Metabolic engineering最新文献_第8页

Metabolic engineering of Corynebacterium glutamicum for increased cis, cis-muconate production from plant-derived p-hydroxycinnamates via deregulated pathway flux and increased CoA intermediate availability 谷氨酸棒状杆菌的代谢工程，通过解除调控的途径通量和增加辅酶a中间体的可用性，增加植物源的对羟基肉桂酸的顺式、顺式肉桂酸的产量。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-01 Epub Date: 2025-08-12 DOI: 10.1016/j.ymben.2025.08.004

Fabia Weiland, Kyoyoung Seo, Franka Janz, Marius Grad, Lea Geldmacher, Michael Kohlstedt, Judith Becker, Christoph Wittmann

Lignocellulosic biomass represents a promising renewable feedstock for sustainable biochemical production, with p-hydroxycinnamates emerging as key aromatic building blocks derived from agricultural residues and grassy plants. C. glutamicum has recently been engineered to produce cis, cis-muconate (MA), a high-value platform chemical used in biobased plastics, resins, and specialty chemicals. However, unlike other aromatics, the metabolism of the p-hydroxycinnamates p-coumarate, ferulate, and caffeate in MA-producing C. glutamicum is inefficient, limiting MA production performance. Here, we discovered that p-hydroxycinnamate metabolism, encoded by the phd operon, is repressed by the local repressor PhdR under glucose-rich conditions, while the global regulator GlxR activates the pathway in the absence of glucose. The deregulated C. glutamicum MA-10 lacking phdR exhibited an up to 98-fold increase in the conversion of p-coumarate, ferulate, and aromatic mixtures derived from plant waste into MA. Transcriptomic and metabolomic analyses revealed strong induction of the phd operon in strain MA-10 and a marked increase in intracellular aromatic CoA-esters and acetyl-CoA, indicating enhanced flux through the p-hydroxycinnamate degradation pathway. ¹³C-tracer studies demonstrated a substantial contribution of aromatic side-chain carbon to central metabolic pathways, supporting biomass formation and enabling MA production even in the absence of sugars. Additionally, MA-10 showed broadened substrate flexibility, degrading cinnamate into MA and methoxylated cinnamates into valuable benzoate derivatives. The strain also successfully converted aromatics from real straw lignin hydrolysates into MA. Our findings reveal the potential of targeted regulatory engineering to optimize C. glutamicum for lignin valorization. The newly developed strain MA-10 provides a robust platform for the biobased production of MA from lignocellulosic feedstocks, paving the way for sustainable and economically viable biorefinery processes.

木质纤维素生物质代表了一种有前途的可持续生化生产的可再生原料，对羟基肉桂酸酯作为从农业残留物和禾草植物中提取的关键芳香基石。C. glutamicum最近被设计用于生产cis， cis-muconate (MA)，这是一种高价值的平台化学品，用于生物基塑料，树脂和特种化学品。然而，与其他芳香烃不同的是，对羟基肉桂酸、对香豆酸、阿魏酸和咖啡酸在产生MA的C. glutamum中的代谢效率很低，限制了MA的生产性能。在这里，我们发现由phd操纵子编码的对羟基肉桂酸代谢在富含葡萄糖的条件下被局部抑制因子PhdR抑制，而全局调节因子GlxR在缺乏葡萄糖的情况下激活该途径。缺乏phdR的不受调控的谷氨酰胺MA-10在从植物废物中提取的对香豆酸盐、阿魏酸盐和芳香混合物转化为MA的能力增加了98倍。转录组学和代谢组学分析显示，菌株MA-10的phd操纵子被强烈诱导，细胞内芳香辅酶a酯和乙酰辅酶a显著增加，表明通过对羟基肉桂酸降解途径的通量增强。13c示踪研究表明芳香侧链碳对中心代谢途径的重要贡献，支持生物量的形成，即使在没有糖的情况下也能产生MA。此外，MA-10表现出更宽的底物柔韧性，将肉桂酸降解为MA，并将甲氧基化的肉桂酸转化为有价值的苯甲酸衍生物。该菌株还成功地将真正的秸秆木质素水解产物中的芳烃转化为MA。我们的研究结果揭示了靶向调控工程优化谷氨酰胺木质素增值的潜力。新开发的菌株MA-10为木质纤维素原料的生物基生产MA提供了一个强大的平台，为可持续和经济上可行的生物炼制工艺铺平了道路。

{"title":"Metabolic engineering of Corynebacterium glutamicum for increased cis, cis-muconate production from plant-derived p-hydroxycinnamates via deregulated pathway flux and increased CoA intermediate availability","authors":"Fabia Weiland, Kyoyoung Seo, Franka Janz, Marius Grad, Lea Geldmacher, Michael Kohlstedt, Judith Becker, Christoph Wittmann","doi":"10.1016/j.ymben.2025.08.004","DOIUrl":"10.1016/j.ymben.2025.08.004","url":null,"abstract":"<div><div>Lignocellulosic biomass represents a promising renewable feedstock for sustainable biochemical production, with <em>p</em>-hydroxycinnamates emerging as key aromatic building blocks derived from agricultural residues and grassy plants. <em>C. glutamicum</em> has recently been engineered to produce <em>cis, cis</em>-muconate (MA), a high-value platform chemical used in biobased plastics, resins, and specialty chemicals. However, unlike other aromatics, the metabolism of the <em>p</em>-hydroxycinnamates <em>p</em>-coumarate, ferulate, and caffeate in MA-producing <em>C. glutamicum</em> is inefficient, limiting MA production performance. Here, we discovered that <em>p</em>-hydroxycinnamate metabolism, encoded by the <em>phd</em> operon, is repressed by the local repressor PhdR under glucose-rich conditions, while the global regulator GlxR activates the pathway in the absence of glucose. The deregulated <em>C. glutamicum</em> MA-10 lacking <em>phdR</em> exhibited an up to 98-fold increase in the conversion of <em>p</em>-coumarate, ferulate, and aromatic mixtures derived from plant waste into MA. Transcriptomic and metabolomic analyses revealed strong induction of the <em>phd</em> operon in strain MA-10 and a marked increase in intracellular aromatic CoA-esters and acetyl-CoA, indicating enhanced flux through the <em>p</em>-hydroxycinnamate degradation pathway. <sup>13</sup>C-tracer studies demonstrated a substantial contribution of aromatic side-chain carbon to central metabolic pathways, supporting biomass formation and enabling MA production even in the absence of sugars. Additionally, MA-10 showed broadened substrate flexibility, degrading cinnamate into MA and methoxylated cinnamates into valuable benzoate derivatives. The strain also successfully converted aromatics from real straw lignin hydrolysates into MA. Our findings reveal the potential of targeted regulatory engineering to optimize <em>C. glutamicum</em> for lignin valorization. The newly developed strain MA-10 provides a robust platform for the biobased production of MA from lignocellulosic feedstocks, paving the way for sustainable and economically viable biorefinery processes.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 262-283"},"PeriodicalIF":6.8,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144850834","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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

