Metabolic engineering最新文献_第6页

Edible fungus Fusarium venenatum: advances, challenges, and engineering strategies for future food production 食用菌镰刀菌：未来食品生产的进展、挑战和工程策略

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-01-01 Epub Date: 2025-09-25 DOI: 10.1016/j.ymben.2025.09.009

Sheng Tong, Qiyu Qiu, Jiaying Gao, Jiali Yu, Yaobo Xu, Zhihua Liao

By 2050, the global population is projected to reach 9.7 billion, necessitating a 70 % increase in traditional agricultural output to meet growing demands. However, critical constraints are emerging as arable land and water resources approach their sustainable utilization thresholds. In this context, ensuring safe, efficient, and sustainable food production has become a pivotal issue intertwined with national economy and people's livelihood. Microbial manufacturing based on microbial chassis and synthetic biology technology represents a transformative approach to future food production. Notably, the edible filamentous fungus Fusarium venenatum serves as an ideal chassis for next-generation future food biomanufacturing. However, there has been a lack of systematic reviews specifically focusing on the development of synthetic biology tools, chassis engineering, and chassis applications for this strain. This paper systematically summarizes the latest significant progress, from the perspectives mentioned above, in the use of F. venenatum for future food biomanufacturing. Furthermore, it discusses potential development directions and challenges, and proposes some available strategies, intending to provide ideas and guidance for the further development of F. venenatum-based future food production systems.

到2050年，全球人口预计将达到97亿，传统农业产量必须提高70%才能满足日益增长的需求。然而，随着耕地和水资源接近其可持续利用阈值，关键的制约因素正在出现。在此背景下，确保安全、高效、可持续的粮食生产已成为关系国计民生的关键问题。基于微生物底盘和合成生物学技术的微生物制造代表了未来食品生产的变革性方法。值得注意的是，可食用丝状真菌镰刀菌是下一代未来食品生物制造的理想基础。然而，对于该菌株的合成生物学工具、底盘工程和底盘应用的开发，一直缺乏系统的综述。本文从以上几个方面系统地总结了维氏霉霉在未来食品生物制造中的应用的最新重大进展。探讨了潜在的发展方向和挑战，并提出了一些可行的策略，旨在为未来以黄曲霉为基础的粮食生产系统的进一步发展提供思路和指导。

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引用次数: 0

Improved arginine production in Escherichia coli by harnessing the intracellular citrulline 利用胞内瓜氨酸提高大肠杆菌精氨酸产量。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-01-01 Epub Date: 2025-09-13 DOI: 10.1016/j.ymben.2025.09.003

Qi Sheng , Shengyang He , Guangjie Liang , Gang Meng , Chunguang Zhao , Aiying Wei , Lining Gou , Jia Liu , Xiaomin Li , Jing Wu , Liming Liu

L-arginine is a high-value amino acid with widely utilized in the food, feed, and pharmaceutical industries. However, its large-scale biosynthesis remains limited by the low efficiency of current microbial strains. In this study, intracellular citrulline accumulation in Escherichia coli-Arg4 was enhanced by 2.45-, 1.90-, and 1.94-fold through supplementation with monosodium glutamate, monosodium aspartate, and glutamine hydrochloride, respectively. Correspondingly, L-arginine titers increased by 47.85 %, 21.18 %, and 10.66 %. Metabolic flux analysis and transcriptomic profiling indicated that exogenous ammonia donors redirected flux through critical metabolic nodes, including oxaloacetate, α-ketoglutarate, and citrulline, thus increasing precursor availability and enhancing L-arginine biosynthesis. Based on these findings, eight key gene targets, such as gdhA, ppc, icd, aspC, glnA, pyrF, gltA, and argF were identified for pathway optimization. Promoter engineering was subsequently employed to modulate their expression, and heterologous gdhA from Salmonella enterica and glnA from Bacillus subtilis were introduced. Consequently, an optimized strain, E. coli-Arg10, was constructed. Following process optimization in a 1000-L fermenter, the titer, yield and productivity of E. coli-Arg10 was achieved 108.33 g/L, 0.54 g/g, and of 2.26 g/L/h, respectively. These results highlight a scalable and efficient approach for microbial L-arginine production.

