Synthetic and Systems Biotechnology最新文献_第7页

Engineering mixed sugar metabolic channels in Pseudomonas putida to produce vanillic acid 改造恶臭假单胞菌混合糖代谢通道生产香草酸

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-10-09 DOI: 10.1016/j.synbio.2025.10.002

Peihan Wu , Haifeng Ding , Zhiqing Xu , Yan Zhang , Yanyan Dang , Bo Gao , Genlin Zhang

Hydrolysis of lignocellulosic biomass produces the mixed sugars of glucose (60–70 %), xylose (20–30 %), and arabinose (2–20 %), etc. Using mixed sugars instead of pure glucose for microbial biosynthesis will reduce the cost of carbon source and maximize utilization of biomass. However, carbon catabolite repression and poor adaptation of metabolic pathways are obstacles in the synergistic utilization of mixed sugars, thus resulting in low carbon utilization efficiency. Here, we engineered the mixed sugar metabolic channels in Pseudomonas putida to achieve their synergistic utilization for producing vanillic acid, a valuable aromatic compound with broad applications in the food, pharmaceuticals, cosmetics, and chemical industries. Expressing O-methyltransferase (OMT) and deleting vanillate-O-demethylase (vanAB) realized vanillic acid accumulation in P. putida from glucose. Introducing the xylose isomerase pathway enabled the strain to produce vanillic acid from xylose. Deleting glucose dehydrogenase (gcd) and transcriptional regulator (hexR), together expressing two critical pentose phosphate pathway enzymes (transketolase and transaldolase) effectively balanced glucose-xylose metabolic channels for vanillic acid production. Further assembling the arabinose oxidation pathway established the efficient metabolism of three sugars. The final engineered strain (VA12) produced 2.75 g/L vanillic acid in fed-batch fermentation with 20 g/L glucose, 10 g/L xylose and 10 g/L arabinose. This study effectively reduces carbon catabolite repression in the synergistic utilization of mixed sugars, and represents the first case utilizing glucose-xylose-arabinose for vanillic acid production, illustrating the capability of transforming lignocellulose hydrolyzate into valuable chemical products.

木质纤维素生物质水解产生葡萄糖（60 - 70%）、木糖（20 - 30%）和阿拉伯糖（2 - 20%）等混合糖。用混合糖代替纯葡萄糖进行微生物合成，可以降低碳源成本，最大限度地利用生物质。然而，碳分解代谢抑制和代谢途径适应性差阻碍了混合糖的协同利用，从而导致碳利用效率低。在此，我们设计了恶臭假单胞菌的混合糖代谢通道，以实现它们的协同利用，以生产香草酸，这是一种有价值的芳香化合物，在食品，制药，化妆品和化学工业中有着广泛的应用。表达o -甲基转移酶（OMT）和删除香草- o -去甲基化酶（vanAB）实现了甘草酸在恶臭p.p . putida中从葡萄糖积累。木糖异构酶途径的引入使该菌株能够以木糖为原料生产香草酸。删除葡萄糖脱氢酶（gcd）和转录调节酶（hexR），共同表达两个关键的戊糖磷酸途径酶（转酮醇酶和转醛醇酶），有效地平衡了葡萄糖-木糖代谢通道，以产生香草酸。进一步组装阿拉伯糖氧化途径建立了三种糖的有效代谢。最终工程菌株VA12以20 g/L葡萄糖、10 g/L木糖和10 g/L阿拉伯糖为补料分批发酵，产生2.75 g/L香草酸。本研究有效降低了混合糖协同利用中的碳分解代谢抑制，并首次利用葡萄糖-木糖-阿拉伯糖生产香草酸，说明了将木质纤维素水解产物转化为有价值的化学产品的能力。

{"title":"Engineering mixed sugar metabolic channels in Pseudomonas putida to produce vanillic acid","authors":"Peihan Wu , Haifeng Ding , Zhiqing Xu , Yan Zhang , Yanyan Dang , Bo Gao , Genlin Zhang","doi":"10.1016/j.synbio.2025.10.002","DOIUrl":"10.1016/j.synbio.2025.10.002","url":null,"abstract":"<div><div>Hydrolysis of lignocellulosic biomass produces the mixed sugars of glucose (60–70 %), xylose (20–30 %), and arabinose (2–20 %), etc. Using mixed sugars instead of pure glucose for microbial biosynthesis will reduce the cost of carbon source and maximize utilization of biomass. However, carbon catabolite repression and poor adaptation of metabolic pathways are obstacles in the synergistic utilization of mixed sugars, thus resulting in low carbon utilization efficiency. Here, we engineered the mixed sugar metabolic channels in <em>Pseudomonas putida</em> to achieve their synergistic utilization for producing vanillic acid, a valuable aromatic compound with broad applications in the food, pharmaceuticals, cosmetics, and chemical industries. Expressing O-methyltransferase (<em>OMT</em>) and deleting vanillate-O-demethylase (<em>vanAB</em>) realized vanillic acid accumulation in <em>P. putida</em> from glucose. Introducing the xylose isomerase pathway enabled the strain to produce vanillic acid from xylose. Deleting glucose dehydrogenase (<em>gcd</em>) and transcriptional regulator (<em>hexR</em>), together expressing two critical pentose phosphate pathway enzymes (transketolase and transaldolase) effectively balanced glucose-xylose metabolic channels for vanillic acid production. Further assembling the arabinose oxidation pathway established the efficient metabolism of three sugars. The final engineered strain (VA12) produced 2.75 g/L vanillic acid in fed-batch fermentation with 20 g/L glucose, 10 g/L xylose and 10 g/L arabinose. This study effectively reduces carbon catabolite repression in the synergistic utilization of mixed sugars, and represents the first case utilizing glucose-xylose-arabinose for vanillic acid production, illustrating the capability of transforming lignocellulose hydrolyzate into valuable chemical products.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 277-286"},"PeriodicalIF":4.4,"publicationDate":"2025-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145325259","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

