Metabolic engineering最新文献_第3页

Engineered non-canonical reductive TCA pathway drives high-yield succinic acid biosynthesis in Yarrowia lipolytica 工程非规范还原TCA途径驱动多脂耶氏菌高产琥珀酸生物合成

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-03-01 Epub Date: 2025-11-06 DOI: 10.1016/j.ymben.2025.11.003

Huilin Tao, Aomei Hao, Xiaoyue Pan, Yutao Zhong, Zhiyong Cui, Qingsheng Qi

The reductive tricarboxylic acid (rTCA) cycle is a crucial metabolic pathway employed in the microbial production of C4-dicarboxylic acids, especially succinic acid (SA). However, the inherent redox constraints associated with this cycle pose significant limitations on the yields of SA. Here, we address this critical bottleneck by engineering a non-canonical reductive TCA (Nc-rTCA) pathway in oleaginous yeast Yarrowia lipolytica. Our approach substitutes the NADH-dependent conversion of oxaloacetate to fumarate in the native rTCA cycle with an engineered cascade utilizing aspartate aminotransferase, aspartate ammonia-lyase, and glutamate dehydrogenase, effectively decoupling SA synthesis from NADH limitations. This NADPH-dependent module resulted in a remarkable 1.28-fold increase in fumarate titer. Further metabolic optimization in the engineered strain Ncr12 minimized malate byproduct formation, achieving an SA titer of 98.16 g/L with a high yield of 0.91 g/g glucose in 5-L bioreactors. Importantly, the Nc-rTCA pathway demonstrated potential for industrial application, yielding 74.34 g/L SA at 0.98 g/g glucose from lignocellulosic hydrolysate and 117.74 g/L SA at 0.74 g/g from glycerol. Our findings address the longstanding redox imbalance issues that have challenged rTCA-based engineering and establish a scalable platform for bio-based C4-dicarboxylic acid production.

还原性三羧酸（rTCA）循环是微生物生产c4 -二羧酸，特别是琥珀酸（SA）的重要代谢途径。然而，与此循环相关的固有氧化还原约束对SA的产率造成了重大限制。在这里，我们通过在产油酵母解脂耶氏菌中设计非规范还原TCA （Nc-rTCA）途径来解决这一关键瓶颈。我们的方法用利用天冬氨酸转氨酶、天冬氨酸解氨酶和谷氨酸脱氢酶的工程级联代替了天然rTCA循环中NADH依赖的草酰乙酸转化为富马酸，有效地将SA合成与NADH限制解耦。这种nadph依赖模块导致富马酸滴度显著增加1.28倍。对工程菌株Ncr12进行了进一步的代谢优化，最大限度地减少了苹果酸副产物的生成，在5-L生物反应器中，SA滴度达到98.16 g/L，葡萄糖产量达到0.91 g/g。重要的是，Nc-rTCA途径显示出了工业应用的潜力，从木质纤维素水解物中以0.98 g/g葡萄糖产生74.34 g/L SA，从甘油中以0.74 g/g产生117.74 g/L SA。我们的研究结果解决了长期存在的氧化还原不平衡问题，该问题挑战了基于rtca的工程，并建立了一个可扩展的生物基c4 -二羧酸生产平台。

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引用次数: 0

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 : 2026-03-01 Epub 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。

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引用次数: 0

Predictive CRISPR-mediated gene downregulation for enhanced production of sustainable aviation fuel precursor in Pseudomonas putida 预测crispr介导的基因下调对恶臭假单胞菌可持续航空燃料前体生产的增强

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-03-01 Epub Date: 2025-11-06 DOI: 10.1016/j.ymben.2025.11.007

Ian S. Yunus , David N. Carruthers , Yan Chen , Jennifer W. Gin , Edward E.K. Baidoo , Christopher J. Petzold , Hector Garcia Martin , Paul D. Adams , Aindrila Mukhopadhyay , Taek Soon Lee

