Metabolic engineering最新文献_第2页

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 : 2026-01-06 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

Establishing heterologous betaxanthin pigment biosynthesis in cyanobacteria 蓝藻中异源β -黄质色素的生物合成

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-01-05 DOI: 10.1016/j.ymben.2026.01.002

Sayali S. Hanamghar , David A. Russo , Silas Busck Mellor , Julie A.Z. Zedler

Betalains are water-soluble pigments with two major classes: red-violet betacyanins and yellow-orange betaxanthins. These pigments are increasingly being sought after as natural replacements for synthetic pigments in the food industry. Traditionally, betalains are extracted from cultivated plants. But due to low concentrations of native pigments, the process is inherently inefficient. Now, an increase in consumer demand calls for the development of scalable and sustainable betalain production routes. To address this challenge, we introduced a heterologous pathway for the production of betaxanthins into cyanobacteria. The pathway consists of an engineered variant of the cytochrome P450 CYP76AD1 (W13L, F309L) and the l-DOPA 4,5-dioxygenase DODA1 from Beta vulgaris (beet). Introduction of the two-enzyme betaxanthin pathway in Synechocystis sp. PCC 6803 did not result in detectable betaxanthins. Subsequent metabolic adjustments to the shikimate pathway, using a feedback resistant AroG^fbr from E. coli, led to an overaccumulation of the aromatic amino acids phenylalanine, tryptophan, and tyrosine, and the production of low levels of phenylalanine-betaxanthin. Optimization of the cultivation conditions (i.e., growth in nutrient-rich medium and CO₂-enriched air) increased titers approximately 165 times and led to the production of phenylalanine-betaxanthin with a final titer of 18.2 ± 5.1 mg L⁻¹. Our work establishes a microbial system for photoautotrophic betaxanthin pigment production without the need for exogenous amino acid supplementation.

甜菜素是水溶性色素，有两大类：紫红色甜菜青素和黄橙色甜菜青素。在食品工业中，这些色素作为合成色素的天然替代品越来越受到追捧。传统上，甜菜碱是从栽培植物中提取的。但由于天然色素浓度低，这一过程本身效率就很低。现在，消费者需求的增加要求开发可扩展和可持续的甜菜生产路线。为了解决这一挑战，我们引入了一种异源途径，使蓝细菌产生β -黄素。该途径由甜菜细胞色素P450 CYP76AD1 （W13L, F309L）和l-DOPA 4,5-双加氧酶DODA1的工程变体组成。在聚囊藻（Synechocystis sp. PCC 6803）中引入双酶途径未检测到betaxanthin。随后使用来自大肠杆菌的反馈抗性AroGfbr对shikimate通路进行代谢调整，导致芳香氨基酸苯丙氨酸、色氨酸和酪氨酸的过度积累，并产生低水平的苯丙氨酸- β黄质。优化培养条件（即在营养丰富的培养基和富含二氧化碳的空气中生长），使其滴度提高了约165倍，最终产生苯丙氨酸- β黄质，最终滴度为18.2±5.1 mg L−1。我们的工作建立了一个不需要外源氨基酸补充的光自养甜菜黄素色素生产的微生物系统。

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引用次数: 0

Proteome constrained metabolic modeling of Sus scrofa muscle stem cells for cultured meat production 蛋白质组学约束下培养肉用Sus scrofa肌干细胞代谢模型的建立

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-01-03 DOI: 10.1016/j.ymben.2026.01.001

Sizhe Qiu , Eliska Kratochvilova , Wei E. Huang , Zhanfeng Cui , Tom Agnew , Aidong Yang , Hua Ye

Cultured meat has recently emerged as a sustainable alternative to traditional livestock farming and gained attention as a promising future protein source. Herein, the Sus scrofa muscle stem cell is a commonly used cell source in the cell proliferation step of cultured meat production. However, a major bottleneck of large-scale cultivation is the inhibition by secreted and accumulated lactate and ammonium in the process of S. scrofa cell proliferation. To simulate the growth and metabolism of S. scrofa muscle stem cells under different lactate and ammonium concentrations, this study constructed the first proteome constrained metabolic model for the core metabolism of S. scrofa muscle stem cells, pcPigMNet 2025. The relationship of lactate and ammonium levels with cellular metabolism was derived from growth and metabolomics data of two culture conditions with low and high initial ammonium concentrations, and then incorporated into metabolic flux simulation. Metabolic flux simulations for experimental conditions, along with perturbation simulations considering stressed non-growth associated maintenance and oxygen supply, demonstrated that pcPigMNet2025 could effectively characterize the response of the S. scrofa muscle stem cell's growth and metabolism to varying environmental conditions, shedding light on model-aided control and optimization of the cultured meat production process.