CRISPR-based synthetic biology toolkit development in Candida viswanthii and functional analysis of the stress responsive Ena1-like protein 基于crispr的viswanthii假丝酵母合成生物学工具包开发及应激反应性ena1样蛋白的功能分析

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-10-01 DOI: 10.1016/j.synbio.2025.09.021

Xin-Yue Li , Kai Li , Feng-Li Zhang , Tomohisa Hasunuma , Akihiko Kondo , Lin Zhang , Xin-Qing Zhao , Feng-Wu Bai

Industrial strains from the Candida genus have been applied in production of enzymes, biochemicals, and single-cell protein. However, the synthetic biology manipulation tools for Candida species remain underdeveloped. In this study, a high-efficiency genome editing strategy for C. viswanathii was established by combining the CRISPR/Cas9 and Cre/loxP systems. This approach achieved 100 % editing efficiency and supported rapid iterative editing cycles within 6 days. The system enables iterative genomic modifications and was successfully applied for multiplex editing and multicopy gene integration up to 7 copies. Leveraging this platform, g144, an Ena1-like protein that exhibited differential expression during dodecanedioic acid (DDA) fermentation, was functionally characterized. The results showed that g144 lacks Na⁺ transport activity, but both the disruption and overexpression strains showed increased sensitivity to alkaline pH and Na⁺ stress, as well as a decrease in DDA production. The genome editing toolkit reported here benefits further applications of Candida strains for sustainable bioproduction.

念珠菌属的工业菌株已被应用于酶、生化和单细胞蛋白的生产。然而，念珠菌的合成生物学操作工具仍然不发达。本研究通过结合CRISPR/Cas9和Cre/loxP系统，建立了一种高效的viswanathii基因组编辑策略。这种方法实现了100%的编辑效率，并支持6天内快速迭代的编辑周期。该系统可实现迭代基因组修饰，并成功应用于最多7个拷贝的多重编辑和多拷贝基因整合。利用这一平台，g144（一种在十二烷二酸（DDA）发酵过程中表现出差异表达的ena1样蛋白）进行了功能表征。结果表明，g144缺乏Na+转运活性，但裂解菌株和过表达菌株对碱性pH和Na+胁迫的敏感性增加，DDA产量下降。本文报道的基因组编辑工具包有利于念珠菌菌株在可持续生物生产中的进一步应用。

{"title":"CRISPR-based synthetic biology toolkit development in Candida viswanthii and functional analysis of the stress responsive Ena1-like protein","authors":"Xin-Yue Li , Kai Li , Feng-Li Zhang , Tomohisa Hasunuma , Akihiko Kondo , Lin Zhang , Xin-Qing Zhao , Feng-Wu Bai","doi":"10.1016/j.synbio.2025.09.021","DOIUrl":"10.1016/j.synbio.2025.09.021","url":null,"abstract":"<div><div>Industrial strains from the <em>Candida</em> genus have been applied in production of enzymes, biochemicals, and single-cell protein. However, the synthetic biology manipulation tools for <em>Candida</em> species remain underdeveloped. In this study, a high-efficiency genome editing strategy for <em>C. viswanathii</em> was established by combining the CRISPR/Cas9 and Cre/loxP systems. This approach achieved 100 % editing efficiency and supported rapid iterative editing cycles within 6 days. The system enables iterative genomic modifications and was successfully applied for multiplex editing and multicopy gene integration up to 7 copies. Leveraging this platform, g144, an Ena1-like protein that exhibited differential expression during dodecanedioic acid (DDA) fermentation, was functionally characterized. The results showed that g144 lacks Na<sup>+</sup> transport activity, but both the disruption and overexpression strains showed increased sensitivity to alkaline pH and Na<sup>+</sup> stress, as well as a decrease in DDA production. The genome editing toolkit reported here benefits further applications of <em>Candida</em> strains for sustainable bioproduction.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 298-308"},"PeriodicalIF":4.4,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145362638","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

An efficient CRISPR-Cas12a tool for iterative genome editing, streamlined minimization, and payload engineering of Pseudomonas phage S1 一种高效的CRISPR-Cas12a工具，用于假单胞菌噬菌体S1的迭代基因组编辑、流线型最小化和有效载荷工程

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-29 DOI: 10.1016/j.synbio.2025.09.019

Yanmei Liu, Zizhen Liang, Yanyun Jing , Yanrui Ye, Xiaofeng Yang, Zhanglin Lin

The ability to precisely engineer phage genomes is critical for advancing phage-based biotechnology and therapeutic development. Here, we present a high-efficiency, scarless CRISPR-Cas12a genome editing method for a virulent Pseudomonas aeruginosa phage vB_PaeM_SCUT-S1 (S1). Using a two-plasmid system, we achieved near-complete efficiency in gene deletion, point mutation, gene insertion, and replacement. Iterative deletions enabled the identification of 27 non-essential, 16 quasi-essential, and seven essential genes, and culminated in the largest genome reduction (13.9 kb) reported for a P. aeruginosa phage. The minimized phage mutant S1_200L retained infectivity and supported integration of large exogenous gene cassettes (e.g., lacZ, lys009). This system offers a versatile and high-throughput approach for phage genome engineering and rational design of functional phages for synthetic biology and antimicrobial applications.

