Metabolic engineering最新文献_第5页

Industrial-scale production of Lacto-N-tetraose in engineered Bacillus subtilis via modular pathway optimization 通过模块化途径优化在工程枯草芽孢杆菌中工业化生产乳酸- n -四糖

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-10-30 DOI: 10.1016/j.ymben.2025.10.012

Chunhua Wang , Shaoru Hu , Shenglong Wang , Jing Du , Ziyi Zhao , Ziyao Zheng , Huihui Qiu , Xiang Ma , Jun Li , Hao Liu , Mingfeng Cao , Weixia Gao

Lacto-N-tetraose (LNT), an important human milk oligosaccharide with prebiotic benefits, was successfully produced de novo in Bacillus subtilis, establishing this Generally Recognized as Safe (GRAS) organism as a suitable platform for infant nutritional ingredients. A detailed enzyme screening identified three key enzymes: β-1,3-galactosyltransferase from Pseudogulbenkiania ferrooxidans, β-1,3-N-acetylglucosaminyltransferase from Neisseria polysaccharea, and β-galactoside permease (LacY) from Escherichia coli. The co-expression of these enzymes in strain BPPY enabled the first complete biosynthesis of LNT in B. subtilis, achieving a yield of 1.42 g/L in shake-flask cultures. Advanced metabolic engineering strategies, such as disrupting competing pathways, enhancing UDP-GlcNAc/Gal precursor flow, and optimizing heterologous pathways, led to the development of strain BPPY31, which produced 7.83 g/L of LNT, a 5.5-fold increase. To efficiently regulate carbon flux, a cost-effective CRISPR interference (CRISPRi) system was created to downregulate essential competing genes (zwf, pfkA, murAB). The engineered strain BD7 yielded 12.51 g/L in flask cultures and an impressive 80.48 g/L in 5-L fed-batch bioreactors, with only 4.43 g/L of the byproduct lacto-N-triose II, achieving lactose and glucose conversion rates of 92.25 % and 24 %, respectively. This study reports the highest documented LNT titer to date using a GRAS-compliant biomanufacturing platform characterized by precise metabolic regulation, scalability, and significant potential for industrial production of human milk oligosaccharides.

乳酸- n -四糖（LNT）是一种重要的具有益生元益处的母乳低聚糖，成功地在枯草芽孢杆菌（Bacillus subtilis）中重新生产，使这种公认安全的（GRAS）生物成为婴儿营养成分的合适平台。详细的酶筛选鉴定了三个关键酶：来自Pseudogulbenkiania ferrooxidans的β-1,3-半乳糖基转移酶，来自Neisseria多糖区的β-1,3- n -乙酰氨基葡萄糖基转移酶和来自大肠杆菌的β-半乳糖苷渗透酶（LacY）。这些酶在BPPY菌株中的共表达使枯草芽孢杆菌首次完成了LNT的生物合成，摇瓶培养的产量达到1.42 g/L。先进的代谢工程策略，如破坏竞争途径，增强UDP-GlcNAc/Gal前体流动，优化异源途径，导致菌株BPPY31的发展，产生7.83 g/L的LNT，增加了5.5倍。为了有效地调节碳通量，我们创建了一种具有成本效益的CRISPR干扰（CRISPRi）系统来下调必要的竞争基因（zwf, pfkA, murAB）。工程菌株BD7在瓶中培养的产量为12.51 g/L，在5-L进料间歇式生物反应器中的产量为80.48 g/L，副产物乳酸- n -三糖II仅为4.43 g/L，乳糖和葡萄糖的转化率分别为92.25%和24%。本研究报告了迄今为止记录最高的LNT滴度，使用符合格拉斯标准的生物制造平台，其特点是精确的代谢调节，可扩展性，以及人乳低聚糖工业生产的巨大潜力。

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引用次数: 0

Microbial production of propionic acid through a novel β-alanine route 微生物通过β-丙氨酸新途径生产丙酸

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-10-30 DOI: 10.1016/j.ymben.2025.10.011