Metabolic flux and flux balance analyses indicate the relevance of metabolic thermogenesis and aerobic glycolysis in cancer cells 代谢通量和通量平衡分析表明，代谢产热和有氧糖酵解在癌细胞中的相关性。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-01 Epub Date: 2025-08-07 DOI: 10.1016/j.ymben.2025.08.002

Nobuyuki Okahashi , Tomoki Shima , Yuya Kondo , Chie Araki , Shuma Tsuji , Akane Sawai , Hikaru Uehara , Susumu Kohno , Hiroshi Shimizu , Chiaki Takahashi , Fumio Matsuda

Adenosine triphosphate (ATP) regeneration by substrate-level phosphorylation is a general feature of cancer metabolism, even under normoxic conditions (aerobic glycolysis). However, it is unclear why cancer cells prefer inefficient aerobic glycolysis over the highly efficient process of oxidative phosphorylation for ATP regeneration. To investigate the metabolic principles underlying aerobic glycolysis, we performed ¹³C-metabolic flux analysis of 12 cultured cancer cell lines and explored the metabolic constraints required to reproduce the results using in silico metabolic simulations. We found that the measured flux distribution can be reproduced by maximizing the ATP consumption in the flux balance analysis considering a limitation of metabolic heat dissipation (enthalpy change). Consistent with the simulation, OXPHOS inhibition induced metabolic redirection to aerobic glycolysis while maintaining the intracellular temperature. Furthermore, the dependency on aerobic glycolysis was partly alleviated upon culturing at low temperatures. Our data suggest that metabolic thermogenesis is an important factor in understanding aerobic glycolysis in cancer cells and that an advantage of aerobic glycolysis is the reduction in metabolic heat generation during ATP regeneration.

通过底物水平磷酸化再生三磷酸腺苷（ATP）是癌症代谢的一个普遍特征，即使在常氧条件下（有氧糖酵解）。然而，目前尚不清楚为什么癌细胞更喜欢低效的有氧糖酵解而不是高效的氧化磷酸化过程来再生ATP。为了研究有氧糖酵解的代谢原理，我们对12株培养的癌细胞进行了13c代谢通量分析，并探索了利用硅代谢模拟重现结果所需的代谢限制。我们发现，考虑到代谢热耗散（焓变）的限制，通量平衡分析中可以通过最大化ATP消耗来再现测量的通量分布。与模拟一致，OXPHOS抑制诱导代谢重定向到有氧糖酵解，同时保持细胞内温度。此外，低温培养部分减轻了对有氧糖酵解的依赖。我们的数据表明，代谢产热是理解癌细胞中有氧糖酵解的一个重要因素，有氧糖酵解的一个优势是在ATP再生过程中代谢产热的减少。

{"title":"Metabolic flux and flux balance analyses indicate the relevance of metabolic thermogenesis and aerobic glycolysis in cancer cells","authors":"Nobuyuki Okahashi , Tomoki Shima , Yuya Kondo , Chie Araki , Shuma Tsuji , Akane Sawai , Hikaru Uehara , Susumu Kohno , Hiroshi Shimizu , Chiaki Takahashi , Fumio Matsuda","doi":"10.1016/j.ymben.2025.08.002","DOIUrl":"10.1016/j.ymben.2025.08.002","url":null,"abstract":"<div><div>Adenosine triphosphate (ATP) regeneration by substrate-level phosphorylation is a general feature of cancer metabolism, even under normoxic conditions (aerobic glycolysis). However, it is unclear why cancer cells prefer inefficient aerobic glycolysis over the highly efficient process of oxidative phosphorylation for ATP regeneration. To investigate the metabolic principles underlying aerobic glycolysis, we performed <sup>13</sup>C-metabolic flux analysis of 12 cultured cancer cell lines and explored the metabolic constraints required to reproduce the results using <em>in silico</em> metabolic simulations. We found that the measured flux distribution can be reproduced by maximizing the ATP consumption in the flux balance analysis considering a limitation of metabolic heat dissipation (enthalpy change). Consistent with the simulation, OXPHOS inhibition induced metabolic redirection to aerobic glycolysis while maintaining the intracellular temperature. Furthermore, the dependency on aerobic glycolysis was partly alleviated upon culturing at low temperatures. Our data suggest that metabolic thermogenesis is an important factor in understanding aerobic glycolysis in cancer cells and that an advantage of aerobic glycolysis is the reduction in metabolic heat generation during ATP regeneration.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 185-193"},"PeriodicalIF":6.8,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144812187","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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

Biomass accumulation in chondrocyte metabolic modelling: Incorporating extracellular matrix proxies to predict tissue engineering outcomes 软骨细胞代谢模型中的生物量积累：结合细胞外基质代理来预测组织工程结果

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-01 Epub Date: 2025-07-13 DOI: 10.1016/j.ymben.2025.07.004

Roberto Tarantino , Halie Mei Jensen , Stephen D. Waldman

Metabolic modeling in chondrocytes plays a pivotal role in advancing our understanding of cellular function. These techniques have been used to study degenerative joint diseases (e.g. osteoarthritis), mechanotransduction, and more recently to optimize strategies for cartilage tissue engineering. Incorporating tissue formation into metabolic flux analysis is inherently challenging due to the complexity of linking metabolic activity to extracellular matrix (ECM) accumulation. Many ECM macromolecules are synthesized using metabolites derived from central carbon metabolism, but direct modeling of their accumulation remains complex. This study establishes a novel methodology for incorporating ECM synthesis into metabolic flux analysis (MFA). By utilizing chondroitin sulfate and hydroxyproline as measurable metabolic proxies for proteoglycan and collagen production, we demonstrate a framework for linking metabolic inputs with tissue formation. Extracellular flux data for glucose, lactate, carbon dioxide, glutamine, and glutamate, along with mass isotopomer distributions, were sourced from previous studies involving three-dimensional high-density cultures of articular cartilage tissue constructs. Additionally, the conditioned culture media used in these studies was used to quantify the production rates of chondroitin sulfate and hydroxyproline. Using a modular network model, proteoglycan and collagen metabolism were assessed independently, and in combination, with sensitivity analyses on ECM retention assumptions. Predicted proteoglycan production aligned well with previously observed trends; however, predicted collagen production was less consistent. These findings offer a novel approach for linking metabolic inputs with ECM production, advancing our ability to predict tissue formation and address key challenges in cartilage tissue engineering.

