Metabolic engineering最新文献_第10页

Engineering Escherichia coli for the production of saturated archaeal lipids 工程大肠杆菌生产饱和古菌脂质。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-12-16 DOI: 10.1016/j.ymben.2025.12.004

Jiayi Jiang , Mirthe Hoekzema , Ruben Andringa , Adriaan J. Minnaard , Arnold J.M. Driessen

Archaeal membrane phospholipids have a different chemical composition than the phospholipids found in bacteria and eukaryotes. Typically, in archaea, phospholipids consist of saturated isoprenoid chains that are ether-bonded to glycerol 1-phosphate whereas in bacteria and eukaryotes, the main phospholipids are fatty acyl chains ester-bonded to glycerol 3-phosphate. This distinct chemical structure of phospholipids is believed to play a crucial role in enabling archaea to survive extreme environments and energy-limited conditions. Escherichia coli has previously been engineered to synthesize archaeal phospholipids next to its endogenous bacterial phospholipids. Cells equipped with these mixed heterochiral membranes were found to be viable with some improvement in robustness. However, a complete biosynthetic pathway for the production of substantial amounts of saturated archaeal lipids has not yet been realized in E. coli. Here, we engineered E. coli for the production of saturated archaeal phospholipids by introducing next to the geranylgeranyl reductase (GGR) and ferredoxin (Fd) from Methanosarcina acetivorans, the pyruvate-ferredoxin oxidoreductase (PFOR) from E. coli to allow for an efficient reduction of Fd. This resulted in a strain where approximately 75 % of the produced archaeal lipids are partially or completely saturated. Importantly, E. coli cells containing this mixed heterochiral membrane showed improved resistance to both heat and cold shock as compared to native E. coli strain. This E. coli strain with saturated archaeal phospholipids can serve as a valuable model for further engineering to incorporate different types of more complex archaeal membrane lipids.

古细菌膜磷脂与细菌和真核生物中的磷脂具有不同的化学组成。通常，在古细菌中，磷脂由饱和的类异戊二烯链组成，这些链与甘油1-磷酸醚键合，而在细菌和真核生物中，主要的磷脂是与甘油3-磷酸酯键合的脂肪酰基链。这种独特的磷脂化学结构被认为在使古细菌能够在极端环境和能量有限的条件下生存方面起着至关重要的作用。大肠杆菌先前已被改造成在其内源性细菌磷脂旁边合成古细菌磷脂。配备这些混合异手性膜的细胞被发现是有活力的，并且在稳健性上有所改善。然而，在大肠杆菌中尚未实现大量饱和古细菌脂质的完整生物合成途径。在这里，我们设计大肠杆菌来生产饱和古细菌磷脂，通过引入来自Methanosarcina acetivorans的香叶基香叶基还原酶（GGR）和铁氧化还蛋白（Fd），从大肠杆菌中引入丙酮酸-铁氧化还蛋白氧化还原酶（PFOR）来实现Fd的有效还原。这导致菌株中大约75%的产生的古菌脂质部分或完全饱和。重要的是，与天然大肠杆菌菌株相比，含有这种混合异手性膜的大肠杆菌细胞对热休克和冷休克的抵抗力都有所提高。这种含有饱和古菌磷脂的大肠杆菌菌株可以作为一个有价值的模型，用于进一步的工程设计，以纳入不同类型的更复杂的古菌膜脂。

{"title":"Engineering Escherichia coli for the production of saturated archaeal lipids","authors":"Jiayi Jiang , Mirthe Hoekzema , Ruben Andringa , Adriaan J. Minnaard , Arnold J.M. Driessen","doi":"10.1016/j.ymben.2025.12.004","DOIUrl":"10.1016/j.ymben.2025.12.004","url":null,"abstract":"<div><div>Archaeal membrane phospholipids have a different chemical composition than the phospholipids found in bacteria and eukaryotes. Typically, in archaea, phospholipids consist of saturated isoprenoid chains that are ether-bonded to glycerol 1-phosphate whereas in bacteria and eukaryotes, the main phospholipids are fatty acyl chains ester-bonded to glycerol 3-phosphate. This distinct chemical structure of phospholipids is believed to play a crucial role in enabling archaea to survive extreme environments and energy-limited conditions. <em>Escherichia coli</em> has previously been engineered to synthesize archaeal phospholipids next to its endogenous bacterial phospholipids. Cells equipped with these mixed heterochiral membranes were found to be viable with some improvement in robustness. However, a complete biosynthetic pathway for the production of substantial amounts of saturated archaeal lipids has not yet been realized in <em>E. coli</em>. Here, we engineered <em>E. coli</em> for the production of saturated archaeal phospholipids by introducing next to the geranylgeranyl reductase (GGR) and ferredoxin (Fd) from <em>Methanosarcina acetivorans</em>, the pyruvate-ferredoxin oxidoreductase (PFOR) from <em>E. coli</em> to allow for an efficient reduction of Fd. This resulted in a strain where approximately 75 % of the produced archaeal lipids are partially or completely saturated. Importantly, <em>E. coli</em> cells containing this mixed heterochiral membrane showed improved resistance to both heat and cold shock as compared to native <em>E. coli</em> strain. This <em>E. coli</em> strain with saturated archaeal phospholipids can serve as a valuable model for further engineering to incorporate different types of more complex archaeal membrane lipids.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 213-222"},"PeriodicalIF":6.8,"publicationDate":"2025-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145777266","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

Lacto-N-tetraose biosynthesis from lactose via metabolically rewired Escherichia coli 通过代谢重组的大肠杆菌从乳糖合成乳酸- n -四糖

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-12-11 DOI: 10.1016/j.ymben.2025.12.001

Dileep Sai Kumar Palur , Shannon R. Pressley , Alex McGill , Yuanyuan Bai , Hai Yu , Xi Chen , Shota Atsumi

Human milk oligosaccharides (HMOs), such as lacto-N-tetraose (LNT), play critical roles in infant health by shaping gut microbiota and modulating immune function. While LNT is already produced at industrial scales, efficient microbial routes to more complex HMOs derived from LNT remain limited. Here, we established a simplified microbial platform in Escherichia coli that produces LNT directly from lactose as the sole carbon and precursor source. A key innovation was construction of a strain library with tunable β-galactosidase (LacZ) activity, enabling controlled lactose hydrolysis to generate glucose and galactose for UDP-sugar biosynthesis while preserving sufficient intact lactose as the scaffold for LNT assembly. Quantitative profiling of intracellular UDP-sugars further guided identification of metabolic bottlenecks. The optimized strain achieved co-production of 2.4 g/L LNT and 2.0 g/L lacto-N-triose II (LNT II) from 10 g/L lactose. This streamlined strategy demonstrates the feasibility of producing LNT from a single substrate and provides a versatile foundation for scalable microbial biosynthesis of more complex HMOs.