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

Propionic acid is a key three carbon platform chemical with broad applications in food preservation, pharmaceuticals, and polymer production. Traditional microbial production of propionic acid employing Propionibacterium species is constrained by slow growth, and limited genetic engineering tools, thereby restricting its industrial use. Here, we report the development of a novel biosynthetic pathway for propionic acid production via the β-alanine route. This pathway was engineered into two modules: an upstream β-alanine-forming module and a downstream propionic acid-forming module. The downstream pathway was first constructed and validated in Escherichia coli W3110. Subsequently, co-expression of the upstream module enabled de novo propionic acid production from glucose. Through enzyme screening, precursor flux enhancement, and optimization of phosphoenolpyruvate carboxylase (PPC) flux, the final engineered E. coli strain achieved 14.8 g/L of propionic acid in fed-batch fermentation. Furthermore, we explored Corynebacterium glutamicum ATCC 13032 as an alternative host due to its superior tolerance to propionic acid. The same downstream pathway was introduced into a previously developed β-alanine-overproducing C. glutamicum strain to enable propionic acid production from glucose. Additional engineering strategies, such as enzyme screening, disruption of competing pathways (ack-pta), and elimination of propionic acid catabolic pathways (prpD2B2C2), led to the production of 47.4 g/L of propionic acid in fed-batch fermentation, representing the highest reported titer of heterologous propionic acid production. This work establishes a novel and vitamin B₁₂-independent strategy for bio-based propionic acid production, offering a sustainable alternative to conventional processes.

丙酸是一种关键的三碳平台化学品，在食品保鲜、制药和聚合物生产中有着广泛的应用。传统的丙酸微生物生产利用丙酸杆菌种类受到限制，生长缓慢，有限的基因工程工具，从而限制了其工业应用。在这里，我们报道了通过β-丙氨酸途径生产丙酸的一种新的生物合成途径的发展。该途径被设计成两个模块：上游β-丙氨酸形成模块和下游丙酸形成模块。下游途径首先在大肠杆菌W3110中构建并验证。随后，上游模块的共表达使葡萄糖从头生成丙酸成为可能。通过酶筛选、前体通量增强和磷酸烯醇丙酮酸羧化酶（PPC）通量优化，最终工程菌株在补料分批发酵中获得了14.8 g/L的丙酸。此外，由于谷氨酸棒状杆菌ATCC 13032对丙酸的耐受性较好，我们探索了其作为替代宿主的可能性。同样的下游途径被引入到先前开发的β-丙氨酸过量生产的C.谷氨酸菌株中，以使葡萄糖产生丙酸。额外的工程策略，如酶筛选、竞争途径的破坏（ack-pta）和丙酸分解代谢途径的消除（prpD2B2C2），导致在补料分批发酵中产生47.4 g/L的丙酸，这是报道的最高滴度的异源丙酸生产。这项工作建立了一种新的、不依赖维生素b12的生物基丙酸生产策略，为传统工艺提供了一种可持续的替代方案。

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引用次数: 0

Demonstration and technoeconomic analysis of dodecanol production from acetate using metabolically engineered Escherichia coli 利用代谢工程大肠杆菌从乙酸酯中生产十二醇的示范和技术经济分析

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-10-27 DOI: 10.1016/j.ymben.2025.10.007

Paul M. Perkovich, Yoel R. Cortés-Peña, Justin J. Baerwald, Thomas H. Graupmann, Theodore A. Chavkin, Shivangi Mishra, William T. Cordell, Victor M. Zavala, Brian F. Pfleger

In a circular bioeconomy, the one-way conversion of petroleum to chemicals and CO₂ is replaced with processes that reduce CO₂ to energy carriers and useful materials that are returned to CO₂ upon combustion. A circular bioeconomy that relies on photosynthesis to generate sugars as the chief energy carrier and precursor to chemical building blocks has yet to overcome many recalcitrant aspects of plant-based photosynthesis, namely, high feedstock costs, arable land scarcity, food competition, and fertilizer overuse. Acetate is a potential sustainable energy carrier because it can be produced from CO₂ either electrocatalytically or by acetogens via the Wood-Ljungdahl pathway. In this work, we conducted a metabolic engineering study of Escherichia coli's ability to convert acetate into dodecanol as a model oleochemical product. We performed techno-economic and life cycle analyses to determine break-even points with alternative fossil fuel-based strategies and identified critical process performance parameters for supporting an industrial acetate-based bioprocess. These analyses showed that oleochemical yield is the primary driver of minimum oleochemical selling price and carbon intensity. Therefore, to increase yield on acetate, we deleted the aceBAK operon, which facilitates funneling of acetate into biomass instead of product. We performed additional strain engineering to increase flux towards dodecanol and increase acetate uptake. Finally, we demonstrated increased yield in controlled bioreactors, improving from 13 % of the maximum theoretical yield to 37 %. Rigorous uncertainty analyses assuming a range of market conditions and future technological performances resulted in 88 % and 37 % of simulated scenarios having lower carbon intensities than fossil fuel-based routes and lower minimum selling prices than the market price.