l -精氨酸是一种高价值氨基酸，广泛应用于食品、饲料、医药等行业。然而，其大规模生物合成仍然受到现有微生物菌株效率低的限制。在本研究中，添加谷氨酸单钠、天冬氨酸单钠和盐酸谷氨酰胺，大肠杆菌- arg4细胞内瓜氨酸积累量分别增加了2.45倍、1.90倍和1.94倍。l -精氨酸滴度相应提高了47.85%、21.18%和10.66%。代谢通量分析和转录组学分析表明，外源性氨供体通过草酰乙酸、α-酮戊二酸和瓜氨酸等关键代谢节点重定向了通量，从而增加了前体利用率，促进了l -精氨酸的生物合成。基于这些发现，我们确定了gdhA、ppc、icd、aspC、glnA、pyrF、gltA和argF等8个关键基因靶点，并进行了途径优化。随后利用启动子工程对其表达进行调控，并引入了来自肠沙门氏菌的外源gdhA和枯草芽孢杆菌的glnA。最终，构建了大肠杆菌- arg10菌株。在1000 L发酵罐中进行工艺优化，大肠杆菌- arg10的滴度为108.33 g/L，产率为0.54 g/g，产率为2.26 g/L/h。这些结果强调了微生物l -精氨酸生产的可扩展和有效的方法。

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引用次数: 0

MDG1-mediated transcriptional reprogramming enhances cellulase production and alters thermal activity in recombinant Saccharomyces cerevisiae mdg1介导的转录重编程增强了重组酿酒酵母菌的纤维素酶生产并改变了热活性。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-01-01 Epub Date: 2025-09-09 DOI: 10.1016/j.ymben.2025.09.002

Chun Wan , Xue-Qing Wang , Hou-Ru Yue , Ming-Ming Zhang , Akihiko Kondo , Riaan den Haan , Tomohisa Hasunuma , Kai Li , Xin-Qing Zhao

The budding yeast Saccharomyces cerevisiae is one of the most widely used microbial cell factories for heterologous protein and enzyme production. However, improving production efficiency and tailoring enzyme properties remain a major challenge. Here we identified MDG1, a gene involved in the pheromone signaling pathway, as a previously unrecognized regulator that significantly enhances cellulase production in recombinant yeast. Overexpression of MDG1 significantly increased the extracellular activities of β-glucosidase I (BGLI), cellobiohydrolase I (CBHI), and endo-glycosidase II (EGII). Intriguingly, MDG1 overexpression also altered the thermal activity profile of BGLI, shifting its peak activity from 50 °C to 37 °C—an inversion relative to the parental strain. Integrated transcriptome analyses revealed that MDG1 regulates the expression of genes involved in the cell cycle and protein folding. Targeted modulation of key cell cycle regulators (CLN1, PCL1, SWI5) further improved BGLI activity, confirming their functional involvement. Secretome analysis and functional assays identified the disulfide isomerase Pdi1p as a key contributor to the enhanced enzyme performance at 37 °C. Our study reveals a novel role of MDG1 in coordinating gene networks to improve enzyme activities and reshape enzymatic properties.

酿酒酵母（Saccharomyces cerevisiae）是生产外源蛋白和酶的最广泛使用的微生物细胞工厂之一。然而，提高生产效率和定制酶的性质仍然是主要的挑战。在这里，我们确定了MDG1，一个参与信息素信号通路的基因，作为一个以前未被识别的调节因子，显著提高重组酵母中纤维素酶的产生。MDG1的过表达显著增加了β-葡萄糖苷酶I （BGLI）、纤维生物水解酶I （chi）和内切糖苷酶II （EGII）的胞外活性。有趣的是，MDG1的过表达也改变了BGLI的热活性分布，将其峰值活性从50°C转移到37°C-相对于亲本菌株而言是倒置的。综合转录组分析显示，MDG1调节参与细胞周期和蛋白质折叠的基因的表达。靶向调节关键细胞周期调节因子（CLN1, PCL1, SWI5）进一步提高了BGLI活性，证实了它们的功能参与。分泌组分析和功能分析发现，二硫异构酶Pdi1p是37°C下酶性能增强的关键因素。我们的研究揭示了MDG1在协调基因网络以改善酶活性和重塑酶特性方面的新作用。