人乳低聚糖（HMOs），如乳酸- n -四糖（LNT），通过塑造肠道微生物群和调节免疫功能在婴儿健康中发挥关键作用。虽然LNT已经在工业规模上生产，但由LNT衍生的更复杂的HMOs的有效微生物途径仍然有限。在这里，我们在大肠杆菌中建立了一个简化的微生物平台，该平台直接从乳糖作为唯一的碳和前体来源产生LNT。一个关键的创新是构建了一个具有可调β-半乳糖苷酶（LacZ）活性的菌株库，可以控制乳糖水解以产生葡萄糖和半乳糖用于udp -糖的生物合成，同时保留足够的完整乳糖作为LNT组装的支架。细胞内udp -糖的定量分析进一步指导了代谢瓶颈的识别。优化菌株以10 g/L乳糖为原料，可同时生产2.4 g/L LNT和2.0 g/L LNT II。这种简化的策略证明了从单一底物生产LNT的可行性，并为更复杂的HMOs的可扩展微生物生物合成提供了一个通用的基础。

{"title":"Lacto-N-tetraose biosynthesis from lactose via metabolically rewired Escherichia coli","authors":"Dileep Sai Kumar Palur , Shannon R. Pressley , Alex McGill , Yuanyuan Bai , Hai Yu , Xi Chen , Shota Atsumi","doi":"10.1016/j.ymben.2025.12.001","DOIUrl":"10.1016/j.ymben.2025.12.001","url":null,"abstract":"<div><div>Human milk oligosaccharides (HMOs), such as lacto-<em>N</em>-tetraose (LNT), play critical roles in infant health by shaping gut microbiota and modulating immune function. While LNT is already produced at industrial scales, efficient microbial routes to more complex HMOs derived from LNT remain limited. Here, we established a simplified microbial platform in <em>Escherichia coli</em> that produces LNT directly from lactose as the sole carbon and precursor source. A key innovation was construction of a strain library with tunable β-galactosidase (LacZ) activity, enabling controlled lactose hydrolysis to generate glucose and galactose for UDP-sugar biosynthesis while preserving sufficient intact lactose as the scaffold for LNT assembly. Quantitative profiling of intracellular UDP-sugars further guided identification of metabolic bottlenecks. The optimized strain achieved co-production of 2.4 g/L LNT and 2.0 g/L lacto-<em>N</em>-triose II (LNT II) from 10 g/L lactose. This streamlined strategy demonstrates the feasibility of producing LNT from a single substrate and provides a versatile foundation for scalable microbial biosynthesis of more complex HMOs.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 182-191"},"PeriodicalIF":6.8,"publicationDate":"2025-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145731784","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

PET-FBA: A lightweight enzyme allocation and thermodynamics-constrained flux analysis approach to explore Escherichia coli metabolic adaptation to intracellular acidification PET-FBA：一种轻量级酶分配和热力学约束通量分析方法，用于探索大肠杆菌对细胞内酸化的代谢适应

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-12-11 DOI: 10.1016/j.ymben.2025.12.003

Chao Wu , Jeffrey N. Law , Onyeka Onyenemezu , Jetendra K. Roy , Peter C. St. John , Robert L. Jernigan , Yannick J. Bomble , Laura Jarboe

Escherichia coli employs diverse strategies to adapt to acidic environments that disrupt enzyme activity and the thermodynamic feasibility of essential reactions. To understand the impact of pH stress on cell metabolism, we present the PET-FBA (pH-, Enzyme protein allocation-, and Thermodynamics-constrained Flux Balance Analysis) framework. PET-FBA extends genome-scale modeling by integrating enzyme protein costs and reaction Gibbs free energy changes. Additionally, by incorporating pH-dependent enzyme kinetics in response to intracellular acidification, this framework enables the simulation of E. coli's metabolic adjustments across varying external pH levels. The model's accuracy is validated by comparing in silico growth simulations with experimental measurements under both anaerobic and aerobic conditions, as well as in silico gene knockouts of essential genes. By explicitly incorporating pH effects, our model accurately replicates the metabolic shift towards lactate production as the primary fermentation product at low pH in anaerobic conditions. This shift is only predicted when enzyme kinetics are dynamically adjusted as a function of pH. Further analysis revealed that this shift can be attributed to the reduced protein efficiency of the acetyl-CoA branch compared to lactate dehydrogenase under acidic stress, which then becomes crucial for maintaining NAD regeneration and cell growth at low pH. Furthermore, we identified strategies for enhancing cell growth under acidic anaerobic conditions by improving the enzyme activity of lactate dehydrogenase and pyruvate formate lyase, which increases NAD production efficiency and reduces enzyme protein allocation costs. Designed as a lightweight yet versatile framework, PET-FBA enables efficient genome-scale metabolic analysis. Using E. coli as a model system, our framework provides a systematic approach to understanding metabolic responses to environmental stress, pinpointing key metabolic bottlenecks, and identifying potential targets for strain optimization. This work also highlights the critical role of intracellular acidification in shaping enzyme performance and microbial adaptation. The PET-FBA framework is implemented as a Python package at https://github.com/Chaowu88/etfba, with detailed documentation provided at https://etfba.readthedocs.io.

大肠杆菌采用多种策略来适应酸性环境，这种环境会破坏酶的活性和基本反应的热力学可行性。为了了解pH胁迫对细胞代谢的影响，我们提出了PET-FBA （pH-，酶蛋白分配-和热力学约束通量平衡分析）框架。PET-FBA通过整合酶蛋白成本和反应吉布斯自由能变化扩展了基因组尺度模型。此外，通过结合pH依赖的酶动力学来响应细胞内酸化，该框架能够模拟大肠杆菌在不同外部pH水平下的代谢调节。通过比较厌氧和有氧条件下的硅生长模拟与实验测量，以及必要基因的硅基因敲除，验证了该模型的准确性。通过明确纳入pH效应，我们的模型准确地复制了厌氧条件下低pH下乳酸生产作为初级发酵产物的代谢转变。只有当酶动力学作为ph的函数被动态调整时，这种转变才会被预测。进一步的分析表明，这种转变可归因于酸性胁迫下乙酰辅酶a分支与乳酸脱氢酶相比蛋白质效率的降低，这对于在低ph下维持NAD再生和细胞生长至关重要。我们确定了在酸性厌氧条件下通过提高乳酸脱氢酶和丙酮酸甲酸裂解酶的酶活性来促进细胞生长的策略，从而提高NAD的生产效率并降低酶蛋白的分配成本。PET-FBA是一种轻量级的通用框架，可实现高效的基因组级代谢分析。使用大肠杆菌作为模型系统，我们的框架提供了一个系统的方法来理解代谢对环境应激的反应，精确定位关键的代谢瓶颈，并确定菌株优化的潜在目标。这项工作还强调了细胞内酸化在塑造酶性能和微生物适应中的关键作用。PET-FBA框架作为Python包在https://github.com/Chaowu88/etfba上实现，详细文档在https://etfba.readthedocs.io上提供。