精确设计噬菌体基因组的能力对于推进基于噬菌体的生物技术和治疗发展至关重要。在这里，我们提出了一种高效、无疤痕的CRISPR-Cas12a基因组编辑方法，用于毒力强的铜绿假单胞菌噬菌体vB_PaeM_SCUT-S1 （S1）。使用双质粒系统，我们在基因缺失、点突变、基因插入和替换方面实现了近乎完全的效率。迭代删除能够鉴定27个非必需基因，16个准必需基因和7个必需基因，并最终在铜绿假单胞菌噬菌体中报道了最大的基因组减少（13.9 kb）。最小化的噬菌体突变体S1_200L保留了传染性，并支持大型外源基因盒的整合（例如lacZ， lys009）。该系统为噬菌体基因组工程和合理设计功能噬菌体提供了一种多功能和高通量的方法，用于合成生物学和抗菌应用。

引用次数: 0

Integrated transcriptomic and metabolomic analyses uncover novel genetic targets for enhanced l-tryptophan production in Corynebacterium glutamicum 整合转录组学和代谢组学分析揭示了谷氨酸棒状杆菌增强l-色氨酸生产的新遗传靶点

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-29 DOI: 10.1016/j.synbio.2025.09.020

Yufei Dong , Rongsheng Gao , Nan Qin , Kunyu Liu , Youmeng Liu , Zhen Chen

Corynebacterium glutamicum is a promising microbial chassis for the industrial production of l-tryptophan, an essential amino acid with diverse applications and high market value. In our previous work, we constructed an l-tryptophan-overproducing C. glutamicum strain, TR26, through multiple rounds of rational metabolic engineering. Here, comparative transcriptomic and metabolomic analyses were conducted between TR26 and its progenitor MB001 to elucidate the underlying molecular mechanisms and identify potential bottlenecks for l-tryptophan production in TR26. Based on the differentially expressed genes identified, systematic gene overexpression and knockdown experiments led to the identification of two novel genetic targets, glnK and sugR. Specifically, the repression of glnK and overexpression of sugR in TR26 increased the l-tryptophan titer by 10.3 % and 16.5 % in fed-batch fermentation, and the yield by 7.2 % and 20.2 %, respectively. Further transcription profiling and intracellular metabolite analysis indicated that these improvements were associated with altered nitrogen metabolism, more efficient allocation of cellular resources, and enhanced supply of phosphoenolpyruvate (PEP), a key precursor in aromatic amino acid biosynthesis. This study expands our understanding of the regulation mechanisms governing l-tryptophan synthesis in C. glutamicum and provides valuable insights for further optimization of industrial cell factories.

l-色氨酸是一种用途广泛、具有较高市场价值的必需氨基酸，谷氨酸棒状杆菌是工业生产l-色氨酸的理想微生物基质。在之前的工作中，我们通过多轮合理的代谢工程，构建了过量生产l-色氨酸的谷氨酸C.菌株TR26。本研究对TR26及其祖基因MB001进行了转录组学和代谢组学比较分析，以阐明TR26产生l-色氨酸的潜在分子机制，并确定TR26产生l-色氨酸的潜在瓶颈。在鉴定出差异表达基因的基础上，通过系统的基因过表达和敲低实验，鉴定出两个新的基因靶点glnK和sugR。具体来说，抑制glnK和TR26过表达糖r可使l-色氨酸滴度提高10.3%和16.5%，产量分别提高7.2%和20.2%。进一步的转录分析和细胞内代谢物分析表明，这些改善与氮代谢的改变、细胞资源的更有效分配和磷酸烯醇丙酮酸（PEP）的供应增加有关，PEP是芳香氨基酸生物合成的关键前体。本研究扩展了我们对谷氨酸酵母l-色氨酸合成调控机制的理解，并为进一步优化工业细胞工厂提供了有价值的见解。