软骨细胞的代谢模型在促进我们对细胞功能的理解方面起着关键作用。这些技术已被用于研究退行性关节疾病（如骨关节炎）、机械转导，以及最近用于优化软骨组织工程的策略。由于将代谢活动与细胞外基质（ECM）积累联系起来的复杂性，将组织形成纳入代谢通量分析本身就具有挑战性。许多ECM是利用源自中心碳代谢的代谢物合成的，但对其积累的直接建模仍然很复杂。本研究建立了一种将ECM合成纳入代谢通量分析（MFA）的新方法。通过利用硫酸软骨素和羟脯氨酸作为蛋白聚糖和胶原蛋白生产的可测量代谢代用物，我们展示了一个将代谢输入与组织形成联系起来的框架。葡萄糖、乳酸、二氧化碳、谷氨酰胺和谷氨酸的细胞外通量数据，以及质量同位素分布，来源于先前涉及关节软骨组织构建的三维高密度培养的研究。此外，在这些研究中使用的条件培养基被用来量化硫酸软骨素和羟脯氨酸的产量。使用模块化网络模型，分别评估蛋白聚糖和胶原代谢，并结合ECM保留假设的敏感性分析。预测的蛋白多糖产量与先前观察到的趋势一致；然而，预测的胶原蛋白生成不太一致。这些发现为将代谢输入与ECM产生联系起来提供了一种新的方法，提高了我们预测组织形成的能力，并解决了软骨组织工程中的关键挑战。

{"title":"Biomass accumulation in chondrocyte metabolic modelling: Incorporating extracellular matrix proxies to predict tissue engineering outcomes","authors":"Roberto Tarantino , Halie Mei Jensen , Stephen D. Waldman","doi":"10.1016/j.ymben.2025.07.004","DOIUrl":"10.1016/j.ymben.2025.07.004","url":null,"abstract":"<div><div>Metabolic modeling in chondrocytes plays a pivotal role in advancing our understanding of cellular function. These techniques have been used to study degenerative joint diseases (e.g. osteoarthritis), mechanotransduction, and more recently to optimize strategies for cartilage tissue engineering. Incorporating tissue formation into metabolic flux analysis is inherently challenging due to the complexity of linking metabolic activity to extracellular matrix (ECM) accumulation. Many ECM macromolecules are synthesized using metabolites derived from central carbon metabolism, but direct modeling of their accumulation remains complex. This study establishes a novel methodology for incorporating ECM synthesis into metabolic flux analysis (MFA). By utilizing chondroitin sulfate and hydroxyproline as measurable metabolic proxies for proteoglycan and collagen production, we demonstrate a framework for linking metabolic inputs with tissue formation. Extracellular flux data for glucose, lactate, carbon dioxide, glutamine, and glutamate, along with mass isotopomer distributions, were sourced from previous studies involving three-dimensional high-density cultures of articular cartilage tissue constructs. Additionally, the conditioned culture media used in these studies was used to quantify the production rates of chondroitin sulfate and hydroxyproline. Using a modular network model, proteoglycan and collagen metabolism were assessed independently, and in combination, with sensitivity analyses on ECM retention assumptions. Predicted proteoglycan production aligned well with previously observed trends; however, predicted collagen production was less consistent. These findings offer a novel approach for linking metabolic inputs with ECM production, advancing our ability to predict tissue formation and address key challenges in cartilage tissue engineering.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 1-12"},"PeriodicalIF":6.8,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144622704","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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

Enhancing ε-poly-L-lysine production in Streptomyces albulus through L-lysine importer engineering 利用l -赖氨酸进口工程提高白球链霉菌的ε-聚l -赖氨酸产量。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-01 Epub Date: 2025-07-25 DOI: 10.1016/j.ymben.2025.07.010

Daojun Zhu , Jiawei Zhang , Shangyu Li, Liang Wang, Hongjian Zhang, Jianhua Zhang, Zhang, Xusheng Chen

ε-Poly-L-lysine (ε-PL) is a homopolymer of L-lysine residues produced by microorganisms, widely utilized in the food, pharmaceutical, and cosmetic industries. However, the development of efficient microbial cell factories (MCFs) for ε-PL production remains challenging. In this study, L-lysine importers were systematically screened, identified, and engineered to enhance ε-PL biosynthesis. First, an ε-PL-producing strain, Streptomyces albulus GS114, efficiently utilizing exogenous L-lysine, was selected. Bioinformatics analysis identified seven putative L-lysine importers in GS114, among which GL6157 was confirmed as the primary importer through molecular docking, transcriptional analysis, and genetic manipulation. Through combinatorial optimization of GL6157 expression coupled with overexpression of ε-poly-L-lysine synthase (pls), we engineered the GS114/pls-GL6157 strain, which achieved a ε-PL of 94.0 g/L in fed-batch fermentation. To our knowledge, this represents the highest reported yield to date. These findings demonstrate that transporter engineering is an effective strategy for enhancing ε-PL biosynthesis in industrial MCFs.

ε-聚l -赖氨酸（ε-PL）是一种由微生物产生的l -赖氨酸残基的均聚物，广泛应用于食品、制药和化妆品等行业。然而，开发高效的微生物细胞工厂（mcf）生产ε-PL仍然具有挑战性。在本研究中，l -赖氨酸导入物被系统筛选、鉴定和改造以促进ε-PL的生物合成。首先，选择了一株能高效利用外源l -赖氨酸的产ε- pl菌株——白链霉菌GS114。生物信息学分析在GS114中鉴定出7个假定的l -赖氨酸进口蛋白，通过分子对接、转录分析和基因操作确定GL6157为主要进口蛋白。通过对GL6157的表达和过表达ε-聚L-赖氨酸合成酶（pls）的组合优化，构建了菌株GS114/pls-GL6157，该菌株在补料分批发酵条件下的ε-PL为94.0 g/L。据我们所知，这是迄今为止报道的最高产量。这些发现表明，转运体工程是促进工业MCFs中ε-PL生物合成的有效策略。