{"title":"PET-FBA: A lightweight enzyme allocation and thermodynamics-constrained flux analysis approach to explore Escherichia coli metabolic adaptation to intracellular acidification","authors":"Chao Wu , Jeffrey N. Law , Onyeka Onyenemezu , Jetendra K. Roy , Peter C. St. John , Robert L. Jernigan , Yannick J. Bomble , Laura Jarboe","doi":"10.1016/j.ymben.2025.12.003","DOIUrl":"10.1016/j.ymben.2025.12.003","url":null,"abstract":"<div><div><em>Escherichia coli</em> employs diverse strategies to adapt to acidic environments that disrupt enzyme activity and the thermodynamic feasibility of essential reactions. To understand the impact of pH stress on cell metabolism, we present the PET-FBA (pH-, Enzyme protein allocation-, and Thermodynamics-constrained Flux Balance Analysis) framework. PET-FBA extends genome-scale modeling by integrating enzyme protein costs and reaction Gibbs free energy changes. Additionally, by incorporating pH-dependent enzyme kinetics in response to intracellular acidification, this framework enables the simulation of <em>E. coli</em>'s metabolic adjustments across varying external pH levels. The model's accuracy is validated by comparing <em>in silico</em> growth simulations with experimental measurements under both anaerobic and aerobic conditions, as well as <em>in silico</em> gene knockouts of essential genes. By explicitly incorporating pH effects, our model accurately replicates the metabolic shift towards lactate production as the primary fermentation product at low pH in anaerobic conditions. This shift is only predicted when enzyme kinetics are dynamically adjusted as a function of pH. Further analysis revealed that this shift can be attributed to the reduced protein efficiency of the acetyl-CoA branch compared to lactate dehydrogenase under acidic stress, which then becomes crucial for maintaining NAD regeneration and cell growth at low pH. Furthermore, we identified strategies for enhancing cell growth under acidic anaerobic conditions by improving the enzyme activity of lactate dehydrogenase and pyruvate formate lyase, which increases NAD production efficiency and reduces enzyme protein allocation costs. Designed as a lightweight yet versatile framework, PET-FBA enables efficient genome-scale metabolic analysis. Using <em>E. coli</em> as a model system, our framework provides a systematic approach to understanding metabolic responses to environmental stress, pinpointing key metabolic bottlenecks, and identifying potential targets for strain optimization. This work also highlights the critical role of intracellular acidification in shaping enzyme performance and microbial adaptation. The PET-FBA framework is implemented as a Python package at <span><span>https://github.com/Chaowu88/etfba</span><svg><path></path></svg></span>, with detailed documentation provided at <span><span>https://etfba.readthedocs.io</span><svg><path></path></svg></span>.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 202-212"},"PeriodicalIF":6.8,"publicationDate":"2025-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145731099","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

Machine learning-driven optimization of metabolic balance for β-carotene production 机器学习驱动的β-胡萝卜素生产代谢平衡优化

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-12-11 DOI: 10.1016/j.ymben.2025.12.002

Weiming Tu , Jiabao Xu , Yongshuo Ma , Constantinos Katsimpouras , Gregory Stephanopoulos

Balancing metabolic pathways is critical for engineering microbial platforms to efficiently and robustly synthesize value-added bioproducts. In the oleaginous yeast Yarrowia lipolytica engineered for β-carotene production, lipid synthesis supports carotenoid storage but also competes with carotenoid synthesis for cellular resources, necessitating precise regulation for optimal resource allocation. In this study, we establish a machine learning framework that captures the complex interactions among three key metabolic modules for β-carotene synthesis: the mevalonate pathway (precursor supply for β-carotene), lipid synthesis (storage capacity), and the β-carotene synthetic cluster (product formation). This computational framework enables the prediction of β-carotene output based on gene combinations and guides iterative gene integration strategies across these interconnected pathways to optimize production. Using this approach, the best-performing strain YLT226 achieved a 7-fold increase in β-carotene titer compared to the initial strain YLT001 through nine rounds of guided gene integration. This work provides a promising strategy for understanding and engineering metabolic flux distributions.

平衡代谢途径是工程微生物平台高效、稳健地合成增值生物产品的关键。在生产β-胡萝卜素的聚脂耶氏酵母中，脂质合成支持类胡萝卜素的储存，但也与类胡萝卜素合成竞争细胞资源，需要精确调节以实现最佳资源分配。在本研究中，我们建立了一个机器学习框架，该框架捕获了β-胡萝卜素合成的三个关键代谢模块之间的复杂相互作用：甲羟戊酸途径（β-胡萝卜素的前体供应），脂质合成（储存能力）和β-胡萝卜素合成簇（产物形成）。该计算框架能够基于基因组合预测β-胡萝卜素的产量，并指导跨这些相互关联途径的迭代基因整合策略以优化产量。利用这种方法，通过9轮引导基因整合，表现最好的菌株YLT226的β-胡萝卜素滴度比初始菌株YLT001提高了7倍。这项工作为理解和工程代谢通量分布提供了一个有前途的策略。

{"title":"Machine learning-driven optimization of metabolic balance for β-carotene production","authors":"Weiming Tu , Jiabao Xu , Yongshuo Ma , Constantinos Katsimpouras , Gregory Stephanopoulos","doi":"10.1016/j.ymben.2025.12.002","DOIUrl":"10.1016/j.ymben.2025.12.002","url":null,"abstract":"<div><div>Balancing metabolic pathways is critical for engineering microbial platforms to efficiently and robustly synthesize value-added bioproducts. In the oleaginous yeast <em>Yarrowia lipolytica</em> engineered for β-carotene production, lipid synthesis supports carotenoid storage but also competes with carotenoid synthesis for cellular resources, necessitating precise regulation for optimal resource allocation. In this study, we establish a machine learning framework that captures the complex interactions among three key metabolic modules for β-carotene synthesis: the mevalonate pathway (precursor supply for β-carotene), lipid synthesis (storage capacity), and the β-carotene synthetic cluster (product formation). This computational framework enables the prediction of β-carotene output based on gene combinations and guides iterative gene integration strategies across these interconnected pathways to optimize production. Using this approach, the best-performing strain YLT226 achieved a 7-fold increase in β-carotene titer compared to the initial strain YLT001 through nine rounds of guided gene integration. This work provides a promising strategy for understanding and engineering metabolic flux distributions.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 192-201"},"PeriodicalIF":6.8,"publicationDate":"2025-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145731098","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 resource balance in the oleaginous yeast Rhodotorula toruloides 产油酵母的代谢通量和资源平衡

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-12-01 DOI: 10.1016/j.ymben.2025.11.012

Eric J. Mooney , Patrick F. Suthers , Wheaton L. Schroeder , Hoang V. Dinh , Xi Li , Yihui Shen , Tianxia Xiao , Catherine M. Call , Heide Baron , Arjuna M. Subramanian , Daniel R. Weilandt , Felix C. Keber , Martin Wühr , Joshua D. Rabinowitz , Costas D. Maranas

The yeast Rhodotorula toruloides is a promising bioproduction organism due to its high lipid yields and ability to grow on cheap and abundant substrates. Quantitative, systems-level assessment of its metabolic activity is accordingly merited. Resource-balance analysis (RBA) models capture not only reaction stoichiometry but also enzyme requirements for catalysis, providing valuable tools for understanding metabolic trade-offs and optimizing metabolic engineering strategies. Here, we present systems-level measurements of R. toruloides metabolic flux based on isotope tracing and metabolic flux analysis. In combination with new proteomic measurements, these flux data are used to parameterize a genome-scale resource balance model rtRBA. We find that S. cerevisiae and R. toruloides grow at nearly indistinguishable rates using similar biosynthetic but dramatically different central metabolic programs. R. toruloides consumes one-fifth as much glucose, which it metabolizes primarily via the pentose phosphate pathway and TCA cycle unlike primarily glycolysis in S. cerevisiae. Overall, across these two divergent yeasts, protein abundances aligned more closely than metabolic flux. Resource balance modeling of these metabolic programs predicts superior theoretical yields but lower productivities in R. toruloides than S. cerevisiae for industrial chemicals, highlighting the value of rapid glucose uptake for productivity but respiratory metabolism for yields.