{"title":"Integrated transcriptomic and metabolomic analyses uncover novel genetic targets for enhanced l-tryptophan production in Corynebacterium glutamicum","authors":"Yufei Dong , Rongsheng Gao , Nan Qin , Kunyu Liu , Youmeng Liu , Zhen Chen","doi":"10.1016/j.synbio.2025.09.020","DOIUrl":"10.1016/j.synbio.2025.09.020","url":null,"abstract":"<div><div><em>Corynebacterium glutamicum</em> is a promising microbial chassis for the industrial production of <span>l</span>-tryptophan, an essential amino acid with diverse applications and high market value. In our previous work, we constructed an <span>l</span>-tryptophan-overproducing <em>C. glutamicum</em> strain, TR26, through multiple rounds of rational metabolic engineering. Here, comparative transcriptomic and metabolomic analyses were conducted between TR26 and its progenitor MB001 to elucidate the underlying molecular mechanisms and identify potential bottlenecks for <span>l</span>-tryptophan production in TR26. Based on the differentially expressed genes identified, systematic gene overexpression and knockdown experiments led to the identification of two novel genetic targets, <em>glnK</em> and <em>sugR</em>. Specifically, the repression of <em>glnK</em> and overexpression of <em>sugR</em> in TR26 increased the <span>l</span>-tryptophan titer by 10.3 % and 16.5 % in fed-batch fermentation, and the yield by 7.2 % and 20.2 %, respectively. Further transcription profiling and intracellular metabolite analysis indicated that these improvements were associated with altered nitrogen metabolism, more efficient allocation of cellular resources, and enhanced supply of phosphoenolpyruvate (PEP), a key precursor in aromatic amino acid biosynthesis. This study expands our understanding of the regulation mechanisms governing <span>l</span>-tryptophan synthesis in <em>C. glutamicum</em> and provides valuable insights for further optimization of industrial cell factories.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 256-267"},"PeriodicalIF":4.4,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145325256","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

Putative membrane protein Cgl2168 and its variant enhance thermotolerance in Corynebacterium glutamicum 推测膜蛋白Cgl2168及其变体增强谷氨酸棒状杆菌的耐热性

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-25 DOI: 10.1016/j.synbio.2025.09.018

Ning Gao , Xiaomeng Ni , Jiao Liu , Jiuzhou Chen , Yu Wang , Ping Zheng , Jibin Sun

Corynebacterium glutamicum ATCC 13032 is widely used as the chassis strain for industrial production of various amino acids, but its thermotolerance limits productivity under high temperatures. Comparative genomic analysis in this study identified cgl2168 as a key determinant of enhanced thermotolerance in a laboratory strain (13032-JN) compared to another stock (13032-TJ). A guanine at Residue 270 (G270) deletion in cgl2168 (cgl2168^Δ270G) in 13032-JN significantly improved growth at 40 °C, while reverting this mutation or deleting cgl2168 impaired thermotolerance. Structural analysis revealed the deletion extends the C-terminus with an additional α-helix. Transcriptomic profiling showed cgl2168^Δ270G increased gene expressions involved in oxidative phosphorylation (e.g., atpABCDEFGH), nitrate metabolism (narIJHK), some heat shock protein genes (e.g., dnaK and groES) and deletion of cgl2168 resulted in upregulating sulfur metabolism (sulfonate transporter protein genes ssuBCD, sulfur-containing amino acid cys and met operons) and central carbon metabolism (gltA, pyc), while downregulating gluconeogenesis (pck) and fermentation (ldh) genes. These changes enhanced ATP synthesis, carbon utilization and oxidative tolerance. Predicted interactors of Cgl2168 linked it to respiratory chain function, stress responses, and cell envelope biogenesis. Thus, Cgl2168 and its variant Cgl2168^Δ270G enhance thermotolerance by coordinating energy production, carbon metabolism, and stress adaptation, aiding industrial applications.

谷氨棒状杆菌ATCC 13032被广泛用作各种氨基酸工业生产的基础菌株，但其耐热性限制了高温下的生产能力。本研究的比较基因组分析发现，cgl2168是实验室菌株（13032-JN）与另一个品系（13032-TJ）相比耐热性增强的关键决定因素。13032-JN中cgl2168 （cgl2168Δ270G）中残基270处的鸟嘌呤（G270）缺失显著改善了40°C下的生长，同时恢复该突变或删除cgl2168损伤了耐热性。结构分析显示，该缺失扩展了c端，增加了一个α-螺旋。转录组学分析显示cgl2168Δ270G参与氧化磷酸化（如atpABCDEFGH）、硝酸盐代谢（narIJHK）、一些热休克蛋白基因（如dnaK和groES）的基因表达增加，cgl2168的缺失导致硫代谢（磺酸转运蛋白基因ssuBCD、含硫氨基酸cys和met操纵子）和中心碳代谢（gltA、pyc）基因表达上调，糖异生（pck）和发酵（ldh）基因表达下调。这些变化增强了ATP合成、碳利用和氧化耐受性。预测的Cgl2168相互作用物将其与呼吸链功能、应激反应和细胞包膜生物发生联系起来。因此，Cgl2168及其变体Cgl2168Δ270G通过协调能量产生、碳代谢和逆境适应来增强热耐受性，有助于工业应用。