人造肉最近成为传统畜牧业的可持续替代品，并作为一种有前途的未来蛋白质来源而受到关注。在这里，Sus scrofa肌干细胞是培养肉生产中细胞增殖步骤中常用的细胞来源。然而，大规模培养的一个主要瓶颈是scrofa细胞增殖过程中分泌和积累的乳酸和铵的抑制作用。为了模拟不同乳酸和铵浓度下黑鲈肌肉干细胞的生长和代谢，本研究构建了首个黑鲈肌肉干细胞核心代谢的蛋白质组约束代谢模型pcPigMNet 2025。根据低、高初始铵浓度两种培养条件下的生长和代谢组学数据，得出乳酸和铵水平与细胞代谢的关系，并将其纳入代谢通量模拟。实验条件下的代谢通量模拟，以及考虑应激非生长相关维持和氧气供应的扰动模拟表明，pcPigMNet2025可以有效表征S. scrofa肌肉干细胞的生长和代谢对不同环境条件的响应，为模型辅助控制和优化培养肉生产过程提供了思路。

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引用次数: 0

Biosynthetic platform for orsellinic acid-derived meroterpenoids in Escherichia coli 大肠杆菌中奥塞利酸衍生巯基萜类化合物的生物合成平台

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-12-30 DOI: 10.1016/j.ymben.2025.12.008

Itsuki Tomita , Takahiro Bamba , Takanobu Yoshida , Lucília Domingues , Ryo Nasuno , Ryota Hidese , Tomohisa Hasunuma

Orsellinic acid (OSA)-derived meroterpenoids, that have an OSA backbone, are plant-derived natural products that have attracted considerable attention as pharmaceutical precursors because of their diverse pharmacological activities. Therefore, developing efficient microbial production methods is highly desirable. However, to date, only a few reports on the microbial production of OSA-derived meroterpenoids are available, and even for the precursor OSA, only minimal production levels (approximately 5 mg/L) have been achieved using engineered microbes. In this study, Escherichia coli was engineered to enable the de novo biosynthesis of OSA to establish an alternative production platform for OSA-derived meroterpenoids. The introduction of type III polyketide synthase and cyclase resulted in 1.4 mg/L production. CRISPR interference aimed at enhancing OSA production revealed that the knockdown of fadR, which is involved in malonyl-CoA consumption, was effective. Metabolome analysis was performed to evaluate the metabolic impact of the engineering strategies revealed malonyl-CoA depletion, indicating that its supply constituted a major bottleneck. Based on this insight, the overexpression of acetyl-CoA carboxylase, pantothenate kinase, and ATP citrate lyase was implemented, which increased OSA production to 202 mg/L under optimized cultivation conditions, representing a 145-fold improvement. Finally, introducing a plant-derived prenyltransferase enabled grifolic acid biosynthesis (2.5 μg/g-DCW), representing the first de novo production of OSA-derived meroterpenoids in E. coli. This study establishes E. coli as a versatile and scalable host for the biosynthesis of pharmacologically valuable meroterpenoids.

Orsellinic acid (OSA)-derived meroterpenoids，是一种具有OSA主干的植物源性天然产物，由于其多种药理活性，作为药物前体受到了广泛关注。因此，开发高效的微生物生产方法是非常必要的。然而，到目前为止，只有少数关于OSA衍生的美罗萜类化合物的微生物生产的报道，甚至对于前体OSA，也只有最小的生产水平（约5毫克/升）已经使用工程微生物实现。在本研究中，我们对大肠杆菌进行了改造，使其能够重新生物合成OSA，从而建立了OSA衍生的巯基萜类化合物的替代生产平台。引入III型聚酮合成酶和环化酶，产量为1.4 mg/L。CRISPR干扰旨在增强OSA的产生，结果表明，抑制参与丙二酰辅酶a消耗的fadR是有效的。代谢组学分析评估了工程策略的代谢影响，发现丙二酰辅酶a耗竭，表明其供应构成了主要瓶颈。在此基础上，对乙酰辅酶a羧化酶、泛酸激酶和ATP柠檬酸裂解酶进行过表达，在优化的培养条件下，OSA产量提高到202 mg/L，提高了145倍。最后，引入一种植物源戊烯基转移酶，实现了沙棘酸的生物合成（2.5 μg/g-DCW），这是首次在大肠杆菌中重新生产沙棘酸衍生的巯基萜类化合物。本研究建立了大肠杆菌作为一个多功能和可扩展的宿主，用于生物合成具有药理价值的美罗萜类化合物。

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引用次数: 0

An automated platform for accelerating and focusing adaptive laboratory evolution 加速和聚焦自适应实验室进化的自动化平台

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-12-25 DOI: 10.1016/j.ymben.2025.12.007

Peter Ruppen , Maximilian Ole Bahls , Michael Sebastian Gerlt , Martin Peter Edelmann , Tania Michelle Roberts , Philippe Marlière , Sven Panke

The rate of change in adaptive laboratory evolution (ALE), in which a population of microorganisms is continuously cultivated under a specific selective pressure, is controlled by the cellular mutagenesis rate and the randomness of where in the genetic material mutations are introduced. The constant selection pressure makes it a crucial, yet slow, method in developing microorganisms with novel phenotypes for which a rational engineering pathway is either too complex or unknown.