酵母是一种很有前途的生物生产生物，因为它具有高脂产量和在廉价和丰富的底物上生长的能力。因此，有必要对其代谢活动进行定量的系统级评估。资源平衡分析（RBA）模型不仅捕获了反应化学计量学，还捕获了催化所需的酶，为理解代谢权衡和优化代谢工程策略提供了有价值的工具。在这里，我们提出了基于同位素示踪和代谢通量分析的系统级测量。结合新的蛋白质组学测量，这些通量数据用于参数化基因组尺度的资源平衡模型rtRBA。我们发现酿酒葡萄球菌和toruloides的生长速度几乎无法区分，使用相似的生物合成，但中心代谢程序截然不同。toruloides消耗的葡萄糖是酵母的五分之一，主要通过戊糖磷酸途径和TCA循环代谢，而酿酒酵母主要通过糖酵解。总的来说，在这两种不同的酵母中，蛋白质丰度比代谢通量更接近。这些代谢程序的资源平衡模型预测，在工业化学品方面，toruloides的理论产量高于S. cerevisiae，但生产率低于S. toruloides，突出了快速葡萄糖摄取对生产率的价值，而呼吸代谢对产量的价值。

{"title":"Metabolic flux and resource balance in the oleaginous yeast Rhodotorula toruloides","authors":"Eric J. Mooney , Patrick F. Suthers , Wheaton L. Schroeder , Hoang V. Dinh , Xi Li , Yihui Shen , Tianxia Xiao , Catherine M. Call , Heide Baron , Arjuna M. Subramanian , Daniel R. Weilandt , Felix C. Keber , Martin Wühr , Joshua D. Rabinowitz , Costas D. Maranas","doi":"10.1016/j.ymben.2025.11.012","DOIUrl":"10.1016/j.ymben.2025.11.012","url":null,"abstract":"<div><div>The yeast <em>Rhodotorula toruloides</em> is a promising bioproduction organism due to its high lipid yields and ability to grow on cheap and abundant substrates. Quantitative, systems-level assessment of its metabolic activity is accordingly merited. Resource-balance analysis (RBA) models capture not only reaction stoichiometry but also enzyme requirements for catalysis, providing valuable tools for understanding metabolic trade-offs and optimizing metabolic engineering strategies. Here, we present systems-level measurements of <em>R. toruloides</em> metabolic flux based on isotope tracing and metabolic flux analysis. In combination with new proteomic measurements, these flux data are used to parameterize a genome-scale resource balance model rtRBA. We find that <em>S. cerevisiae</em> and <em>R. toruloides</em> grow at nearly indistinguishable rates using similar biosynthetic but dramatically different central metabolic programs. <em>R. toruloides</em> consumes one-fifth as much glucose, which it metabolizes primarily via the pentose phosphate pathway and TCA cycle unlike primarily glycolysis in <em>S. cerevisiae.</em> Overall, across these two divergent yeasts, protein abundances aligned more closely than metabolic flux. Resource balance modeling of these metabolic programs predicts superior theoretical yields but lower productivities in <em>R. toruloides</em> than <em>S. cerevisiae</em> for industrial chemicals, highlighting the value of rapid glucose uptake for productivity but respiratory metabolism for yields.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 169-181"},"PeriodicalIF":6.8,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145657141","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

Pilot production of P(3HB-co-4HB) by engineered Halomonas bluephagenesis harboring an endogenous plasmid grown on glucose 在葡萄糖上培养内源性质粒的工程蓝芽胞菌中试生产P（3HB-co-4HB）

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-30 DOI: 10.1016/j.ymben.2025.11.017

Rou Wen , Yiling Chen , Jiale Wang , Weinan Yang , Fang Yang , Qiong Wu , Fuqing Wu , Xu Yan , Guo-Qiang Chen

Poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (P (3HB-co-4HB) or P34HB) is a promising biopolyester for applications in food packaging, medical sutures, drug delivery, and tissue engineering due to its tunable thermomechanical properties. However, industrial-scale production of P34HB from glucose remains challenging. In this study, scalable P34HB production by engineered Halomonas bluephagenesis was developed. A de novo 4HB synthesis pathway was introduced into an endogenous toxin-antitoxin plasmid, enabling stable expression in the absence of antibiotics. Promoter engineering and pathway optimization fine-tuned 4HB molar ratio in P34HB from 18 to 39 mol%. The engineered H. bluephagenesis WR3 demonstrated successful scale-up from 7-L to 100-L and 5000-L bioreactors, achieving a maximum cell dry weight (CDW) of 72 g/L and 84% P (3HB-co-30 mol% 4HB) from the cultures in 7-L bioreactor. In further scale-up studies, H. bluephagenesis WR3 maintained comparable 4HB ratios, producing 69 g/L and 71 g/L CDW containing 74% and 61% P34HB copolymer in 100-L and 5000-L scale bioreactors, respectively. The amorphous P (3HB-co-30 mol% 4HB) exhibited high ductility, with an elongation at break of over 800% and a Young's modulus of 164 MPa. Additionally, morphology engineering and a controllable cell lysis were applied to enhance downstream processing efficiency. The optimized H. bluephagenesis WR25 produced 97 g/L CDW containing 83% P (3HB-co-20 mol% 4HB) in 7-L bioreactor, and 83 g/L CDW with 80% P34HB in 100-L bioreactor, while maintaining a consistently high glucose to P34HB conversion efficiency of 37%. This study provides a robust and cost-effective platform for industrial P34HB production from glucose harboring the toxin-antitoxin stable plasmid encoded with the 4HB pathway.