{"title":"Putative membrane protein Cgl2168 and its variant enhance thermotolerance in Corynebacterium glutamicum","authors":"Ning Gao , Xiaomeng Ni , Jiao Liu , Jiuzhou Chen , Yu Wang , Ping Zheng , Jibin Sun","doi":"10.1016/j.synbio.2025.09.018","DOIUrl":"10.1016/j.synbio.2025.09.018","url":null,"abstract":"<div><div><em>Corynebacterium glutamicum</em> ATCC 13032 is widely used as the chassis strain for industrial production of various amino acids, but its thermotolerance limits productivity under high temperatures. Comparative genomic analysis in this study identified <em>cgl2168</em> as a key determinant of enhanced thermotolerance in a laboratory strain (13032-JN) compared to another stock (13032-TJ). A guanine at Residue 270 (G270) deletion in <em>cgl2168</em> (<em>cgl2168</em><sup>Δ270G</sup>) in 13032-JN significantly improved growth at 40 °C, while reverting this mutation or deleting <em>cgl2168</em> impaired thermotolerance. Structural analysis revealed the deletion extends the C-terminus with an additional α-helix. Transcriptomic profiling showed <em>cgl2168</em><sup>Δ270G</sup> increased gene expressions involved in oxidative phosphorylation (e.g., <em>atpABCDEFGH</em>), nitrate metabolism (<em>narIJHK</em>), some heat shock protein genes (e.g., <em>dnaK</em> and <em>groES</em>) and deletion of <em>cgl2168</em> resulted in upregulating sulfur metabolism (sulfonate transporter protein genes <em>ssuBCD</em>, sulfur-containing amino acid <em>cys</em> and <em>met</em> operons) and central carbon metabolism (<em>gltA</em>, <em>pyc</em>), while downregulating gluconeogenesis (<em>pck</em>) and fermentation (<em>ldh</em>) genes. These changes enhanced ATP synthesis, carbon utilization and oxidative tolerance. Predicted interactors of Cgl2168 linked it to respiratory chain function, stress responses, and cell envelope biogenesis. Thus, Cgl2168 and its variant Cgl2168<sup>Δ270G</sup> enhance thermotolerance by coordinating energy production, carbon metabolism, and stress adaptation, aiding industrial applications.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 287-297"},"PeriodicalIF":4.4,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145325258","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

Structural and functional insights into the iminium ion intermediate in AsKslB-mediated Pictet-Spengler reaction askslb介导的Pictet-Spengler反应中微量离子中间体的结构和功能研究

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-25 DOI: 10.1016/j.synbio.2025.09.017

Yan-Bin Teng , Zhi Qiao , Chunya Xie , Xiaona Yang , Xinyu Liu , Zhengrong Zou , Yunchang Xie , Xuan Zhang , Qi Chen

Pictet-Spengler (PS) reactions are pivotal in the biosynthesis of bioactive alkaloids and pharmaceuticals, yet key structural details underlying their enzymatic catalysis remain insufficiently understood. We identified AsKslB from Actinosynnema sp. ALI-1.44 as a Pictet-Spenglerase with broad substrate scope that catalyzes the stereoselective condensation of l-tryptophan (l-Trp) and α-ketoglutarate (α-KG) to form kitasetalic acid (KA), a tetrahydro-β-carboline (THβC). High-resolution crystal structures of apo, substrate-, intermediate-, and product-bound forms elucidate the full catalytic trajectory and key residues. Crucially, the elusive iminium ion intermediate (IM-1) and a synchronously released water molecule are captured, providing direct structural evidence for the initiating cyclization step of Pictet-Spengler reaction. Glu276 undergoes conformational changes essential for catalysis. These findings offer detailed mechanistic insights into Pictet-Spenglerase function and establish AsKslB as a promising biocatalyst for stereoselective N-heterocycle synthesis.

Pictet-Spengler （PS）反应在生物活性生物碱和药物的生物合成中是至关重要的，但其酶催化的关键结构细节仍未得到充分的了解。我们从Actinosynnema sp. i -1.44中鉴定出AsKslB是一种具有广泛底物范围的picet - spenglerase，它催化l-色氨酸（l-Trp）和α-酮戊二酸（α-KG）的立体选择性缩合，形成四氢β-碳碱（th -β c） kitasetalic acid （KA）。载脂蛋白、底物结合、中间结合和产物结合形式的高分辨率晶体结构阐明了完整的催化轨迹和关键残基。关键是，捕获了难以捉摸的im1和同步释放的水分子，为Pictet-Spengler反应的起始环化步骤提供了直接的结构证据。Glu276经历了催化所必需的构象变化。这些发现为Pictet-Spenglerase功能提供了详细的机制见解，并确立了AsKslB作为立体选择性n -杂环合成生物催化剂的前景。

{"title":"Structural and functional insights into the iminium ion intermediate in AsKslB-mediated Pictet-Spengler reaction","authors":"Yan-Bin Teng , Zhi Qiao , Chunya Xie , Xiaona Yang , Xinyu Liu , Zhengrong Zou , Yunchang Xie , Xuan Zhang , Qi Chen","doi":"10.1016/j.synbio.2025.09.017","DOIUrl":"10.1016/j.synbio.2025.09.017","url":null,"abstract":"<div><div>Pictet-Spengler (PS) reactions are pivotal in the biosynthesis of bioactive alkaloids and pharmaceuticals, yet key structural details underlying their enzymatic catalysis remain insufficiently understood. We identified AsKslB from <em>Actinosynnema</em> sp. ALI-1.44 as a Pictet-Spenglerase with broad substrate scope that catalyzes the stereoselective condensation of <span>l</span>-tryptophan (<span>l</span>-Trp) and α-ketoglutarate (α-KG) to form kitasetalic acid (KA), a tetrahydro-β-carboline (THβC). High-resolution crystal structures of apo, substrate-, intermediate-, and product-bound forms elucidate the full catalytic trajectory and key residues. Crucially, the elusive iminium ion intermediate (IM-1) and a synchronously released water molecule are captured, providing direct structural evidence for the initiating cyclization step of Pictet-Spengler reaction. Glu276 undergoes conformational changes essential for catalysis. These findings offer detailed mechanistic insights into Pictet-Spenglerase function and establish AsKslB as a promising biocatalyst for stereoselective <em>N</em>-heterocycle synthesis.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 247-255"},"PeriodicalIF":4.4,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145324732","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 smart gene circuit for precise regulation of tumor cell behavior 精确调控肿瘤细胞行为的智能基因回路