{"title":"Enhancing ε-poly-L-lysine production in Streptomyces albulus through L-lysine importer engineering","authors":"Daojun Zhu , Jiawei Zhang , Shangyu Li, Liang Wang, Hongjian Zhang, Jianhua Zhang, Zhang, Xusheng Chen","doi":"10.1016/j.ymben.2025.07.010","DOIUrl":"10.1016/j.ymben.2025.07.010","url":null,"abstract":"<div><div>ε-Poly-L-lysine (ε-PL) is a homopolymer of L-lysine residues produced by microorganisms, widely utilized in the food, pharmaceutical, and cosmetic industries. However, the development of efficient microbial cell factories (MCFs) for ε-PL production remains challenging. In this study, L-lysine importers were systematically screened, identified, and engineered to enhance ε-PL biosynthesis. First, an ε-PL-producing strain, <em>Streptomyces albulus</em> GS114, efficiently utilizing exogenous L-lysine, was selected. Bioinformatics analysis identified seven putative L-lysine importers in GS114, among which GL6157 was confirmed as the primary importer through molecular docking, transcriptional analysis, and genetic manipulation. Through combinatorial optimization of <em>GL6157</em> expression coupled with overexpression of ε-poly-L-lysine synthase (<em>pls</em>), we engineered the GS114/pls-GL6157 strain, which achieved a ε-PL of 94.0 g/L in fed-batch fermentation. To our knowledge, this represents the highest reported yield to date. These findings demonstrate that transporter engineering is an effective strategy for enhancing ε-PL biosynthesis in industrial MCFs.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 102-112"},"PeriodicalIF":6.8,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144720261","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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

Genome-scale overexpression screen reveals membrane homeostasis as a key determinant for free fatty acids overproduction in Escherichia coli 基因组尺度的过表达筛选揭示了膜稳态是大肠杆菌游离脂肪酸过量产生的关键决定因素。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-01 Epub Date: 2025-07-10 DOI: 10.1016/j.ymben.2025.07.002

Lixia Fang , Xiaolei Liu , Zhongxiu Chen , Jiaqi Zhang , Lian Wang , Yingxiu Cao

Genome-scale target identification is essential for optimizing microbial biosynthesis due to the highly complex and interconnected nature of cellular metabolism. Free fatty acids (FFAs), valuable precursors for biofuels and industrial chemicals, have been extensively studied in Escherichia coli. However, genome-wide exploration of beneficial targets that promote FFAs production remains limited, hindering efforts to fully unlock the potential of microbial biosynthetic capabilities. In this study, we performed genome-scale screening of upregulation targets for FFAs overproduction in E. coli by leveraging the ASKA (A Complete Set of E. coli K-12 ORF Archive) library in combination with fluorescence-activated cell sorting (FACS) and next-generation sequencing (NGS). We found that overexpression of rfaY, encoding a lipopolysaccharide (LPS) core heptose II phosphokinase, led to a 207.8% increase in FFAs production by enhancing membrane stability, as evidenced by reduced permeability and improved integrity. Further investigation revealed that overexpressing additional LPS biosynthesis-related genes similarly improved membrane robustness and FFAs production. Through iterative screening, yafL and rimM were identified as synergistic partners with rfaY, and subsequent integration of fadR overexpression ultimately yielded the optimal strain rfaY⁺-yafL⁺-fadR⁺, which achieved a FFAs titer of 39.6 g/L under fed-batch fermentation—the highest reported to date in E. coli. This study highlights the significance of genome-scale mining potential genetic determinants for enhancing the biosynthesis of desired products.

由于细胞代谢的高度复杂和相互关联的性质，基因组尺度的目标鉴定对于优化微生物生物合成是必不可少的。游离脂肪酸（FFAs）是生物燃料和工业化学品的宝贵前体，在大肠杆菌中得到了广泛的研究。然而，对促进FFAs产生的有益靶点的全基因组探索仍然有限，阻碍了充分释放微生物生物合成能力潜力的努力。在这项研究中，我们利用ska （A Complete Set of E. coli K-12 ORF Archive）文库，结合荧光活化细胞分选（FACS）和下一代测序（NGS），对大肠杆菌中FFAs过量产生的上调靶点进行了基因组规模的筛选。我们发现，编码脂多糖（LPS）核心heptose II磷酸激酶的rfaY过表达通过增强膜稳定性导致FFAs产量增加207.8%，这可以通过降低通透性和提高完整性来证明。进一步的研究表明，过表达额外的LPS生物合成相关基因同样提高了膜的健壮性和FFAs的产生。通过反复筛选，yafL和rimM被确定为与rfaY具有协同作用的伙伴，随后整合fadR过表达最终获得了最佳菌株rfaY+-yafL+-fadR+，该菌株在补料分批发酵下的FFAs滴度达到了39.6 g/L，这是迄今为止在大肠杆菌中报道的最高滴度。这项研究强调了基因组规模挖掘潜在遗传决定因素对增强所需产品的生物合成的重要性。

{"title":"Genome-scale overexpression screen reveals membrane homeostasis as a key determinant for free fatty acids overproduction in Escherichia coli","authors":"Lixia Fang , Xiaolei Liu , Zhongxiu Chen , Jiaqi Zhang , Lian Wang , Yingxiu Cao","doi":"10.1016/j.ymben.2025.07.002","DOIUrl":"10.1016/j.ymben.2025.07.002","url":null,"abstract":"<div><div>Genome-scale target identification is essential for optimizing microbial biosynthesis due to the highly complex and interconnected nature of cellular metabolism. Free fatty acids (FFAs), valuable precursors for biofuels and industrial chemicals, have been extensively studied in <em>Escherichia coli</em>. However, genome-wide exploration of beneficial targets that promote FFAs production remains limited, hindering efforts to fully unlock the potential of microbial biosynthetic capabilities. In this study, we performed genome-scale screening of upregulation targets for FFAs overproduction in <em>E. coli</em> by leveraging the ASKA (A Complete Set of <em>E. coli</em> K-12 ORF Archive) library in combination with fluorescence-activated cell sorting (FACS) and next-generation sequencing (NGS). We found that overexpression of <em>rfaY</em>, encoding a lipopolysaccharide (LPS) core heptose II phosphokinase, led to a 207.8% increase in FFAs production by enhancing membrane stability, as evidenced by reduced permeability and improved integrity. Further investigation revealed that overexpressing additional LPS biosynthesis-related genes similarly improved membrane robustness and FFAs production. Through iterative screening, <em>yafL</em> and <em>rimM</em> were identified as synergistic partners with <em>rfaY</em>, and subsequent integration of <em>fadR</em> overexpression ultimately yielded the optimal strain <em>rfaY</em><sup>+</sup>-<em>yafL</em><sup>+</sup>-<em>fadR</em><sup>+</sup>, which achieved a FFAs titer of 39.6 g/L under fed-batch fermentation—the highest reported to date in <em>E. coli</em>. This study highlights the significance of genome-scale mining potential genetic determinants for enhancing the biosynthesis of desired products.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 13-21"},"PeriodicalIF":6.8,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144613064","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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 metabolic engineering strategy for producing poly-(3-hydroxyoctanoic acid) in Escherichia coli from glycerol 利用甘油在大肠杆菌中生产聚- 3-羟基辛酸的代谢工程策略