聚（3-羟基丁酸-co-4-羟基丁酸）（P （3HB-co-4HB）或P34HB）是一种很有前途的生物聚酯，由于其可调节的热机械性能，可用于食品包装，医疗缝合，药物输送和组织工程。然而，从葡萄糖中提取P34HB的工业规模生产仍然具有挑战性。在这项研究中，开发了可扩展的工程盐单胞菌蓝发生产P34HB。将一种新的4HB合成途径引入内源性毒素-抗毒素质粒中，使其在没有抗生素的情况下稳定表达。启动子工程和途径优化将P34HB中的4HB摩尔比从18 mol%调整到39 mol%。经改造的蓝发芽孢杆菌WR3成功地从7-L生物反应器放大到100-L和5000-L生物反应器，在7-L生物反应器中培养的细胞最大干重（CDW）为72 g/L， P （3HB-co-30 mol% 4HB）为84%。在进一步的放大研究中，H. bluephagenesis WR3保持了相当的4HB比率，在100-L和5000-L规模的生物反应器中分别产生69 g/L和71 g/L含有74%和61% P34HB共聚物的CDW。无定形P （3hb -co- 30mol % 4HB）具有较高的延展性，断裂伸长率超过800%，杨氏模量为164mpa。此外，形态学工程和可控的细胞裂解技术可以提高下游加工效率。优化后的H. bluephagenesis WR25在7-L的生物反应器中产生含有83% P （3HB-co-20 mol% 4HB）的CDW 97 g/L，在100-L的生物反应器中产生含有80% P34HB的CDW 83 g/L，同时保持了37%的高葡萄糖到P34HB的转化效率。该研究为从葡萄糖中提取含有4HB途径编码的毒素-抗毒素稳定质粒的P34HB工业生产提供了一个强大而经济的平台。

{"title":"Pilot production of P(3HB-co-4HB) by engineered Halomonas bluephagenesis harboring an endogenous plasmid grown on glucose","authors":"Rou Wen , Yiling Chen , Jiale Wang , Weinan Yang , Fang Yang , Qiong Wu , Fuqing Wu , Xu Yan , Guo-Qiang Chen","doi":"10.1016/j.ymben.2025.11.017","DOIUrl":"10.1016/j.ymben.2025.11.017","url":null,"abstract":"<div><div>Poly (3-hydroxybutyrate<em>-co</em>-4-hydroxybutyrate) (P (3HB-<em>co</em>-4HB) or P34HB) is a promising biopolyester for applications in food packaging, medical sutures, drug delivery, and tissue engineering due to its tunable thermomechanical properties. However, industrial-scale production of P34HB from glucose remains challenging. In this study, scalable P34HB production by engineered <em>Halomonas bluephagenesis</em> was developed. A <em>de novo</em> 4HB synthesis pathway was introduced into an endogenous toxin-antitoxin plasmid, enabling stable expression in the absence of antibiotics. Promoter engineering and pathway optimization fine-tuned 4HB molar ratio in P34HB from 18 to 39 mol%. The engineered <em>H. bluephagenesis</em> WR3 demonstrated successful scale-up from 7-L to 100-L and 5000-L bioreactors, achieving a maximum cell dry weight (CDW) of 72 g/L and 84% P (3HB-<em>co</em>-30 mol% 4HB) from the cultures in 7-L bioreactor. In further scale-up studies, <em>H. bluephagenesis</em> WR3 maintained comparable 4HB ratios, producing 69 g/L and 71 g/L CDW containing 74% and 61% P34HB copolymer in 100-L and 5000-L scale bioreactors, respectively. The amorphous P (3HB-<em>co</em>-30 mol% 4HB) exhibited high ductility, with an elongation at break of over 800% and a Young's modulus of 164 MPa. Additionally, morphology engineering and a controllable cell lysis were applied to enhance downstream processing efficiency. The optimized <em>H. bluephagenesis</em> WR25 produced 97 g/L CDW containing 83% P (3HB-<em>co</em>-20 mol% 4HB) in 7-L bioreactor, and 83 g/L CDW with 80% P34HB in 100-L bioreactor, while maintaining a consistently high glucose to P34HB conversion efficiency of 37%. This study provides a robust and cost-effective platform for industrial P34HB production from glucose harboring the toxin-antitoxin stable plasmid encoded with the 4HB pathway.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 153-168"},"PeriodicalIF":6.8,"publicationDate":"2025-11-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145619676","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

Unlocking the potential of unique genes in cyanobacterial alkane synthesis 解锁独特的基因在蓝藻烷烃合成的潜力

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-27 DOI: 10.1016/j.ymben.2025.11.016

Humaira Parveen , Vineesha Garg , Piyush Pachauri , Mohd Azeem Khan , Syed Shams Yazdani

<div><div>Alkanes are considered among the most promising candidates for next-generation biofuels. Amongst various pathways discovered for alkane production, the cyanobacterial AAR (acyl ACP reductase) - ADO (aldehyde deformylating oxygenase) pathway has been the most studied pathway. Considering that cyanobacteria have the innate ability to produce alkanes, they can serve as an excellent chassis for sustainable biofuel production. In the first report of the AAR-ADO pathway, it was recorded that there are 17 unique genes present in the genome of only alkane-producing cyanobacteria. However, except for the role of AAR and ADO, none of the other genes have been implicated in alkane production so far. In this study, we performed overexpression and/or deletion of all 17 unique genes in <em>Synechococcus elongatus</em> PCC7942 and evaluated their role in growth, photosynthetic efficiency and alkane production. Based on the essentiality feature of genes in the cell survival of PCC7942, 9 essential genes were overexpressed, and 8 non-essential genes were knocked out in PCC7942. Among the essential genes that made a significant impact on alkane production, the overexpression of <em>Synpcc7942_1772</em> (encoding small subunit ribosomal protein) and <em>Synpcc7942_2212</em> (encoding large subunit ribosomal protein) led to ∼3.3-fold and ∼4.1-fold increased alkane production, respectively, suggesting a previously unrecognized link between translational machinery and metabolic pathway, while co-expression of <em>aar</em> and <em>ado</em> together increased alkane production by ∼5-fold. For the non-essential genes, the deletion of <em>Synpcc7942_0452</em> (encoding hypothetical protein), <em>Synpcc7942_1223</em> (encoding DevC), and <em>Synpcc7942_1918</em> (encoding UDP‐glucose: tetrahydro biopterin glucosyltransferase) led to the complete abolition of alkane production, indicating their critical roles. On the other hand, the deletion of <em>Synpcc7942_0544</em> and <em>Synpcc7942_0619,</em> both encoding hypothetical proteins, led to a ∼5.6-fold and ∼4.4-fold increase in intracellular alkane production, respectively. Measurement of photosynthetic efficiency via Dual PAM (Pulse-Amplitude Modulated) fluorometry revealed a correlation between higher alkane production and increased photosynthetic efficiency, which the genome-scale metabolic model of PCC7942 also validated. Upon further detailed investigation of the genes making a large impact on alkane production, we identified <em>Synpcc7942_0619</em> and <em>Synpcc7942_1223</em> gene products as potential transporters based on the AlphaFold structure model and TMHMM (Transmembrane Hidden Markov Model) plot. The <em>Synpcc7942_0619</em> encodes a DedA family transporter whose overexpression led to ∼1.5-fold higher extracellular alkane production, while deletion had a reverse effect. On the other hand, <em>Synpcc7942_1223</em> encodes the DevC component of the tripartite efflux system, whose overexpression inc