CRISPR interference (CRISPRi) has emerged as a valuable tool for redirecting metabolic flux to enhance bioproduction. However, its application is often constrained by two challenges: (i) rationally identifying effective gene targets for downregulation and (ii) efficiently constructing multiplexed CRISPRi systems. In this study, we address both challenges by integrating a computational prioritization tool with a versatile assembly method for building multiplexed CRISPRi systems. FluxRETAP (Flux-Reaction Target Prioritization) accurately identified gene targets whose knockdown led to substantial increase of isoprenol titers in Pseudomonas putida KT2440, outperforming a conventional non-computational, pathway-guided target selection. The highest isoprenol titer of nearly 1.5 g/L was achieved by knocking down PP_4118 (a gene encoding α-ketoglutarate dehydrogenase). The use of VAMMPIRE (Versatile Assembly Method for MultiPlexing CRISPRi-mediated downREgulation) enabled accurate assembly of CRISPRi constructs containing up to five sgRNA arrays, reducing context dependency and achieving uniform, position-independent gene downregulation. The integration of FluxRETAP and VAMMPIRE has the potential to advance metabolic engineering by rapidly identifying CRISPRi-mediated knockdowns and knockdown combinations that enhance bioproduction titers, with potential applicability to other microbial systems.

CRISPR干扰（CRISPRi）已成为重定向代谢通量以增强生物生产的一种有价值的工具。然而，它的应用往往受到两个挑战的制约：(1)合理地识别有效的基因下调靶点；(2)高效地构建多路CRISPRi系统。在本研究中，我们通过将计算优先级工具与构建多路CRISPRi系统的通用组装方法集成来解决这两个挑战。FluxRETAP （Flux-Reaction Target priority）准确地鉴定了敲除导致恶臭假单胞菌KT2440异戊二醇滴度大幅增加的基因靶标，优于传统的非计算、途径引导的靶标选择。通过敲除PP_4118 （α-酮戊二酸脱氢酶基因），获得了最高的异戊二醇滴度，接近1.5 g/L。使用VAMMPIRE (Versatile Assembly Method for MultiPlexing crispr -mediated downREgulation)，可以精确组装包含多达5个sgRNA阵列的CRISPRi构建体，减少上下文依赖性，实现一致的、不依赖于位置的基因下调。FluxRETAP和VAMMPIRE的整合有可能通过快速识别crispr介导的敲低和敲低组合来推进代谢工程，从而提高生物生产滴度，并可能适用于其他微生物系统。

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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 : 2026-03-01 Epub 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。

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引用次数: 0

Corrigendum to “Microbial production of propionic acid through a novel β-alanine route” [Metabol. Eng. (2026) 219–231 93] “微生物通过一种新的β-丙氨酸途径生产丙酸”的勘误表[代谢]。Eng。（2026） 219-231 93]

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-03-01 Epub Date: 2025-11-12 DOI: 10.1016/j.ymben.2025.11.008

Da-Hee Ahn , Yoo-Sung Ko , Cindy Pricilia Surya Prabowo , Sang Yup Lee

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 : 2026-03-01 Epub 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。

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引用次数: 0

Metabolic engineering of a plasmid-free, non-auxotrophic Escherichia coli for efficient glycolate production 无质粒、非营养型大肠杆菌的代谢工程，用于高效的乙醇酸生产

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-03-01 Epub Date: 2026-01-06 DOI: 10.1016/j.ymben.2025.12.006

Jinyu Cheng , Xinyi Jiang , Xiaomin Li , Wanqing Wei , Jia Liu , Wei Song , Guipeng Hu , Cong Gao , Liming Liu