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-01 Epub Date: 2025-08-22 DOI: 10.1016/j.ymben.2025.08.009

Shivangi Mishra, Ke Xu, Madeline K. Kuckuk, William T. Cordell, Néstor J. Hernández-Lozada, Brian F. Pfleger

Poly(3-hydroxyoctanoate) (PHO) is a medium-chain-length PHA with low crystallinity and high elongation to break ratio, unlike the brittle short-chain-PHAs like PHB. These properties make PHO a promising candidate for industrial and biomedical applications. In this study, we demonstrated the production of PHO in Escherichia coli from a renewable and inexpensive glycerol feedstock by engineering fatty acid synthesis and β-oxidation to create a pool of 2,3-octenoyl-CoAs. In this base strain, E. coli ΔfadRABIJ, an (R)-specific enoyl-CoA hydratase (phaJ) and a PHA synthase (phaC) were expressed to produce PHO. Bioprospecting phaJ and phaC homologs from Pseudomonas aeruginosa and fadD homolog from Pseudomonas putida implicated a combination of phaJ2, phaC2, and ^PpfadD genes yielded the highest PHO content from exogenously fed octanoate. Finally, when a single copy of a previously described C₈-specific thioesterase mutant CpFatB1.2-M4-287 was integrated into the chromosome of E. coli ΔfadRABIJ, the resulting E. coli strain NHL18 was capable of producing 3.69 ± 0.146 g/L of octanoic acid. Subsequently, the integration of PHA synthesis genes in NHL18 resulting in strain SM23 allowed the cell to accumulate 15 % cell dry weight of PHO with a final titer of 1.54 ± 0.234 g/L from glycerol in fed-batch fermentation.

聚3-羟基辛酸酯（PHO）是一种低结晶度、高断裂伸长率的中链PHA，不同于PHB等脆性短链PHA。这些特性使PHO成为工业和生物医学应用的有前途的候选者。在这项研究中，我们证明了大肠杆菌通过工程脂肪酸合成和β-氧化产生2,3-辛烯酰辅酶a池，从可再生和廉价的甘油原料生产PHO。在大肠杆菌ΔfadRABIJ中，表达了一种(R)特异性烯酰辅酶a水合酶（phaJ）和一种PHA合成酶（phaC）来产生PHO。从铜绿假单胞菌的phaJ和phaC同源物和恶臭假单胞菌的fadD同源物中发现，外源性辛酸盐中PHO含量最高的是phaJ2、phaC2和PpfadD基因的组合。最后，将先前描述的c8特异性硫酯酶突变体CpFatB1.2-M4-287的单个拷贝整合到大肠杆菌ΔfadRABIJ的染色体上，得到的大肠杆菌菌株NHL18能够产生3.69±0.146 g/L的辛酸。随后，在NHL18中整合PHA合成基因，产生菌株SM23，使细胞在补料分批发酵中积累了15%细胞干重的PHO，最终滴度为1.54±0.234 g/L。

{"title":"A metabolic engineering strategy for producing poly-(3-hydroxyoctanoic acid) in Escherichia coli from glycerol","authors":"Shivangi Mishra, Ke Xu, Madeline K. Kuckuk, William T. Cordell, Néstor J. Hernández-Lozada, Brian F. Pfleger","doi":"10.1016/j.ymben.2025.08.009","DOIUrl":"10.1016/j.ymben.2025.08.009","url":null,"abstract":"<div><div>Poly(3-hydroxyoctanoate) (PHO) is a medium-chain-length PHA with low crystallinity and high elongation to break ratio, unlike the brittle short-chain-PHAs like PHB. These properties make PHO a promising candidate for industrial and biomedical applications. In this study, we demonstrated the production of PHO in <em>Escherichia coli</em> from a renewable and inexpensive glycerol feedstock by engineering fatty acid synthesis and β-oxidation to create a pool of 2,3-octenoyl-CoAs. In this base strain, <em>E. coli</em> Δ<em>fadRABIJ,</em> an (R)-specific enoyl-CoA hydratase (<em>phaJ</em>) and a PHA synthase (<em>phaC</em>) were expressed to produce PHO. Bioprospecting <em>phaJ</em> and <em>phaC</em> homologs from <em>Pseudomonas aeruginosa</em> and <em>fadD</em> homolog from <em>Pseudomonas putida</em> implicated a combination of <em>phaJ2</em>, <em>phaC2</em>, and <sup>Pp</sup><em>fadD</em> genes yielded the highest PHO content from exogenously fed octanoate. Finally, when a single copy of a previously described C<sub>8</sub>-specific thioesterase mutant <em>CpFatB1.2-M4-287</em> was integrated into the chromosome of <em>E. coli</em> Δ<em>fadRABIJ,</em> the resulting <em>E. coli</em> strain NHL18 was capable of producing 3.69 ± 0.146 g/L of octanoic acid. Subsequently, the integration of PHA synthesis genes in NHL18 resulting in strain SM23 allowed the cell to accumulate 15 % cell dry weight of PHO with a final titer of 1.54 ± 0.234 g/L from glycerol in fed-batch fermentation.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 232-240"},"PeriodicalIF":6.8,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144895573","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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

Pan-reactome analysis of Streptomyces strains reveals association and disconnection between primary and secondary metabolism 链霉菌菌株的泛反应组分析揭示了初级和次级代谢之间的联系和脱节

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-01 Epub Date: 2025-08-20 DOI: 10.1016/j.ymben.2025.08.005

Byung Tae Lee , Omkar S. Mohite , Mun Su Kwon , Hahk-Soo Kang , Tilmann Weber , Sang Yup Lee , Hyun Uk Kim

Secondary metabolites have crucial medicinal and industrial applications, but their alignment with primary metabolism remains unclear. As secondary metabolism depends on primary metabolism for precursor supply, we present a pan-reactome analysis of 242 Streptomyces strains to investigate their association and disconnection. This analysis includes phylogenetic grouping of the strains using genome data, and uniform manifold approximation and projection (UMAP) analysis of their genome-scale metabolic models (GEMs) and biosynthetic gene cluster (BGC) data, which represent biochemical reactions in primary and secondary metabolism. Subsequent correlation analysis of the preprocessed GEM and BGC data showed a Pearson correlation coefficient of 0.54, revealing both metabolic association and disconnection. In particular, among 47 precursors of polyketides, nonribosomal peptides, and hybrids, nine precursors required by these BGCs were predicted to be non-producible due to missing genes in primary metabolism or BGCs. The pan-reactome analysis facilitates the identification of precursor availability and metabolic gaps, providing insights into secondary metabolite biosynthesis.