烷烃被认为是下一代生物燃料最有希望的候选者之一。在已发现的各种烷烃生成途径中，蓝藻的AAR（酰基ACP还原酶）- ADO（醛去甲酰基加氧酶）途径是研究最多的途径。考虑到蓝藻具有产生烷烃的先天能力，它们可以作为可持续生物燃料生产的优秀基础。在AAR-ADO通路的第一篇报道中，记录到仅产烷烃蓝藻的基因组中就存在17个独特的基因。然而，除了AAR和ADO的作用，到目前为止，没有其他基因参与烷烃的产生。在这项研究中，我们对长聚球菌PCC7942中所有17个独特基因进行了过表达和/或缺失，并评估了它们在生长、光合效率和烷烃生产中的作用。基于基因在PCC7942细胞存活中的本质特征，PCC7942中有9个必需基因过表达，8个非必需基因被敲除。在对烷烃生成产生显著影响的必要基因中，Synpcc7942_1772（编码小亚基核糖体蛋白）和Synpcc7942_2212（编码大亚基核糖体蛋白）的过表达分别导致烷烃生成增加~ 3.3倍和~ 4.1倍，这表明翻译机制和代谢途径之间存在先前未被认识到的联系，而aar和ado的共表达使烷烃生成增加~ 5倍。对于非必需基因，Synpcc7942_0452（编码假想蛋白）、Synpcc7942_1223（编码DevC）和Synpcc7942_1918（编码UDP‐glucose: tetrahydro biopterin glucosyltransferase）的缺失导致烷烃生产完全取消，表明它们的关键作用。另一方面，编码假想蛋白的Synpcc7942_0544和Synpcc7942_0619的缺失分别导致细胞内烷烃产量增加5.6倍和4.4倍。通过双脉冲振幅调制（Dual PAM）荧光法测量光合效率，发现更高的烷烃产量与更高的光合效率之间存在相关性，PCC7942的基因组尺度代谢模型也验证了这一点。在对影响烷烃产生的基因进行进一步详细研究后，我们基于AlphaFold结构模型和TMHMM（跨膜隐马尔可夫模型）图确定了Synpcc7942_0619和Synpcc7942_1223基因产物是潜在的转运体。Synpcc7942_0619编码一个DedA家族转运蛋白，其过表达导致细胞外烷烃产量增加约1.5倍，而缺失具有相反的效果。另一方面，Synpcc7942_1223编码了三边外排系统的DevC组分，其过表达使胞内烷烃增加了约13倍，缺失导致胞外和胞内烷烃消失，表明其与烷烃合成途径核心酶的重要相互作用。这些发现强调了翻译、光合作用、转运和烷烃生物合成之间的复杂相互作用，并为通过系统水平的代谢工程开发具有提高烷烃产量的基因工程菌株提供了基础。

{"title":"Unlocking the potential of unique genes in cyanobacterial alkane synthesis","authors":"Humaira Parveen , Vineesha Garg , Piyush Pachauri , Mohd Azeem Khan , Syed Shams Yazdani","doi":"10.1016/j.ymben.2025.11.016","DOIUrl":"10.1016/j.ymben.2025.11.016","url":null,"abstract":"<div><div>Alkanes are considered among the most promising candidates for next-generation biofuels. Amongst various pathways discovered for alkane production, the cyanobacterial AAR (acyl ACP reductase) - ADO (aldehyde deformylating oxygenase) pathway has been the most studied pathway. Considering that cyanobacteria have the innate ability to produce alkanes, they can serve as an excellent chassis for sustainable biofuel production. In the first report of the AAR-ADO pathway, it was recorded that there are 17 unique genes present in the genome of only alkane-producing cyanobacteria. However, except for the role of AAR and ADO, none of the other genes have been implicated in alkane production so far. In this study, we performed overexpression and/or deletion of all 17 unique genes in <em>Synechococcus elongatus</em> PCC7942 and evaluated their role in growth, photosynthetic efficiency and alkane production. Based on the essentiality feature of genes in the cell survival of PCC7942, 9 essential genes were overexpressed, and 8 non-essential genes were knocked out in PCC7942. Among the essential genes that made a significant impact on alkane production, the overexpression of <em>Synpcc7942_1772</em> (encoding small subunit ribosomal protein) and <em>Synpcc7942_2212</em> (encoding large subunit ribosomal protein) led to ∼3.3-fold and ∼4.1-fold increased alkane production, respectively, suggesting a previously unrecognized link between translational machinery and metabolic pathway, while co-expression of <em>aar</em> and <em>ado</em> together increased alkane production by ∼5-fold. For the non-essential genes, the deletion of <em>Synpcc7942_0452</em> (encoding hypothetical protein), <em>Synpcc7942_1223</em> (encoding DevC), and <em>Synpcc7942_1918</em> (encoding UDP‐glucose: tetrahydro biopterin glucosyltransferase) led to the complete abolition of alkane production, indicating their critical roles. On the other hand, the deletion of <em>Synpcc7942_0544</em> and <em>Synpcc7942_0619,</em> both encoding hypothetical proteins, led to a ∼5.6-fold and ∼4.4-fold increase in intracellular alkane production, respectively. Measurement of photosynthetic efficiency via Dual PAM (Pulse-Amplitude Modulated) fluorometry revealed a correlation between higher alkane production and increased photosynthetic efficiency, which the genome-scale metabolic model of PCC7942 also validated. Upon further detailed investigation of the genes making a large impact on alkane production, we identified <em>Synpcc7942_0619</em> and <em>Synpcc7942_1223</em> gene products as potential transporters based on the AlphaFold structure model and TMHMM (Transmembrane Hidden Markov Model) plot. The <em>Synpcc7942_0619</em> encodes a DedA family transporter whose overexpression led to ∼1.5-fold higher extracellular alkane production, while deletion had a reverse effect. On the other hand, <em>Synpcc7942_1223</em> encodes the DevC component of the tripartite efflux system, whose overexpression inc","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 136-152"},"PeriodicalIF":6.8,"publicationDate":"2025-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145611806","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

Model-guided metabolic engineering of 2-phenylethanol in Arabidopsis 模型引导的2-苯乙醇在拟南芥中的代谢工程

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-21 DOI: 10.1016/j.ymben.2025.11.011

Joseph H. Lynch , Shaunak Ray , Clint Chapple , Natalia Dudareva , John A. Morgan

2-Phenylethanol (2-PE) is a natural aromatic compound with properties that make it a potential biological oxygenate for petroleum-derived gasoline. In plants, 2-PE biosynthesis competes with the phenylpropanoid pathway for the common precursor, phenylalanine (Phe). The phenylpropanoid pathway directs significant carbon flux towards the formation of lignin, a major biopolymer in plant cell walls that impedes the process of biofuel production. Prior genetic engineering in Arabidopsis used acetaldehyde synthase (AAS) in tandem with phenylacetaldehyde reductase (PAR) to redirect part of the carbon flux from lignin towards 2-PE production as a value-added product. To identify the bottleneck(s) in 2-PE biosynthesis, we established a baseline by generating transgenic Arabidopsis overexpressing AAS and PAR. Next, fluxes to 2-PE and lignin were calculated based on the time course of isotopic enrichment of downstream metabolites after feeding with ¹³C₆-ring labeled Phe, which revealed a limitation in Phe precursor availability. To increase substrate availability in plants, we tested two independent strategies by crossing lines with the highest AAS/PAR expression to: (1) Arabidopsis overexpressing a feedback-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase known to increase Phe production, and (2) the pal1/pal2 double mutant known to reduce the activity of the competing enzyme, phenylalanine ammonia lyase (PAL). Although both strategies increased 2-PE production in both Arabidopsis stem and leaves, the second strategy had a higher impact. To identify additional metabolic targets, we performed a metabolic control analysis, which revealed that the plastidial Phe transporter limits flux towards 2-PE formation. To avoid this transport step, the PAR/AAS tandem construct was fused to a sequence encoding a chloroplast signal peptide to target 2-PE biosynthesis to plastids. The direct availability of Phe to AAS in plastids combined with the lack of competition with cytosolic PAL resulted in significantly elevated 2-PE levels. Thus, integrating metabolic control analysis with experimental validation of model predictions establishes a foundation for the rational engineering of 2-PE in plants.