Glycolate, an α-hydroxycarboxylic acid, is widely used in industries such as bioplastics, food, and pharmaceuticals. However, current microbial production methods are limited by the use of plasmids and chemical inducers, hindering their industrial scalability. In this study, a stable and efficient Escherichia coli platform was developed for glycolate production. The glycolate biosynthetic pathway was reconstructed through the identification of a highly efficient glyoxylate reductase (GhrA) from Acetobacter aceti. Carbon flux toward glycolate synthesis was optimized through strategies including enhancing precursor supply, blocking competing pathways, and fine-tuning gene copy numbers. Cofactor engineering was employed by engineering GhrA cofactor preference from NADPH to NADH. Additionally, a non-auxotrophic strain (eliminating exogenous nutrient requirements) for glycolate production was engineered by implementing a growth-stage-dependent molecular switch to dynamically regulate the expression of isocitrate dehydrogenase. Through fermentation optimization, the engineered strain E. coli GA26 achieved a glycolate titer of 81.5 g/L, a yield of 0.49 g/g glucose, and a productivity of 1.9 g/L/h in a 5-L bioreactor, representing the highest reported glycolate titer from glucose to date. These results pave the way for sustainable and cost-effective industrial glycolate production.

乙醇酸是一种α-羟基羧酸，广泛应用于生物塑料、食品和制药等行业。然而，目前的微生物生产方法受到质粒和化学诱导剂使用的限制，阻碍了它们的工业可扩展性。在本研究中，建立了一个稳定高效的生产乙醇酸的大肠杆菌平台。通过鉴定乙酰杆菌中高效的乙醛酸还原酶（GhrA），重构了乙醇酸生物合成途径。通过增加前体供应、阻断竞争途径和微调基因拷贝数等策略，优化了乙醇酸合成的碳通量。辅助因子工程是将GhrA辅助因子的偏好从NADPH转移到NADH。此外，通过实施生长阶段依赖的分子开关来动态调节异柠檬酸脱氢酶的表达，设计了一种用于乙醇酸生产的非营养不良菌株（消除了外源营养需求）。通过发酵优化，工程菌株E. coli GA26在5-L生物反应器中乙醇酸滴度为81.5 g/L，葡萄糖产率为0.49 g/g，产率为1.9 g/L/h，是迄今为止报道的葡萄糖乙醇酸滴度最高的菌株。这些结果为可持续和具有成本效益的工业乙醇酸生产铺平了道路。

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引用次数: 0

Metabolic engineering of Corynebacterium glutamicum for vitamin B12-independent production of 3-hydroxypropionic acid 不依赖维生素b12生产3-羟基丙酸的谷氨酸棒状杆菌代谢工程

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-03-01 Epub Date: 2025-11-17 DOI: 10.1016/j.ymben.2025.11.013

Cheon Woo Moon , Mohammad Rifqi Ghiffary , Cindy Pricilia Surya Prabowo , Hyun Uk Kim , Sang Yup Lee

3-Hydroxypropionic acid (3-HP) is a versatile platform chemical with broad applications, serving as a precursor for the synthesis of value-added chemicals as well as the biodegradable polymers. However, current industrial production of 3-HP relies on chemical synthesis, which requires harmful raw materials and harsh reaction conditions. As a sustainable alternative, microbial biosynthesis of 3-HP has gained increasing attention. Yet, most reported pathways remain constrained by their dependence on vitamin B₁₂, a costly cofactor that limits scalability in industrial applications. Here, we report the development of a Corynebacterium glutamicum strain capable of high-level fermentative production of 3-HP from glucose via the introduction of a vitamin B₁₂-independent, β-alanine-derived pathway. Candidate genes for the conversion of β-alanine to 3-HP were first screened, and the optimized pathway was subsequently introduced into a previously developed β-alanine-overproducing BAL10 strain. By eliminating competing pathways to increase precursor availability, redirecting carbon flux through the pentose phosphate pathway to improve cofactor balance, strengthening the β-alanine biosynthetic pathway, and identifying a previously uncharacterized 3-HP transporter followed by fine-tuning its expression, the final engineered strain produced 126.3 g/L of 3-HP in high-inoculum fed-batch fermentation, with a yield of 0.36 g/g glucose and an overall productivity of 1.75 g/L/h. These results demonstrate the feasibility of a vitamin B₁₂-independent pathway for high-level 3-HP production, highlighting its potential for sustainable and scalable industrial application.