A variety of targeted genome editing methods to accelerate evolution and facilitate the engineering of complex novel traits are available. However, these protocols require (nearly) as many successive transformation steps as loci they target, leaving the actual engineering process quite labor-intense, cumbersome, and at odds with the continuous nature of ALE. Here, we provide a fully integrated microfluidic platform that automates and accelerates bacterial transformation by electroporation to the mere push of a button. We demonstrate the functionality and effect by using oligonucleotide-directed mutagenesis in an ALE experiment to accelerate the engineering of riboflavin prototrophy into Escherichia coli.

在适应性实验室进化（ALE）中，微生物种群在特定的选择压力下持续培养，其变化率由细胞诱变率和遗传物质突变引入的随机性控制。持续的选择压力使其成为开发具有新表型的微生物的关键但缓慢的方法，因为合理的工程途径要么太复杂，要么未知。多种靶向基因组编辑方法可以加速进化并促进复杂新性状的工程设计。然而，这些协议需要（几乎）与它们所针对的位点一样多的连续转换步骤，这使得实际的工程过程非常费力、繁琐，并且与ALE的连续特性不一致。在这里，我们提供了一个完全集成的微流体平台，只需按一下按钮，就可以通过电穿孔自动加速细菌转化。我们通过在ALE实验中使用寡核苷酸定向诱变来证明其功能和效果，以加速大肠杆菌的核黄素原生化工程。

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引用次数: 0

Systematic metabolic engineering of an industrial Penicillium citrinum for one-step pravastatin production 一步法生产普伐他汀的工业柠檬酸青霉的系统代谢工程。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-12-16 DOI: 10.1016/j.ymben.2025.12.005

Mengyi Xiong , Zhiqiang Du , Zehao Fan , Beibei Wang , Wenjiao Diao , Min Wang , Xuenian Huang , Xuefeng Lu

Pravastatin is a widely prescribed cholesterol-lowering drug known for its superior water solubility and favorable pharmacokinetics. However, its industrial production remains constrained by an inefficient two-step fermentation process, particularly the second biotransformation step involving Streptomyces fermentation. In this study, we engineered the industrial mevastatin-producing strain Penicillium citrinum MEFC10 to achieve efficient one-step pravastatin biosynthesis. Through systematic screening and integration of optimal cytochrome P450-redox partner modules, a one-step pravastatin production cell factory was constructed in industrial Penicillium citrinum MEFC10. Next, NADP⁺-dependent g6pd3 was overexpressed to increase statin biosynthesis via NADPH regeneration. Further manipulation of pathway transcriptional regulator, self-resistance gene and minimization of byproduct formation, a high-performance Pra2.0 strain was constructed. The Pra2.0 strain produced 8.48 g/L pravastatin and 15.06 g/L total statins in a 50-L bioreactor under fed-batch fermentation. This work established a one-step fermentation process for pravastatin production with markedly improved efficiency over the conventional methods. This work not only establishes an efficient, green production route for pravastatin but also provides a versatile engineering framework for the sustainable biosynthesis of other complex fungal polyketides.

普伐他汀是一种广泛使用的降胆固醇药物，因其优越的水溶性和良好的药代动力学而闻名。然而，其工业生产仍然受到低效的两步发酵过程的限制，特别是涉及链霉菌发酵的第二步生物转化步骤。在本研究中，我们设计了生产甲伐他汀的工业菌株柑橘青霉MEFC10，以实现高效的一步合成普伐他汀。通过系统筛选和整合最佳细胞色素p450 -氧化还原伙伴模块，在工业柠檬酸青霉MEFC10中构建一步法普伐他汀生产细胞工厂。接下来，NADP+依赖性g6pd3过表达，通过NADPH再生增加他汀类药物的生物合成。进一步对途径转录调控因子、自抗基因和最小化副产物的形成进行调控，构建了高性能的Pra2.0菌株。Pra2.0菌株在50 L生物反应器中分批补料发酵，产普伐他汀8.48 g/L，总他汀15.06 g/L。本工作建立了一步发酵生产普伐他汀的工艺，其效率明显高于传统的方法。这项工作不仅建立了高效、绿色的普伐他汀生产路线，而且为其他复杂真菌聚酮的可持续生物合成提供了一个多功能的工程框架。

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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 : 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

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。

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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。

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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。

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