在循环生物经济中，将石油单向转化为化学品和二氧化碳的过程被减少二氧化碳为能量载体和有用材料的过程所取代，这些物质在燃烧时又返回为二氧化碳。依靠光合作用产生糖作为主要的能量载体和化学成分的前体的循环生物经济尚未克服植物光合作用的许多顽固性方面，即原料成本高、可耕地稀缺、食品竞争和肥料过度使用。醋酸盐是一种潜在的可持续能源载体，因为它可以通过电催化或通过Wood-Ljungdahl途径由二氧化碳产生。在这项工作中，我们对大肠杆菌将醋酸酯转化为十二醇作为模型油脂化学产物的能力进行了代谢工程研究。我们进行了技术经济和生命周期分析，以确定替代化石燃料战略的盈亏平衡点，并确定了支持工业醋酸酯生物工艺的关键工艺性能参数。分析表明，油脂化产量是油脂化最低销售价格和碳强度的主要驱动因素。因此，为了提高醋酸酯的产率，我们删除了aceBAK操纵子，这有利于醋酸酯进入生物质而不是产品。我们进行了额外的菌株工程来增加对十二醇的通量和增加乙酸的吸收。最后，我们证明了在受控生物反应器中提高了产率，从最大理论产率的13%提高到37%。严格的不确定性分析假设了一系列的市场条件和未来的技术性能，结果显示88%和37%的模拟情景的碳强度低于基于化石燃料的路线，最低销售价格低于市场价格。

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引用次数: 0

Harnessing the MEP pathway for heterologous (+)-nootkatone biosynthesis in a green microalga under fine-tuned light and carbon regimes 利用MEP途径在微调光和碳制度下在绿色微藻中进行异源(+)-诺卡酮生物合成

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-10-27 DOI: 10.1016/j.ymben.2025.10.010

Merve Saudhof , Jona Brückner , Timo Sürmene , Anke Rattenholl , Thomas Baier , Olaf Kruse

The high-value sesquiterpenoid (+)-nootkatone has important applications in food, agriculture, and pharmaceutical industries. Extraction from plant material, however, is technically challenging and inefficient due to inherent low concentrations in native sources. Over the last decade, the eukaryotic green microalga Chlamydomonas reinhardtii has emerged as a powerful alternative for heterologous terpenoid production, due to a natively high carbon flux through its MEP pathway. This study describes strategic fusion protein designs of different valencene and farnesyl pyrophosphate (FPP) synthases, which allowed efficient (+)-valencene biosynthesis in the C. reinhardtii cytosol and also found the algal chloroplast to be highly suitable for heterologous production. Successful co-expression of cytochrome P450 monooxygenases resulted in a two-step oxidation towards (+)-nootkatone at a comparably high conversion rate of 76 % and was independent of recombinant reductase activity. In addition, the 1-deoxyxylulose-5-phosphate synthase (DXS) was found to be rate-limiting for increased sesquiterpenoid production.

Currently available photobioreactors suffer from limitations in light availability, which can hinder phototrophic growth, especially at higher cell densities. C. reinhardtii harbours the ability to use acetic acid as a carbon source, and fine-tuned cultivation regimes under photo-, mixo-, and heterotrophic conditions were tested to optimize heterologous sesquiterpene production. Customized scale-up cultivations in 2.5 L showed efficient volumetric production of 148 mg/L under phototrophic conditions and a maximal gravimetric production of 76 mg/g_CDW under heterotrophic cultivation regimes, which displays a first industrially relevant (+)-nootkatone production concept in a green cell factory.

高价值倍半萜(+)-诺卡酮在食品、农业和制药工业中有着重要的应用。然而，由于天然来源中固有的低浓度，从植物材料中提取是具有技术挑战性和效率低下的。在过去的十年中，真核绿色微藻莱茵衣藻（Chlamydomonas reinhardtii）由于其MEP途径具有天然的高碳通量，已成为异源萜类化合物生产的强大替代品。本研究描述了不同价二烯和法尼酰焦磷酸合成酶（FPP）的战略性融合蛋白设计，使C. reinhardtii细胞质中高效的(+)-价二烯生物合成成为可能，并发现藻类叶绿体非常适合异种生产。细胞色素P450单加氧酶的成功共表达导致了(+)-诺卡酮的两步氧化，转化率高达76%，并且不依赖于重组还原酶的活性。此外，1-脱氧醛糖-5-磷酸合成酶（DXS）对倍半萜类化合物产量的增加具有限速作用。目前可用的光生物反应器受到光可用性的限制，这可能会阻碍光营养生长，特别是在较高的细胞密度下。C. reinhardtii具有利用乙酸作为碳源的能力，并在光、混合和异养条件下进行了微调培养，以优化异源倍半萜的生产。2.5 L的定制放大培养在光养条件下的有效体积产量为148 mg/L，在异养培养制度下的最大重量产量为76 mg/gCDW，这显示了绿色细胞工厂中第一个与工业相关的(+)-诺卡酮生产概念。