次生代谢产物具有重要的医学和工业应用，但它们与初级代谢的一致性尚不清楚。由于次级代谢依赖于初级代谢提供前体，我们对242株链霉菌进行了泛反应组分析，以研究它们的关联和分离。该分析包括利用基因组数据对菌株进行系统发育分组，并对它们的基因组尺度代谢模型（GEMs）和生物合成基因簇（BGC）数据进行均匀流形近似和投影（UMAP）分析，这些数据代表了初级和次级代谢中的生化反应。随后对预处理后的GEM和BGC数据进行相关分析，Pearson相关系数为0.54，表明代谢相关和分离。特别是，在聚酮、非核糖体肽和杂交体的47种前体中，由于初级代谢或bgc中缺失基因，预计这些bgc所需的9种前体无法产生。泛反应组分析有助于识别前体可利用性和代谢间隙，为次级代谢物的生物合成提供见解。

{"title":"Pan-reactome analysis of Streptomyces strains reveals association and disconnection between primary and secondary metabolism","authors":"Byung Tae Lee , Omkar S. Mohite , Mun Su Kwon , Hahk-Soo Kang , Tilmann Weber , Sang Yup Lee , Hyun Uk Kim","doi":"10.1016/j.ymben.2025.08.005","DOIUrl":"10.1016/j.ymben.2025.08.005","url":null,"abstract":"<div><div>Secondary metabolites have crucial medicinal and industrial applications, but their alignment with primary metabolism remains unclear. As secondary metabolism depends on primary metabolism for precursor supply, we present a pan-reactome analysis of 242 <em>Streptomyces</em> strains to investigate their association and disconnection. This analysis includes phylogenetic grouping of the strains using genome data, and uniform manifold approximation and projection (UMAP) analysis of their genome-scale metabolic models (GEMs) and biosynthetic gene cluster (BGC) data, which represent biochemical reactions in primary and secondary metabolism. Subsequent correlation analysis of the preprocessed GEM and BGC data showed a Pearson correlation coefficient of 0.54, revealing both metabolic association and disconnection. In particular, among 47 precursors of polyketides, nonribosomal peptides, and hybrids, nine precursors required by these BGCs were predicted to be non-producible due to missing genes in primary metabolism or BGCs. The pan-reactome analysis facilitates the identification of precursor availability and metabolic gaps, providing insights into secondary metabolite biosynthesis.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 241-251"},"PeriodicalIF":6.8,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144895574","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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

Improving recombinant antibody production using FcBAR: An in situ approach to detect and amplify protein-protein interactions 利用FcBAR提高重组抗体的生产：一种原位检测和扩增蛋白质相互作用的方法。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-01 Epub Date: 2025-07-23 DOI: 10.1016/j.ymben.2025.07.006

Mina Ying Min Wu , Frances Rocamora , Mojtaba Samoudi , Caressa M. Robinson , Chih-Chung Kuo , Nuša Pristovšek , Lise Marie Grav , Helene Faustrup Kildegaard , Gyun Min Lee , Alexandre Rosa Campos , Nathan E. Lewis

Recombinant proteins, in particular monoclonal antibodies and related molecules, have become dominant therapeutics. As they are produced in mammalian cells, they require the concerted function of hundreds of host cell proteins in the protein secretion pathway. However, the comprehensive set of host cell machinery involved remains unclear. Thus, it is often unknown why some recombinant proteins fail to express well. Here we present and deploy an approach called Fc-targeting Biotinylation by Antibody Recognition (FcBAR), which allows for the in situ detection of protein-protein interactions for any recombinant protein with Fc domain. Briefly, cells are permeabilized and incubated with an anti-Fc antibody, conjugated with horseradish peroxidase. All proteins interacting with Fc-bearing proteins are then biotinylated, pulled down and identified via mass spectrometry. We applied this method on a panel of rituximab-producing CHO-S clones with a range of productivity levels. Through analysis of FcBAR protein-protein interactions and RNA-Seq, we identified protein interactions positively correlated with rituximab secretion, and tested 7 of these targets. We found overexpression of AGPAT4, EPHX1, and NSDHL significantly increased rituximab production. Thus, FcBAR provides an unbiased approach to measure PPIs supporting recombinant antibody production in situ, and can guide efforts to boost production of biotherapeutics and biosimilars by addressing production bottlenecks.

重组蛋白，特别是单克隆抗体和相关分子，已经成为主要的治疗方法。由于它们是在哺乳动物细胞中产生的，因此在蛋白质分泌途径中需要数百种宿主细胞蛋白协同作用。然而，宿主细胞机制的综合机制仍不清楚。因此，通常不知道为什么一些重组蛋白不能很好地表达。在这里，我们提出并部署了一种称为Fc靶向生物素化抗体识别（FcBAR）的方法，该方法允许原位检测任何具有Fc结构域的重组蛋白的蛋白质相互作用。简单地说，细胞渗透和培养抗fc抗体，结合辣根过氧化物酶。然后，所有与含fc蛋白相互作用的蛋白质都被生物素化，并通过质谱法进行鉴定。我们将这种方法应用于一组具有不同生产水平的产生利妥昔单抗的CHO-S克隆。通过FcBAR蛋白-蛋白相互作用和RNA-Seq分析，我们发现蛋白相互作用与利妥昔单抗分泌呈正相关，并对其中7个靶点进行了检测。我们发现过表达AGPAT4、EPHX1和NSDHL显著增加了利妥昔单抗的产量。因此，FcBAR提供了一种公正的方法来测量支持原位重组抗体生产的PPIs，并可以通过解决生产瓶颈来指导促进生物治疗药物和生物仿制药的生产。