3-羟基丙酸（3-HP）是一种用途广泛的多用途平台化学品，可作为合成增值化学品和生物可降解聚合物的前体。然而，目前3-HP的工业生产依赖于化学合成，这需要有害的原料和恶劣的反应条件。微生物合成3-HP作为一种可持续的替代方法，越来越受到人们的关注。然而，大多数报道的途径仍然受到维生素B12依赖的限制，维生素B12是一种昂贵的辅助因子，限制了工业应用的可扩展性。在这里，我们报道了一种谷氨酸棒状杆菌菌株的发展，该菌株能够通过引入维生素b12独立的β-丙氨酸衍生途径从葡萄糖中高水平发酵生产3-HP。首先筛选β-丙氨酸转化为3-HP的候选基因，然后将优化后的途径引入先前开发的β-丙氨酸过量产生的BAL10菌株中。通过消除竞争途径以提高前体利用率，通过戊糖磷酸途径重定向碳通量以改善辅助因子平衡，加强β-丙氨酸生物合成途径，并确定先前未被表征的3-HP转运体并对其表达进行微调，最终工程菌株在高接种量补料分批发酵中产生126.3 g/L的3-HP，产量为0.36 g/g葡萄糖，总产量为1.75 g/L/h。这些结果证明了不依赖维生素b12的高水平3-HP生产途径的可行性，突出了其可持续和可扩展的工业应用潜力。

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引用次数: 0

Engineering artificial biosynthetic pathways for efficient microbial production of psilocybin and psilocin 高效微生物生产裸盖菇素和裸盖菇素的工程非天然生物合成途径

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-03-01 Epub Date: 2025-11-05 DOI: 10.1016/j.ymben.2025.11.002

Cui Guo , Nguyen N.T. Luu , Maryem M. Adwer , Hemen Hosseinzadeh , Venkatesh Balan , Yajun Yan , Yuheng Lin

Psychedelic-assisted therapy is emerging as a highly promising approach for treating depression, with psilocybin, a psychoactive compound in magic mushrooms, gaining the most recognition for its efficacy in treating post-traumatic stress disorder and treatment-resistant depression. However, its low natural abundance makes extraction costly, necessitating alternative production methods. While engineered microbial production has been explored, dependence on the CYP450 hydroxylase (PsiH) in the natural biosynthetic pathway remains a major bottleneck, limiting production efficiency. Here, we report the design, validation, and optimization of artificial biosynthetic pathways in Escherichia coli that bypass PsiH, enabling efficient psilocybin and psilocin production. De novo biosynthesis of psilocybin achieved record titers of 557.91 mg/L in shake flasks and 2.00 g/L in a bioreactor, outperforming previous microbial engineering efforts. This work demonstrates the great commercial potential of microbial psilocybin production via combinatorial metabolic engineering and synthetic biology approaches.

迷幻剂辅助疗法正在成为一种非常有前景的治疗抑郁症的方法，迷幻蘑菇中的一种精神活性化合物裸盖菇素（psilocybin）因其在治疗创伤后应激障碍和难治性抑郁症方面的功效而获得了最多的认可。然而，它的天然丰度低，开采成本高，需要其他生产方法。虽然已经探索了工程微生物生产，但对天然生物合成途径中CYP450羟化酶（PsiH）的依赖仍然是限制生产效率的主要瓶颈。在这里，我们报道了大肠杆菌中绕过PsiH的人工生物合成途径的设计、验证和优化，使裸盖菇素和裸盖菇素的高效生产成为可能。全新生物合成裸盖菇素的滴度在摇瓶中达到557.91 mg/L，在生物反应器中达到2.00 g/L，优于之前的微生物工程成果。这项工作证明了通过组合代谢工程和合成生物学方法生产微生物裸盖菇素的巨大商业潜力。

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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 : 2026-03-01 Epub 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。

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