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引用次数: 0

Genome-wide CRISPR screening identifies genes in recombinant human embryonic kidney 293 cells for increased ammonia resistance 全基因组CRISPR筛选鉴定重组人胚胎肾293细胞中增加氨抗性的基因

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-10-21 DOI: 10.1016/j.ymben.2025.10.008

Sang Yoon Lee , Hyun Seung Kim , Yeon Gu Kim , Seunghyeon Shin , Seokchan Kweon , Jae Jun Lee , Gyun Min Lee

Ammonia, a byproduct of glutamine metabolism, inhibits cell growth and reduces product yield and quality in mammalian cell culture. To identify novel genes associated with ammonia resistance, a genome-wide CRISPR knockout screening was conducted in monoclonal antibody (mAb)-producing human embryonic kidney 293 (HEK-mAb) cells using a virus-free, recombinase-mediated cassette exchange-based gRNA interrogation method. The knockout cell library was subcultured for five consecutive passages under 20 mM NH₄Cl, enriching cells with a sgRNA that conferred a proliferation advantage under high-ammonia conditions. Next-generation sequencing analysis of the enriched population identified three target genes -WNT3, TSPAN1, and CYHR1-among 19,114 genes. Knockout of these genes in HEK-mAb cells resulted in a 1.33- to 1.56-fold increase in maximum viable cell concentration and a 1.28- to 1.58-fold increase in maximum mAb concentration under 20 mM NH₄Cl. Notably, WNT3 knockout maintained N-glycan galactosylation proportions of mAb despite ammonia stress. These findings highlight the effectiveness of genome-wide CRISPR knockout screening in identifying novel gene targets for ammonia-resistant HEK293 cell, offering a promising strategy for improving mAb production.

氨是谷氨酰胺代谢的副产物，在哺乳动物细胞培养中抑制细胞生长，降低产品产量和质量。为了鉴定与氨抗性相关的新基因，采用无病毒、重组酶介导的基于盒式交换的gRNA询问方法，在产生单克隆抗体（mAb）的人胚胎肾293 （HEK-mAb）细胞中进行了全基因组CRISPR敲除筛选。将敲除细胞文库在20 mM NH4Cl下连续传代5代，富集具有高氨条件下增殖优势的sgRNA细胞。下一代测序分析富集群体在19,114个基因中鉴定出三个靶基因- wnt3， TSPAN1和CYHR1。在20 mM NH4Cl条件下，敲除HEK-mAb细胞中的这些基因导致最大活细胞浓度增加1.33- 1.56倍，最大mAb浓度增加1.28- 1.58倍。值得注意的是，尽管氨胁迫，敲除WNT3仍能维持单抗的n -聚糖半乳糖化比例。这些发现强调了全基因组CRISPR敲除筛选在鉴定氨抗性HEK293细胞的新基因靶点方面的有效性，为提高单克隆抗体的生产提供了一种有希望的策略。

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引用次数: 0

Emerging hosts for metabolic engineering 代谢工程的新宿主。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-10-21 DOI: 10.1016/j.ymben.2025.10.009

Hal S. Alper

引用次数: 0

Multiplex base editing of RBSs rewires Bacillus subtilis metabolism for lycopene overproduction RBSs的多重碱基编辑重组枯草芽孢杆菌的代谢以过量生产番茄红素。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-10-17 DOI: 10.1016/j.ymben.2025.10.005

Yang Liu , Xianhai Cao , Xiaojuan Wang , Ruirui Chen , Boxin Yuan , Shixin Li , Yu Wang , Xiaoping Liao , Xiaomeng Ni , Lixian Wang , Yanmei Guo , Hongwu Ma , Meng Wang