{"title":"Improving recombinant antibody production using FcBAR: An in situ approach to detect and amplify protein-protein interactions","authors":"Mina Ying Min Wu , Frances Rocamora , Mojtaba Samoudi , Caressa M. Robinson , Chih-Chung Kuo , Nuša Pristovšek , Lise Marie Grav , Helene Faustrup Kildegaard , Gyun Min Lee , Alexandre Rosa Campos , Nathan E. Lewis","doi":"10.1016/j.ymben.2025.07.006","DOIUrl":"10.1016/j.ymben.2025.07.006","url":null,"abstract":"<div><div>Recombinant proteins, in particular monoclonal antibodies and related molecules, have become dominant therapeutics. As they are produced in mammalian cells, they require the concerted function of hundreds of host cell proteins in the protein secretion pathway. However, the comprehensive set of host cell machinery involved remains unclear. Thus, it is often unknown why some recombinant proteins fail to express well. Here we present and deploy an approach called Fc-targeting Biotinylation by Antibody Recognition (FcBAR), which allows for the <em>in situ</em> detection of protein-protein interactions for any recombinant protein with Fc domain. Briefly, cells are permeabilized and incubated with an anti-Fc antibody, conjugated with horseradish peroxidase. All proteins interacting with Fc-bearing proteins are then biotinylated, pulled down and identified via mass spectrometry. We applied this method on a panel of rituximab-producing CHO-S clones with a range of productivity levels. Through analysis of FcBAR protein-protein interactions and RNA-Seq, we identified protein interactions positively correlated with rituximab secretion, and tested 7 of these targets. We found overexpression of AGPAT4, EPHX1, and NSDHL significantly increased rituximab production. Thus, FcBAR provides an unbiased approach to measure PPIs supporting recombinant antibody production <em>in situ</em>, and can guide efforts to boost production of biotherapeutics and biosimilars by addressing production bottlenecks.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 174-184"},"PeriodicalIF":6.8,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144718113","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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 genome-scale CRISPR deletion screen in Chinese hamster ovary cells reveals essential regions of the coding and non-coding genome 中国仓鼠卵巢细胞基因组级CRISPR缺失筛选揭示了编码和非编码基因组的重要区域

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-01 Epub Date: 2025-08-18 DOI: 10.1016/j.ymben.2025.08.007

Federico De Marco , Ivy Rose Sebastian , Antonino Napoleone , Alexander Molin , Markus Riedl , Nina Bydlinski , Krishna Motheramgari , Mohamed K. Hussein , Lovro Kramer , Thomas Kelly , Thomas Jostock , Nicole Borth

The biopharmaceutical sector relies on CHO cells to investigate biological processes and as the preferred host for production of biotherapeutics. Simultaneously, advancements in CHO cell genome assembly have provided insights for developing sophisticated genetic engineering strategies. While the majority of these efforts have focused on coding genes, with some interest in transcribed non-coding RNAs (e.g., microRNAs and lncRNAs), there remains a lack of genome-wide systematic studies that precisely examine the remaining 90 % of the genome and its impact on cellular phenotypes. This unannotated “dark matter” includes regulatory elements and other poorly characterized genomic features that may be potentially critical for cell behaviour. In this study, we deployed a genome-scale CRISPR screening platform with 112,272 paired guide RNAs targeting 14,034 genomic regions for complete deletion of 150 kb long sections. This platform enabled the execution of a negative screen that selectively identified dying cells to determine regions essential for cell survival. By using paired gRNAs, we overcame the intrinsic limitations of traditional frameshift strategies, which will likely have little or no effect on the non-coding genome. This study revealed 427 regions essential for CHO cell survival, many of which currently lack gene annotation or known functions. For these regions, we present their annotation status, transcriptional activity and annotated chromatin states. Selected regions, particularly those lacking all of the above, were individually deleted to confirm their essentiality. This work sheds a novel light on a substantial portion of the mammalian genome that has been traditionally difficult to investigate and therefore neglected. Notably, the fact that the deletion of some of these regions is lethal to cells suggests they encode critical regulatory functions. A better genome-wide understanding of these functions could open new avenues for engineering cells with improved bioprocess relevant properties.

生物制药部门依靠CHO细胞来研究生物过程，并作为生产生物治疗药物的首选宿主。同时，CHO细胞基因组组装的进展为开发复杂的基因工程策略提供了见解。虽然这些研究大多集中在编码基因上，对转录的非编码rna（如microrna和lncrna）也有一些兴趣，但仍然缺乏精确检查其余90%基因组及其对细胞表型影响的全基因组系统研究。这个未注释的“暗物质”包括调控元件和其他可能对细胞行为至关重要的基因组特征。在这项研究中，我们部署了一个基因组规模的CRISPR筛选平台，其中有112,272对引导rna，针对14,034个基因组区域，完全删除150 kb长的片段。该平台能够执行阴性筛选，选择性地鉴定死亡细胞，以确定细胞存活所必需的区域。通过使用配对grna，我们克服了传统移码策略的内在局限性，传统移码策略可能对非编码基因组影响很小或没有影响。这项研究揭示了427个对CHO细胞存活至关重要的区域，其中许多区域目前缺乏基因注释或已知功能。对于这些区域，我们给出了它们的注释状态、转录活性和注释的染色质状态。个别删除了选定的区域，特别是不具备上述所有条件的区域，以确认其重要性。这项工作为哺乳动物基因组的很大一部分提供了新的视角，这些基因组在传统上很难研究，因此被忽视了。值得注意的是，其中一些区域的缺失对细胞是致命的，这表明它们编码了关键的调节功能。对这些功能更好的全基因组理解可以为改进生物过程相关特性的工程细胞开辟新的途径。

{"title":"A genome-scale CRISPR deletion screen in Chinese hamster ovary cells reveals essential regions of the coding and non-coding genome","authors":"Federico De Marco , Ivy Rose Sebastian , Antonino Napoleone , Alexander Molin , Markus Riedl , Nina Bydlinski , Krishna Motheramgari , Mohamed K. Hussein , Lovro Kramer , Thomas Kelly , Thomas Jostock , Nicole Borth","doi":"10.1016/j.ymben.2025.08.007","DOIUrl":"10.1016/j.ymben.2025.08.007","url":null,"abstract":"<div><div>The biopharmaceutical sector relies on CHO cells to investigate biological processes and as the preferred host for production of biotherapeutics. Simultaneously, advancements in CHO cell genome assembly have provided insights for developing sophisticated genetic engineering strategies. While the majority of these efforts have focused on coding genes, with some interest in transcribed non-coding RNAs (e.g., microRNAs and lncRNAs), there remains a lack of genome-wide systematic studies that precisely examine the remaining 90 % of the genome and its impact on cellular phenotypes. This unannotated “dark matter” includes regulatory elements and other poorly characterized genomic features that may be potentially critical for cell behaviour. In this study, we deployed a genome-scale CRISPR screening platform with 112,272 paired guide RNAs targeting 14,034 genomic regions for complete deletion of 150 kb long sections. This platform enabled the execution of a negative screen that selectively identified dying cells to determine regions essential for cell survival. By using paired gRNAs, we overcame the intrinsic limitations of traditional frameshift strategies, which will likely have little or no effect on the non-coding genome. This study revealed 427 regions essential for CHO cell survival, many of which currently lack gene annotation or known functions. For these regions, we present their annotation status, transcriptional activity and annotated chromatin states. Selected regions, particularly those lacking all of the above, were individually deleted to confirm their essentiality. This work sheds a novel light on a substantial portion of the mammalian genome that has been traditionally difficult to investigate and therefore neglected. Notably, the fact that the deletion of some of these regions is lethal to cells suggests they encode critical regulatory functions. A better genome-wide understanding of these functions could open new avenues for engineering cells with improved bioprocess relevant properties.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 194-207"},"PeriodicalIF":6.8,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144885422","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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