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-22 DOI: 10.1016/j.synbio.2025.09.016

Hao Fu , Zhiwei Zhou , Lan Fang , Qing Wang , Xin Tang , Wei Su , Xuancai Chen , Yachun Tang , Qun Zhou

Personalized gene circuit is a robust mode of cellular regulation that can manipulate intracellular gene expression to achieve desired functional regulation. However, the construction of logic circuits that automatically sense the characteristics of a particular environment within a cell is often difficult and lacking in sensitivity. Here, we synthesize from scratch specific promoters capable of sensing in cells, and use the combination of different types of promoters to construct smart gene circuits that can regulate gene expression in specific cell types sensitively. In detail, the tumour-specific promoter and the prostate tissue-specific promoter were constructed to be combined together into generating an artificial AND-gate gene circuit using the CRISPR-Cas9 system which could identify prostate cancer selectively. We then utilized this artificial gene circuit to drive targeted genes expression, such as P21, E-cadherin and Bax, to inhibit multifunctional prostate cancer cells but not other cells. Moreover, we applied gene circuits to redirect endogenous genes within cells and significantly and specifically suppressed the tumor growth of prostate cancer in vivo. Overall, these results highlight the clinical potential of these gene circuits as specific tools for prostate cancer detection and treatment, which is a new method for specifically reprogramming prostate cancer cells in vivo and may serve as a promising treatment strategy.

个性化基因回路是一种强大的细胞调控模式，可以操纵细胞内基因表达以实现所需的功能调控。然而，在细胞内自动感知特定环境特征的逻辑电路的构建通常是困难的，而且缺乏灵敏度。在这里，我们从无到有地合成了能够在细胞中感知的特异性启动子，并利用不同类型启动子的组合构建了智能基因回路，可以敏感地调节特定细胞类型中的基因表达。具体而言，构建肿瘤特异性启动子和前列腺组织特异性启动子，利用CRISPR-Cas9系统将它们组合在一起，形成一个人工and -gate基因回路，可以选择性地识别前列腺癌。然后，我们利用这种人工基因回路驱动P21、E-cadherin和Bax等靶向基因的表达，抑制多功能前列腺癌细胞而非其他细胞。此外，我们利用基因回路在细胞内重定向内源性基因，在体内显著特异性抑制前列腺癌的肿瘤生长。总之，这些结果突出了这些基因回路作为前列腺癌检测和治疗的特异性工具的临床潜力，这是一种在体内特异性重编程前列腺癌细胞的新方法，可能是一种有前景的治疗策略。

{"title":"A smart gene circuit for precise regulation of tumor cell behavior","authors":"Hao Fu , Zhiwei Zhou , Lan Fang , Qing Wang , Xin Tang , Wei Su , Xuancai Chen , Yachun Tang , Qun Zhou","doi":"10.1016/j.synbio.2025.09.016","DOIUrl":"10.1016/j.synbio.2025.09.016","url":null,"abstract":"<div><div>Personalized gene circuit is a robust mode of cellular regulation that can manipulate intracellular gene expression to achieve desired functional regulation. However, the construction of logic circuits that automatically sense the characteristics of a particular environment within a cell is often difficult and lacking in sensitivity. Here, we synthesize from scratch specific promoters capable of sensing in cells, and use the combination of different types of promoters to construct smart gene circuits that can regulate gene expression in specific cell types sensitively. In detail, the tumour-specific promoter and the prostate tissue-specific promoter were constructed to be combined together into generating an artificial AND-gate gene circuit using the CRISPR-Cas9 system which could identify prostate cancer selectively. We then utilized this artificial gene circuit to drive targeted genes expression, such as P21, E-cadherin and Bax, to inhibit multifunctional prostate cancer cells but not other cells. Moreover, we applied gene circuits to redirect endogenous genes within cells and significantly and specifically suppressed the tumor growth of prostate cancer in vivo. Overall, these results highlight the clinical potential of these gene circuits as specific tools for prostate cancer detection and treatment, which is a new method for specifically reprogramming prostate cancer cells <em>in vivo</em> and may serve as a promising treatment strategy.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 237-246"},"PeriodicalIF":4.4,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145220420","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

Enhanced microbial production of pyridoxine (Vitamin B6) in Bacillus subtilis via pathway and process optimization 优化枯草芽孢杆菌产吡哆醇（维生素B6）的途径和工艺

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-16 DOI: 10.1016/j.synbio.2025.09.014

Ai-Tong Jiang , Guang-Qing Du , Xu-Yang Huang , Zheng-Zi Ji , Si-Riguleng Qian , Lin-Xia Liu , Da-Wei Zhang