Bacillus subtilis is a GRAS-certified chassis, yet scalable and precise multi-gene regulation remains a bottleneck in its metabolic engineering. Here, we present bsBETTER, a base editor-guided, template-free system enabling high-diversity expression tuning across multiple genomic loci. By editing ribosome binding sites (RBSs) of 12 lycopene biosynthetic genes, we generated thousands of combinatorial variants in situ, achieving up to 255 of 256 theoretical RBS combinations per gene. High-throughput screening identified variants exhibiting up to a 6.2-fold increase in lycopene production relative to strains carrying direct genomic overexpression of MEP pathway genes. RBS strength measurements revealed strong context dependence, highlighting the importance of genome-integrated expression optimization. Multi-omics analysis showed extensive transcriptional and metabolic rewiring, including enhanced MEP pathway flux and NADPH-generating capacity. bsBETTER offers a scalable, high-resolution approach for metabolic pathway engineering in B. subtilis, providing a generalizable framework for combinatorial gene expression modulation and metabolic pathway optimization.

枯草芽孢杆菌（Bacillus subtilis）是grass认证的“底盘”，但可扩展和精确的多基因调控仍然是其代谢工程的瓶颈。在这里，我们提出了bsBETTER，这是一个碱基编辑器引导的无模板系统，可以跨多个基因组位点进行高多样性表达调节。通过编辑12个番茄红素生物合成基因的核糖体结合位点（RBSs），我们原位生成了数千个组合变体，每个基因获得256个理论RBS组合中的255个。高通量筛选发现，与携带MEP通路基因直接基因组过表达的菌株相比，番茄红素产量增加了6.2倍。RBS强度测量显示了强烈的上下文依赖性，突出了基因组整合表达优化的重要性。多组学分析显示广泛的转录和代谢重新布线，包括增强的MEP途径通量和nadph生成能力。bsBETTER为枯草芽孢杆菌的代谢途径工程提供了一个可扩展的、高分辨率的方法，为组合基因表达调节和代谢途径优化提供了一个可推广的框架。

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引用次数: 0

Corrigendum to “Metabolic and enzyme rewiring enables high-production of vanillin in unconventional yeast” [Metabol. Eng. 93 (2026) 158–167] “代谢和酶重新布线使非常规酵母能够高产香兰素”的勘误表[代谢。工程学报，2009(3):387 - 387。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-10-17 DOI: 10.1016/j.ymben.2025.10.006

Yan Guo , Liyang Zhou , Wanshu Lai , Zhilan Qian , Haishuang Yu , Menghao Cai

引用次数: 0

Feedstock-efficient conversion through hydrogen and formate-driven metabolism in Escherichia coli 在大肠杆菌中通过氢和甲酸驱动代谢实现原料高效转化。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-10-15 DOI: 10.1016/j.ymben.2025.10.003

Robert L. Bertrand , Justin Panich , Aidan E. Cowan , Jacob B. Roberts , Lesley J. Rodriguez , Juliana Artier , Emili Toppari , Edward E.K. Baidoo , Yan Chen , Christopher J. Petzold , Graham A. Hudson , Patrick M. Shih , Steven W. Singer , Jay D. Keasling

Product yields for biomanufacturing processes are often constrained by the tight coupling of cellular energy generation and carbon metabolism in sugar-based fermentation systems. To overcome this limitation, we engineered Escherichia coli to utilize hydrogen gas (H₂) and formate (HCOO⁻) as alternative sources of energy and reducing equivalents, thereby decoupling energy generation from carbon metabolism. This approach enabled precise suppression of decarboxylative oxidation during acetate growth, with 86.6 ± 1.6 % of electrons from hydrogen gas (via soluble hydrogenase from Cupriavidus necator H16) and 98.4 ± 3.6 % of electrons from formate (via formate dehydrogenase from Pseudomonas sp. 101) offsetting acetate oxidation. Hydrogen gas supplementation led to a titratable and stoichiometric reduction in CO₂ evolution in acetate-fed cultures. Metabolomic analysis suggests that this metabolic decoupling redirects carbon flux through the glyoxylate shunt, partially bypassing two decarboxylative steps in the TCA cycle. We demonstrated the utility of this strategy by applying it to mevalonate biosynthesis, where formate supplementation during glucose fermentation increased titers by 57.6 % in our best-performing strain. Flux balance analysis further estimated that 99.0 ± 2.8 % of electrons from formate were used to enhance mevalonate production. These findings highlight a broadly applicable strategy for enhancing biomanufacturing efficiency by leveraging external reducing power to optimize feedstock and energy use.