Retrofitting Escherichia coli for de novo production of rare L-sorbose from abundant D-glucose 改造大肠杆菌，使其从丰富的d -葡萄糖中重新生产稀有的l -山梨糖。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-01 Epub Date: 2025-08-16 DOI: 10.1016/j.ymben.2025.08.006

Jayce E. Taylor , Trevor Gannalo , Bryant Luu , Dileep Sai Kumar Palur , Augustine Arredondo , Ian C. Anderson , Twisha Dasgupta , John Didzbalis , Justin B. Siegel , Shota Atsumi

Monosaccharides exist in either “D” or “L” conformations, with L-sugars being much less abundant in nature and therefore classified as “rare sugars.” Rare sugars hold significant potential due to their unique interactions with biological systems, offering health, food, and crop benefits. One such sugar, L-sorbose, serves as a critical precursor to Vitamin C and offers a low-calorie, moderately sweet alternative to table sugar, being 60–70 % as sweet but with only 25 % of the caloric value. However, the broader study and application of rare sugars, including L-sorbose, are constrained by their high cost and limited availability. To address this challenge, we developed a biosynthetic strategy to convert the abundant and inexpensive D-sugar D-glucose into the rare L-sugar L-sorbose using microbial production. By utilizing phosphorylation and dephosphorylation steps to thermodynamically drive carbon flux, efficient production of 14.5 g L⁻¹ L-sorbose was achieved under test tube conditions. Additionally, this pathway results in the co-production of D-sedoheptulose, a non-sweet, rare sugar shown to inhibit C6 sugar consumption in humans by modulating energy metabolism. The dual production of L-sorbose and D-sedoheptulose presents unique opportunities for applications in food and health sciences. This study demonstrates microbial production as a promising platform for rare L-sugar biosynthesis and provides a generalizable strategy for converting abundant D-sugars into underexplored L-sugars. Expanding access to L-sugars enables deeper investigations into their biological functions, metabolic pathways, and industrial applications. By advancing both fundamental sugar metabolism research and microbial production strategies, this study broadens the scope of rare sugar utilization.

单糖以“D”或“L”构象存在，L糖在自然界中的含量要少得多，因此被归类为“稀有糖”。稀有糖具有巨大的潜力，因为它们与生物系统的独特相互作用，提供健康，食品和作物效益。其中一种糖，l -山梨糖，是维生素C的关键前体，提供了一种低热量、中等甜味的替代食糖，甜味为60-70%，但热量只有食糖的25%。然而，包括l -山梨糖在内的稀有糖的广泛研究和应用受到其高成本和有限可用性的限制。为了解决这一挑战，我们开发了一种生物合成策略，利用微生物生产将丰富而廉价的d -糖d -葡萄糖转化为稀有的l -糖L-sorbose。利用磷酸化和去磷酸化步骤热力学驱动碳通量，在试管条件下实现了14.5 g L-1 l -海马糖的高效生产。此外，这一途径导致D-sedoheptulose的共同产生，D-sedoheptulose是一种非甜的稀有糖，通过调节能量代谢来抑制人体对C6糖的消耗。l -山梨糖和d -糖庚糖的双重生产为食品和健康科学的应用提供了独特的机会。本研究证明微生物生产是稀有l糖生物合成的一个有前途的平台，并为将丰富的d糖转化为未开发的l糖提供了一种通用策略。扩大对l糖的获取，可以更深入地研究它们的生物学功能、代谢途径和工业应用。本研究通过推进糖代谢基础研究和微生物生产策略，拓宽了稀有糖利用的范围。

{"title":"Retrofitting Escherichia coli for de novo production of rare L-sorbose from abundant D-glucose","authors":"Jayce E. Taylor , Trevor Gannalo , Bryant Luu , Dileep Sai Kumar Palur , Augustine Arredondo , Ian C. Anderson , Twisha Dasgupta , John Didzbalis , Justin B. Siegel , Shota Atsumi","doi":"10.1016/j.ymben.2025.08.006","DOIUrl":"10.1016/j.ymben.2025.08.006","url":null,"abstract":"<div><div>Monosaccharides exist in either “D” or “L” conformations, with L-sugars being much less abundant in nature and therefore classified as “rare sugars.” Rare sugars hold significant potential due to their unique interactions with biological systems, offering health, food, and crop benefits. One such sugar, L-sorbose, serves as a critical precursor to Vitamin C and offers a low-calorie, moderately sweet alternative to table sugar, being 60–70 % as sweet but with only 25 % of the caloric value. However, the broader study and application of rare sugars, including L-sorbose, are constrained by their high cost and limited availability. To address this challenge, we developed a biosynthetic strategy to convert the abundant and inexpensive D-sugar D-glucose into the rare L-sugar L-sorbose using microbial production. By utilizing phosphorylation and dephosphorylation steps to thermodynamically drive carbon flux, efficient production of 14.5 g L<sup>−1</sup> L-sorbose was achieved under test tube conditions. Additionally, this pathway results in the co-production of D-sedoheptulose, a non-sweet, rare sugar shown to inhibit C6 sugar consumption in humans by modulating energy metabolism. The dual production of L-sorbose and D-sedoheptulose presents unique opportunities for applications in food and health sciences. This study demonstrates microbial production as a promising platform for rare L-sugar biosynthesis and provides a generalizable strategy for converting abundant D-sugars into underexplored L-sugars. Expanding access to L-sugars enables deeper investigations into their biological functions, metabolic pathways, and industrial applications. By advancing both fundamental sugar metabolism research and microbial production strategies, this study broadens the scope of rare sugar utilization.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"92 ","pages":"Pages 208-216"},"PeriodicalIF":6.8,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144873966","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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