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引用次数: 0

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 : 2026-01-01 Epub 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

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 : 2026-01-01 Epub 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

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 : 2026-01-01 Epub 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

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 : 2026-01-01 Epub 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

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 : 2026-01-01 Epub 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

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 : 2026-01-01 Epub 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

Development of a thermophilic l-arabinose-inducible system in Acetivibrio thermocellus (Clostridium thermocellum) 热细胞活动弧菌（Clostridium thermocellum）嗜热l-阿拉伯糖诱导体系的建立。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-01-01 Epub Date: 2025-09-19 DOI: 10.1016/j.ymben.2025.09.008

Fenghua Liu , Chao Chen , Ya-Jun Liu , Edward A. Bayer , Itzhak Mizrahi , Yingang Feng

Inducible genetic operation systems constitute essential tools in microbial synthetic biology and metabolic engineering. However, inducible systems in non-model microbes, particularly thermophiles, are rarely reported. Acetivibrio thermocellus (previously termed Clostridium thermocellum), a representative strain of thermophilic non-model microbes, currently serves as a promising chassis organism in biorefinery. Although various genetic tools are available for A. thermocellus, superior thermophilic inducible systems are in high demand. In this study, we developed a thermostable l-arabinose-inducible system (ThermoARAi) in A. thermocellus by utilizing the inducible promoter P_abnE and repressor AraR from Geobacillus stearothermophilus T-6. Through systematic promoter engineering and optimization of induction conditions using a thermostable β-glucuronidase as reporter, the system exhibited dynamic range improvement from a 5.4-fold induction to a 175-fold induction with negligible leakage. Furthermore, the ThermoARAi system was appropriate for use in metabolic engineering, as validated by its applications in whole-cell saccharification of cellulosic substrates and degradation of amorphous polyethylene terephthalate films. The ThermoARAi system significantly expands the genetic toolkit for precise gene expression modulation, metabolic engineering, and biotechnological applications in A. thermocellus. Importantly, this approach may also serve as a foundation for developing genetic tools in other Clostridia that play key roles in diverse ecosystems, including the gut.

诱导型遗传操作系统是微生物合成生物学和代谢工程的重要工具。然而，非模式微生物，特别是嗜热菌的诱导系统很少被报道。热细胞活动弧菌（以前称为热细胞梭菌）是一种具有代表性的嗜热非模式微生物，目前在生物炼制中是一种很有前途的基础生物。虽然有多种遗传工具可用于热细胞芽孢杆菌，但对优良的嗜热诱导系统的需求很大。在这项研究中，我们利用嗜热脂肪地杆菌T-6的诱导启动子PabnE和抑制子AraR，在a . thermocellus中建立了一个耐热的l-阿拉伯糖诱导体系（ThermoARAi）。通过系统启动子工程和以耐热β-葡萄糖醛酸酶为报告因子的诱导条件优化，系统的动态范围从5.4倍诱导提高到175倍诱导，且泄漏可以忽略不计。此外，ThermoARAi系统适用于代谢工程，其在纤维素底物的全细胞糖化和无定形聚对苯二甲酸乙二醇酯膜降解中的应用验证了这一点。ThermoARAi系统极大地扩展了热细胞拟南芥精确基因表达调控、代谢工程和生物技术应用的遗传工具包。重要的是，这种方法也可以作为开发其他梭状芽孢杆菌遗传工具的基础，这些梭状芽孢杆菌在包括肠道在内的各种生态系统中发挥关键作用。

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