生物制造过程的产品产量通常受到糖基发酵系统中细胞能量产生和碳代谢的紧密耦合的限制。为了克服这一限制，我们设计了大肠杆菌，利用氢气（H2）和甲酸（HCOO-）作为替代能源和还原物，从而将能量产生与碳代谢解耦。这种方法能够精确抑制乙酸生长过程中的脱羧氧化，86.6±1.6%的电子来自氢气（通过Cupriavidus necator H16的可溶性氢化酶）和98.4±3.6%的电子来自甲酸（通过Pseudomonas sp. 101的甲酸脱氢酶）抵消乙酸氧化。氢气的补充导致醋酸盐培养物中CO2演化的可滴定和化学计量减少。代谢组学分析表明，这种代谢解耦通过乙醛酸分流重定向碳通量，部分绕过TCA循环中的两个脱羧步骤。我们通过将其应用于甲羟戊酸生物合成证明了这一策略的实用性，在葡萄糖发酵期间补充甲酸盐使我们表现最好的菌株的滴度提高了57.6%。通量平衡分析进一步估计，甲酸盐中99.0±2.8%的电子被用于提高甲羟戊酸的产量。这些发现强调了一种广泛适用的策略，通过利用外部减少功率来优化原料和能源使用，从而提高生物制造效率。

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引用次数: 0

Evolution-assisted engineering of formate assimilation via the formyl phosphate route in Escherichia coli 在大肠杆菌中通过磷酸甲酰基途径进行甲酸同化的进化辅助工程。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-10-15 DOI: 10.1016/j.ymben.2025.10.004

Jenny Bakker , Maximilian Boinot , Karin Schann , Jörg Kahnt , Timo Glatter , Tobias J. Erb , Maren Nattermann , Sebastian Wenk

The transition towards a sustainable bioeconomy requires the use of alternative feedstocks, with CO₂-derived formate emerging as a promising candidate for industrial biotechnology. Despite its beneficial characteristics as a feedstock, microbial assimilation of formate is limited by the inefficiency of naturally evolved formate-fixing pathways. To overcome this limitation, synthetic formate reduction cascades could enable formate assimilation via formaldehyde, a key intermediate of several existing one carbon assimilation pathways. Recently, the formyl phosphate route, combining ATP-dependent activation of formate to formyl phosphate, followed by its reduction to formaldehyde, was developed through enzyme engineering and characterized in vitro. In this work, we successfully established the formyl phosphate route in vivo by developing a selection strategy that couples formate reduction to growth in a threonine/methionine auxotrophic Escherichia coli. Through adaptive laboratory evolution, we achieved formate-dependent growth via this novel pathway. Evolved strains were capable of growing robustly with formate concentrations between 20 mM and 100 mM with glucose in the co-feed. Genomic and proteomic analyses together with activity assays uncovered that formate activation was limiting in vivo. This discovery guided the rational engineering of a strain capable of efficient formate assimilation through the formyl phosphate route. By demonstrating that novel enzyme activities can link formate reduction to cell growth, our study shows how synthetic metabolic routes can be functionally established inside the cell, paving the way for the engineering of more complex synthetic pathways.

向可持续生物经济的过渡需要使用替代原料，二氧化碳衍生的甲酸酯正在成为工业生物技术的有希望的候选者。尽管甲酸作为一种原料具有有益的特性，但由于自然进化的甲酸固定途径效率低下，微生物对甲酸的同化受到限制。为了克服这一限制，合成甲酸还原级联可以通过甲醛进行甲酸同化，甲醛是几种现有的一碳同化途径的关键中间体。近年来，通过酶工程的方法开发了甲酸盐atp依赖性活化生成磷酸甲酰，再还原为甲醛的磷酸甲酰途径，并对其进行了体外表征。在这项工作中，我们通过开发一种选择策略，将甲酸还原与苏氨酸/蛋氨酸营养不良的大肠杆菌的生长结合起来，成功地在体内建立了磷酸甲酰途径。通过适应性实验室进化，我们通过这种新途径实现了甲酸依赖性生长。进化菌株在甲酸盐浓度为20 ~ 100 mM、共饲料中有葡萄糖的条件下能够稳定生长。基因组学和蛋白质组学分析以及活性分析表明，甲酸盐在体内的激活是有限的。这一发现指导了合理设计一种能够通过磷酸甲酰基途径有效同化甲酸盐的菌株。通过证明新的酶活性可以将甲酸还原与细胞生长联系起来，我们的研究显示了如何在细胞内功能性地建立合成代谢途径，为更复杂的合成途径的工程铺平了道路。

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引用次数: 0