Vitamin B₆ refers to a family of water-soluble B vitamin, which is essential for various physiological functions, including amino acid metabolism, neurotransmitter synthesis, and hemoglobin synthesis. The biosynthesis of pyridoxine (PN), a commercial form of vitamin B₆, through microbial fermentation has garnered widespread attention owing to its environmentally friendly and safe production methods, as well as its mild reaction conditions. However, the low yield of natural strains limits their application. This study focused on constructing a high-yielding strain of PN through pathway engineering and process optimization. Firstly, five key deoxyxylulose-5-phosphate-dependent pathway genes (epd, pdxB, serC, pdxA, and pdxJ) were overexpressed in Bacillus subtilis ARTP, which improved the PN titer by 3.2-fold to 2.9 mg/L. Subsequently, pdxST genes involved in the DXP-independent pathway were screened from various strains. Ribosome binding site (RBS) sequences were optimized to regulate their expression, which further increased the PN titer to 24.6 mg/L. Finally, systematic medium optimization was identified as a critical strategy for enhancing PN biosynthesis, leading to a remarkable 1.8-fold improvement in PN production. Under optimized fermentation conditions, the engineered strain achieved a PN titer of 174.6 mg/L in fed-batch fermentation, which represents the highest level reported to date in B. subtilis. Overall, this study presents an effective strategy combining pathway engineering and medium optimization for significantly improving PN production, offering valuable insights for the industrial development of PN biosynthesis.

维生素B6是指水溶性B族维生素，对氨基酸代谢、神经递质合成、血红蛋白合成等多种生理功能至关重要。吡哆醇（pyridoxine， PN）是维生素B6的一种商业形式，由于其生产方法环保、安全、反应条件温和而受到广泛关注。然而，天然菌株的低产量限制了它们的应用。本研究主要通过途径工程和工艺优化来构建高产的PN菌株。首先，枯草芽孢杆菌ARTP中5个关键的脱氧葡糖-5-磷酸依赖通路基因（epd、pdxB、serC、pdxA和pdxJ）过表达，使PN效价提高3.2倍，达到2.9 mg/L。随后，从不同菌株中筛选了参与dxp独立通路的pdxST基因。通过优化核糖体结合位点（RBS）序列调控其表达，使PN效价进一步提高至24.6 mg/L。最后，系统培养基优化被确定为促进PN生物合成的关键策略，导致PN产量显著提高1.8倍。在优化的发酵条件下，工程菌株在补料分批发酵中获得了174.6 mg/L的PN滴度，这是迄今为止报道的枯草芽孢杆菌的最高水平。总的来说，本研究提出了一种有效的途径工程和培养基优化相结合的策略，可以显著提高PN的产量，为PN生物合成的工业发展提供有价值的见解。

{"title":"Enhanced microbial production of pyridoxine (Vitamin B6) in Bacillus subtilis via pathway and process optimization","authors":"Ai-Tong Jiang , Guang-Qing Du , Xu-Yang Huang , Zheng-Zi Ji , Si-Riguleng Qian , Lin-Xia Liu , Da-Wei Zhang","doi":"10.1016/j.synbio.2025.09.014","DOIUrl":"10.1016/j.synbio.2025.09.014","url":null,"abstract":"<div><div>Vitamin B<sub>6</sub> refers to a family of water-soluble B vitamin, which is essential for various physiological functions, including amino acid metabolism, neurotransmitter synthesis, and hemoglobin synthesis. The biosynthesis of pyridoxine (PN), a commercial form of vitamin B<sub>6</sub>, through microbial fermentation has garnered widespread attention owing to its environmentally friendly and safe production methods, as well as its mild reaction conditions. However, the low yield of natural strains limits their application. This study focused on constructing a high-yielding strain of PN through pathway engineering and process optimization. Firstly, five key deoxyxylulose-5-phosphate-dependent pathway genes (<em>epd</em>, <em>pdxB</em>, <em>serC</em>, <em>pdxA</em>, and <em>pdxJ</em>) were overexpressed in <em>Bacillus subtilis</em> ARTP, which improved the PN titer by 3.2-fold to 2.9 mg/L. Subsequently, <em>pdxST</em> genes involved in the DXP-independent pathway were screened from various strains. Ribosome binding site (RBS) sequences were optimized to regulate their expression, which further increased the PN titer to 24.6 mg/L. Finally, systematic medium optimization was identified as a critical strategy for enhancing PN biosynthesis, leading to a remarkable 1.8-fold improvement in PN production. Under optimized fermentation conditions, the engineered strain achieved a PN titer of 174.6 mg/L in fed-batch fermentation, which represents the highest level reported to date in <em>B</em>. <em>subtilis</em>. Overall, this study presents an effective strategy combining pathway engineering and medium optimization for significantly improving PN production, offering valuable insights for the industrial development of PN biosynthesis.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 216-225"},"PeriodicalIF":4.4,"publicationDate":"2025-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145220362","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

Development of a CRISPR/Cas12a genome editing toolbox in Kluyveromyces marxianus and its application in succinic acid biosynthesis 马氏克卢维菌CRISPR/Cas12a基因组编辑工具箱的开发及其在琥珀酸生物合成中的应用

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-16 DOI: 10.1016/j.synbio.2025.09.015

Hao Zha , Yanjie Li , Zhongmei Hu, Jiacheng Li, Yujie Xie, Mingtao Zhao, Lili Ren, Biao Zhang

