R</em>)-1-Boc-3-aminopiperidine with >99 % enantiomeric excess. To efficiently enhance its catalytic performance, a Substrate–Protein Interaction Network (SPIN) strategy was implemented, integrating structure-guided analysis, molecular docking, virtual saturation mutagenesis, and dual energy–distance filtering. SPIN first screened and constructed a mutational library covering 70 amino acid positions, which was subsequently narrowed to 9 key residues, ultimately yielding 15 candidate mutants for experimental validation. Experimental results showed that five mutants exhibited higher catalytic activity than the wild-type enzyme, among which R126A was the most effective, displaying approximately a 4-fold increase in catalytic activity and a 13-fold enhancement in catalytic efficiency (<em>k</em><sub><em>ca</em></sub><em><sub>t</sub>/K</em><sub><em>m</em></sub> = 2.05 s<sup>−1</sup> mM<sup>−1</sup>). Molecular dynamics simulations revealed that the R126A mutation expanded the active-site cavity, alleviated steric hindrance, and strengthened hydrophobic interactions, thereby improving substrate binding and catalytic turnover. Furthermore, substrate profiling demonstrated that FalAT possesses moderate substrate promiscuity. Overall, the SPIN strategy significantly improved the catalytic performance of FalAT while markedly reducing experimental workload, providing an efficient and generalizable approach for the directed evolution of (<em>R</em>)-amine transaminases for the green synthesis of chiral amines.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"12 ","pages":"Pages 229-237"},"PeriodicalIF":4.4,"publicationDate":"2025-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145883628","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 : 2025-12-29DOI: 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}
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