2-苯乙醇（2-PE）是一种天然芳香化合物，其特性使其成为石油衍生汽油的潜在生物氧合物。在植物中，2-PE的生物合成与苯丙素途径竞争共同前体苯丙氨酸（Phe）。苯丙烷途径将大量的碳通量导向木质素的形成，木质素是植物细胞壁中阻碍生物燃料生产过程的主要生物聚合物。先前的拟南芥基因工程使用乙醛合成酶（AAS）与苯乙醛还原酶（PAR）串联，将木质素的部分碳通量定向到2-PE生产中，作为增值产品。为了确定2-PE生物合成的瓶颈，我们通过培养过表达AAS和PAR的转基因拟南芥建立了基线。接下来，根据喂食13c6环标记的苯丙氨酸后下游代谢物同位素富集的时间过程计算了对2-PE和木质素的通量，这揭示了苯丙氨酸前体可利用性的局限性。为了提高植物的底物利用度，我们通过杂交AAS/PAR表达最高的系，测试了两种独立的策略：(1)拟南芥过度表达一种反馈不敏感的3-脱氧-d -阿拉伯-heptulosonate 7-磷酸（DAHP）合成酶，已知会增加Phe的产生；(2)已知会降低竞争酶苯丙氨酸解氨酶（PAL）活性的pal1/pal2双突变体。虽然这两种策略都增加了拟南芥茎和叶片的2-PE产量，但第二种策略的影响更大。为了确定其他代谢靶点，我们进行了代谢控制分析，结果显示，质体Phe转运蛋白限制了2-PE形成的通量。为了避免这一运输步骤，PAR/AAS串联结构被融合到编码叶绿体信号肽的序列中，以靶向质体的2-PE生物合成。在质体中对AAS的Phe直接可用性，加上缺乏与细胞质PAL的竞争，导致2-PE水平显著升高。因此，将代谢控制分析与模型预测的实验验证相结合，为植物2-PE的合理工程设计奠定了基础。

{"title":"Model-guided metabolic engineering of 2-phenylethanol in Arabidopsis","authors":"Joseph H. Lynch , Shaunak Ray , Clint Chapple , Natalia Dudareva , John A. Morgan","doi":"10.1016/j.ymben.2025.11.011","DOIUrl":"10.1016/j.ymben.2025.11.011","url":null,"abstract":"<div><div>2-Phenylethanol (2-PE) is a natural aromatic compound with properties that make it a potential biological oxygenate for petroleum-derived gasoline. In plants, 2-PE biosynthesis competes with the phenylpropanoid pathway for the common precursor, phenylalanine (Phe). The phenylpropanoid pathway directs significant carbon flux towards the formation of lignin, a major biopolymer in plant cell walls that impedes the process of biofuel production. Prior genetic engineering in Arabidopsis used acetaldehyde synthase (AAS) in tandem with phenylacetaldehyde reductase (PAR) to redirect part of the carbon flux from lignin towards 2-PE production as a value-added product. To identify the bottleneck(s) in 2-PE biosynthesis, we established a baseline by generating transgenic Arabidopsis overexpressing AAS and PAR. Next, fluxes to 2-PE and lignin were calculated based on the time course of isotopic enrichment of downstream metabolites after feeding with <sup>13</sup>C<sub>6</sub>-ring labeled Phe, which revealed a limitation in Phe precursor availability. To increase substrate availability in plants, we tested two independent strategies by crossing lines with the highest <em>AAS/PAR</em> expression to: (1) Arabidopsis overexpressing a feedback-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase known to increase Phe production, and (2) the <em>pal1/pal2</em> double mutant known to reduce the activity of the competing enzyme, phenylalanine ammonia lyase (PAL). Although both strategies increased 2-PE production in both Arabidopsis stem and leaves, the second strategy had a higher impact. To identify additional metabolic targets, we performed a metabolic control analysis, which revealed that the plastidial Phe transporter limits flux towards 2-PE formation. To avoid this transport step, the PAR/AAS tandem construct was fused to a sequence encoding a chloroplast signal peptide to target 2-PE biosynthesis to plastids. The direct availability of Phe to AAS in plastids combined with the lack of competition with cytosolic PAL resulted in significantly elevated 2-PE levels. Thus, integrating metabolic control analysis with experimental validation of model predictions establishes a foundation for the rational engineering of 2-PE in plants.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 124-135"},"PeriodicalIF":6.8,"publicationDate":"2025-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145567417","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

Engineering amino acid-derived malonyl-CoA pathways to boost polyketide production in Yarrowia lipolytica 工程氨基酸衍生的丙二酰辅酶a途径促进聚脂耶氏菌的聚酮生产

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-19 DOI: 10.1016/j.ymben.2025.11.015

Jinpeng Wang , Yuxiang Hong , Zizhao Wu , Ayelet Fishman , Peng Xu

Malonyl-CoA is a central precursor involved in the synthesis of various bio-based chemicals, including polyketides, fatty acids, and flavonoids. However, the production of these chemicals is often limited by the availability of malonyl-CoA. Based on retrosynthesis principles, we designed two thermodynamically favorable malonyl-CoA pathways using L-glutamate and L-aspartate as substrates. The novel pathways leverage oxidative deamination and decarboxylation reactions and efficiently channel metabolic flux toward malonyl-CoA, resulting in increased production of total polyketides beyond the capacity of the native acetyl-CoA carboxylase route using glucose as substrate. We also discovered a new-to-nature polyketide (4-hydroxy-6-hydroxyethyl-2-pyrone) derived from the side activity of the TAL pathway, reaching 6.4 g/L in Y. lipolytica. This work highlights the utility of the novel malonyl-CoA pathways in enhancing polyketide production, and the possibility of upcycling abundant amino acids or protein waste in the animal farming or meat industry to produce high-value nonnatural polyketides.