Kluyveromyces marxianus is a promising thermotolerant yeast for industrial biotechnology, but lacks efficient genome engineering tools. A CRISPR/Cas12a genome editing toolbox for K. marxianus was developed for the first time in this study. A plasmid-free transient system achieved single-gene knockout efficiencies of about 50 %–100 % in Δku70 strain. Even with homology arms as short as 35 bp, the knockout efficiency remained 66.67 %. Chromosomal integration of Cas12a enabled single-to-triple fragment knock-ins efficiency of 82.93–85.70 % and 94.50 % for large fragment (>5 kb) integrations. Applying this system, the roles of succinate dehydrogenase (SDH) genes SDH1-SDH5 were elucidated. Combinatorial SDH genes knockouts redirected carbon flux toward succinic acid (SA), but increased glycerol/acetate byproducts. Subsequent GPD1/ACH1/ADH2A co-knockout in a Δsdh1,3,5,4A,2 strain with NDE1 overexpression (YZH43) yielded a chassis producing 32.38 g/L SA from glucose at 37 °C, which is the highest reported titer in K. marxianus, while reducing ethanol, acetate, and glycerol by 60.79 %, 89.24 %, and 67.5 %, respectively. At 46 °C, YZH43 produced 20.51 g/L SA through simultaneous saccharification and fermentation (SSF) using cellulose as substrate. This work provides a high-efficiency CRISPR/Cas12a platform for K. marxianus, enabling rapid metabolic engineering for value-added chemical production, and demonstrates its utility in developing thermotolerant SA-overproducing strains.

马氏克鲁维酵母是一种很有应用前景的耐热酵母，但缺乏有效的基因组工程工具。本研究首次构建了马氏乳杆菌CRISPR/Cas12a基因组编辑工具箱。无质粒瞬时系统在Δku70菌株中实现了约50% - 100%的单基因敲除效率。即使同源臂短至35 bp，敲除效率仍为66.67%。Cas12a染色体整合使单片段到三片段的敲入效率为82.93 - 85.70%，大片段（>5 kb）整合的敲入效率为94.50%。利用该系统，研究了琥珀酸脱氢酶（SDH）基因SDH1-SDH5的作用。组合SDH基因敲除将碳通量转向琥珀酸（SA），但增加了甘油/乙酸副产物。随后，在NDE1过表达（YZH43）的Δsdh1、3、5、4A、2菌株（YZH43）中共敲除GPD1/ACH1/ADH2A，在37℃下从葡萄糖中产生32.38 g/L SA，这是在马氏K. marxianus中报道的最高滴度，同时乙醇、乙酸和甘油分别减少了60.79%、89.24%和67.5%。在46℃下，YZH43以纤维素为底物，通过同时糖化发酵（SSF）产生20.51 g/L SA。这项工作为K. marxianus提供了一个高效的CRISPR/Cas12a平台，实现了增值化学品生产的快速代谢工程，并证明了其在培养耐热sa过量菌株方面的实用性。

{"title":"Development of a CRISPR/Cas12a genome editing toolbox in Kluyveromyces marxianus and its application in succinic acid biosynthesis","authors":"Hao Zha , Yanjie Li , Zhongmei Hu, Jiacheng Li, Yujie Xie, Mingtao Zhao, Lili Ren, Biao Zhang","doi":"10.1016/j.synbio.2025.09.015","DOIUrl":"10.1016/j.synbio.2025.09.015","url":null,"abstract":"<div><div><em>Kluyveromyces marxianus</em> is a promising thermotolerant yeast for industrial biotechnology, but lacks efficient genome engineering tools. A CRISPR/Cas12a genome editing toolbox for <em>K</em>. <em>marxianus</em> was developed for the first time in this study. A plasmid-free transient system achieved single-gene knockout efficiencies of about 50 %–100 % in <em>Δku70</em> strain. Even with homology arms as short as 35 bp, the knockout efficiency remained 66.67 %. Chromosomal integration of Cas12a enabled single-to-triple fragment knock-ins efficiency of 82.93–85.70 % and 94.50 % for large fragment (>5 kb) integrations. Applying this system, the roles of succinate dehydrogenase (<em>SDH</em>) genes <em>SDH1-SDH5</em> were elucidated. Combinatorial <em>SDH</em> genes knockouts redirected carbon flux toward succinic acid (SA), but increased glycerol/acetate byproducts. Subsequent <em>GPD1</em>/<em>ACH1/ADH2A</em> co-knockout in a <em>Δsdh1,3,5,4A,2</em> strain with <em>NDE1</em> overexpression (YZH43) yielded a chassis producing 32.38 g/L SA from glucose at 37 °C, which is the highest reported titer in <em>K. marxianus</em>, while reducing ethanol, acetate, and glycerol by 60.79 %, 89.24 %, and 67.5 %, respectively. At 46 °C, YZH43 produced 20.51 g/L SA through simultaneous saccharification and fermentation (SSF) using cellulose as substrate. This work provides a high-efficiency CRISPR/Cas12a platform for <em>K. marxianus</em>, enabling rapid metabolic engineering for value-added chemical production, and demonstrates its utility in developing thermotolerant SA-overproducing strains.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 193-204"},"PeriodicalIF":4.4,"publicationDate":"2025-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145158419","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

Corrigendum to “Overproduction of endusamycin in Streptomyces endus subsp. aureus” [Synth Syst Biotechnol 10 (2) (2025) 523–531] “endusamycin在endus链霉菌亚种中的过量生产”的勘误。[Synth系统生物技术10 (2)(2025)523-531]

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-15 DOI: 10.1016/j.synbio.2025.09.001

Yingying Chang , Zhen Liu , Zixin Deng , Tiangang Liu

引用次数: 0