丙二酰辅酶a是多种生物基化学物质合成的中心前体，包括聚酮、脂肪酸和类黄酮。然而，这些化学品的生产往往受到丙二醇辅酶a供应的限制。基于反合成原理，我们以谷氨酸和天冬氨酸为底物设计了两种热力学有利的丙二酰辅酶a途径。新途径利用氧化脱胺和脱羧反应，有效地将代谢通量导向丙二酰辅酶a，导致总聚酮的产量增加，超出了以葡萄糖为底物的天然乙酰辅酶a羧化酶途径的能力。我们还发现了一种新的天然聚酮（4-羟基-6-羟乙基-2-吡咯酮），来自TAL途径的副活性，在脂肪菌中达到6.4 g/L。这项工作强调了新的丙二酰辅酶a途径在提高聚酮生产中的效用，以及在动物养殖或肉类工业中对丰富的氨基酸或蛋白质废物进行升级循环以生产高价值非天然聚酮的可能性。

{"title":"Engineering amino acid-derived malonyl-CoA pathways to boost polyketide production in Yarrowia lipolytica","authors":"Jinpeng Wang , Yuxiang Hong , Zizhao Wu , Ayelet Fishman , Peng Xu","doi":"10.1016/j.ymben.2025.11.015","DOIUrl":"10.1016/j.ymben.2025.11.015","url":null,"abstract":"<div><div>Malonyl-CoA is a central precursor involved in the synthesis of various bio-based chemicals, including polyketides, fatty acids, and flavonoids. However, the production of these chemicals is often limited by the availability of malonyl-CoA. Based on retrosynthesis principles, we designed two thermodynamically favorable malonyl-CoA pathways using L-glutamate and L-aspartate as substrates. The novel pathways leverage oxidative deamination and decarboxylation reactions and efficiently channel metabolic flux toward malonyl-CoA, resulting in increased production of total polyketides beyond the capacity of the native acetyl-CoA carboxylase route using glucose as substrate. We also discovered a new-to-nature polyketide (4-hydroxy-6-hydroxyethyl-2-pyrone) derived from the side activity of the TAL pathway, reaching 6.4 g/L in <em>Y. lipolytica</em>. This work highlights the utility of the novel malonyl-CoA pathways in enhancing polyketide production, and the possibility of upcycling abundant amino acids or protein waste in the animal farming or meat industry to produce high-value nonnatural polyketides.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 99-109"},"PeriodicalIF":6.8,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145553880","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

Elucidating the itaconic acid pathway dynamics in Saccharomyces cerevisiae 衣康酸在酿酒酵母中的途径动力学研究

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-19 DOI: 10.1016/j.ymben.2025.11.010

Roy Eerlings , Tobias Karmainski , Andreas Müsgens , Philipp Demling , Samira van den Bogaard , Makarius Baier , Alexander Deitert , Amila Vejzovic , Vanessa Veccari , Lillith Yöndem , Tobias Alter , Lars M. Blank

Itaconic acid, a versatile platform chemical, has garnered significant attention due to its broad use in polymers, resins, and bio-based materials. Although fungi, especially Aspergillus and Ustilago, are the main producers of itaconic acid, reprogramming yeast species like Saccharomyces cerevisiae and Yarrowia lipolytica as alternative production platforms offers advantages for industrial bioproduction, including rapid growth, accessible genetic tools, and well-established fermentation methods. In this study, we systematically investigated the fungal itaconic acid pathway dynamics in S. cerevisiae, identifying the Ustilago metabolic route and mitochondrial transport mechanism as the most efficient. Notably, the heterologous produced itaconic acid was predominantly secreted by the yeast transformants. With in silico methods, we confirmed the role of Aqr1p, Dtr1p, and Qdr3p in itaconic acid transport in S. cerevisiae. Significant increases in itaconic acid biosynthesis were obtained when the main promiscuous itaconic acid transporter Dtr1p was swapped with the specialized Ustilago itaconic acid transporter Itp1, reaching titers of up to 1.3 g/L in shake flask cultivations. Transferring to 3.8 L bioreactor fermentations achieved a final titer of 4.7 g/L, the highest itaconic acid titer reported in S. cerevisiae to date. Although current yeast production levels are still below those of natural fungal producers, the molecular and mechanistic insights gained here are useful for improving itaconic acid biosynthesis, both in yeast and in the existing fungal production systems.

衣康酸是一种多功能平台化学品，由于其在聚合物、树脂和生物基材料中的广泛应用而引起了极大的关注。虽然真菌，尤其是曲霉和黑霉是衣康酸的主要生产者，但像酿酒酵母和脂解耶氏酵母这样的重编程酵母物种作为替代生产平台，为工业生物生产提供了优势，包括快速生长，易于获取的遗传工具和完善的发酵方法。在本研究中，我们系统地研究了酿酒酵母的衣康酸途径动态，确定了黑穗病菌的代谢途径和线粒体运输机制是最有效的。值得注意的是，外源产生的衣康酸主要由酵母转化子分泌。通过计算机方法，我们证实了Aqr1p、Dtr1p和Qdr3p在酿酒酵母衣康酸转运中的作用。当主要的混杂衣康酸转运蛋白Dtr1p与特殊的衣康酸转运蛋白Itp1交换时，衣康酸的生物合成显著增加，在摇瓶培养中滴度高达1.3 g/L。转入3.8 L生物反应器发酵，最终滴度达到4.7 g/L，这是迄今为止酿酒酵母中报道的最高衣康酸滴度。虽然目前酵母菌的生产水平仍低于天然真菌生产者，但在此获得的分子和机制见解对于改善酵母和现有真菌生产系统中的衣康酸生物合成是有用的。

{"title":"Elucidating the itaconic acid pathway dynamics in Saccharomyces cerevisiae","authors":"Roy Eerlings , Tobias Karmainski , Andreas Müsgens , Philipp Demling , Samira van den Bogaard , Makarius Baier , Alexander Deitert , Amila Vejzovic , Vanessa Veccari , Lillith Yöndem , Tobias Alter , Lars M. Blank","doi":"10.1016/j.ymben.2025.11.010","DOIUrl":"10.1016/j.ymben.2025.11.010","url":null,"abstract":"<div><div>Itaconic acid, a versatile platform chemical, has garnered significant attention due to its broad use in polymers, resins, and bio-based materials. Although fungi, especially <em>Aspergillus</em> and <em>Ustilago</em>, are the main producers of itaconic acid, reprogramming yeast species like <em>Saccharomyces cerevisiae</em> and <em>Yarrowia lipolytica</em> as alternative production platforms offers advantages for industrial bioproduction, including rapid growth, accessible genetic tools, and well-established fermentation methods. In this study, we systematically investigated the fungal itaconic acid pathway dynamics in <em>S. cerevisiae</em>, identifying the <em>Ustilago</em> metabolic route and mitochondrial transport mechanism as the most efficient. Notably, the heterologous produced itaconic acid was predominantly secreted by the yeast transformants. With <em>in silico</em> methods, we confirmed the role of Aqr1p, Dtr1p, and Qdr3p in itaconic acid transport in <em>S. cerevisiae</em>. Significant increases in itaconic acid biosynthesis were obtained when the main promiscuous itaconic acid transporter Dtr1p was swapped with the specialized <em>Ustilago</em> itaconic acid transporter Itp1, reaching titers of up to 1.3 g/L in shake flask cultivations. Transferring to 3.8 L bioreactor fermentations achieved a final titer of 4.7 g/L, the highest itaconic acid titer reported in <em>S. cerevisiae</em> to date. Although current yeast production levels are still below those of natural fungal producers, the molecular and mechanistic insights gained here are useful for improving itaconic acid biosynthesis, both in yeast and in the existing fungal production systems.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 110-123"},"PeriodicalIF":6.8,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145559771","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