Metabolic engineering最新文献_第6页

Metabolic and enzyme rewiring enables high-production of vanillin in unconventional yeast 代谢和酶重组使非常规酵母的香兰素高产成为可能。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-10-09 DOI: 10.1016/j.ymben.2025.10.002

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

Vanillin is an aromatic flavor compound widely used in the food, pharmaceutical, and cosmetic industries. Microbial biosynthesis offers a sustainable alternative to traditional plant extraction and chemical synthesis; however, the susceptibility of vanillin to redox reactions and the weak enzyme activity in cells severely limit the vanillin production capacity by microbial biosynthesis. This study presents the first successful attempt at de novo synthesis of vanillin in the unconventional yeast Komagataella phaffii. The initial titer was quite low (0.5 mg/L), but removal of 14 endogenous oxidoreductases to block vanillin conversion resulted in an 11.1-fold improvement in vanillin production. The combination of pathway rewiring and cofactor (nicotinamide adenine dinucleotide phosphate [NADPH] and S-adenosylmethionine) regeneration redirected the metabolic flux toward vanillin synthesis and achieved a further 19.9-fold improvement in vanillin production. Rational rewiring of the rate-limiting enzyme, caffeic acid O-methyltransferase (NtCOMT), generated a dominant mutant NtCOMT^N312A/H315N from 70 variants, which promoted activity by 49.7 % and prevented intermediate accumulation. These strategies eventually enabled the co-coupling of de novo biosynthesis and caffeic acid conversion, achieving the highest reported production of vanillin (1055.9 mg/L) by K. phaffii fermentation in a bioreactor. These findings highlight the potential of unconventional yeast as a chassis host for aromatic aldehyde synthesis and the construction of a versatile microbial platform for the production of carbonyl compounds.

香兰素是一种芳香香料化合物，广泛应用于食品、制药和化妆品行业。微生物生物合成为传统的植物提取和化学合成提供了可持续的替代方案；然而，由于香兰素对氧化还原反应的敏感性和细胞内酶活性较弱，严重限制了微生物合成香兰素的生产能力。本研究首次成功尝试在非常规酵母法菲酵母中重新合成香兰素。初始滴度很低（0.5 mg/L），但去除14个内源性氧化还原酶以阻断香兰素转化，使香兰素产量提高了11.1倍。途径重组和辅助因子（烟酰胺腺嘌呤二核苷酸磷酸[NADPH]和s -腺苷蛋氨酸）再生的结合将代谢通量转向香兰素合成，并使香兰素产量进一步提高19.9倍。通过对限速酶咖啡酸o -甲基转移酶（NtCOMT）的合理重新连接，从70个突变体中产生了显性突变体NtCOMTN312A/H315N，其活性提高了49.7%，并阻止了中间积累。这些策略最终实现了新生物合成和咖啡酸转化的共偶联，在生物反应器中通过K. phaffii发酵实现了最高的香兰素产量（1055.9 mg/L）。这些发现突出了非传统酵母作为芳香醛合成的基础宿主和构建生产羰基化合物的多功能微生物平台的潜力。

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引用次数: 0

Metabolic engineering of Acinetobacter baylyi ADP1 for efficient utilization of pentose sugars and production of glutamic acid 贝氏不动杆菌ADP1代谢工程对戊糖的高效利用和谷氨酸的生产。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-10-09 DOI: 10.1016/j.ymben.2025.10.001

Jin Luo, Elena Efimova, Ville Santala, Suvi Santala

Efficient utilization of pentose sugars is critical for advancing sustainable biomanufacturing using lignocellulose. However, many host strains capable of consuming glucose and lignin-derived monomers are unable to utilize pentose sugars. Here, we engineered Acinetobacter baylyi ADP1 for the utilization of D-xylose and L-arabinose. We first modelled different pentose utilization pathways using flux balance analysis to choose the most optimal pathway. A marker-free approach combining transformation and selection facilitated the integration of the pentose catabolic gene clusters of the selected Weimberg pathway into the A. baylyi genome, generating strains capable of efficiently utilizing both D-xylose and L-arabinose as sole carbon sources without any additional engineering or adaptation. For D-xylose, the cells achieved the highest growth rate (μ = 0.73 h⁻¹) reported to date for non-native hosts engineered for pentose utilization. For L-arabinose, a growth rate of μ = 0.40 h⁻¹ was achieved, which also surpassed the growth rate on a native substrate of A. baylyi, glucose (μ = 0.37 h⁻¹). Importantly, pentose utilization occurred simultaneously with glucose utilization. We then applied metabolic flux analysis using ¹³C labeled xylose to reveal D-xylose metabolism in the engineered strain. To demonstrate the potential for bioproduction, L-glutamate was selected as a target compound. Deletion of sucAB and gabT, and the overexpression of gdhA enabled L-glutamate production. With the engineered strain, a carbon yield of 34 % during co-utilization with succinate and 70 % via whole-cell catalysis using resting cells was achieved. Notably, L-glutamate production directly from industrially relevant hemicellulose hydrolysate was demonstrated. This study establishes a robust platform for pentose utilization and bioproduction in A. baylyi ADP1 and highlights the potential for metabolic optimization.

戊糖的有效利用对于推进木质纤维素的可持续生物制造至关重要。然而，许多能够消耗葡萄糖和木质素衍生单体的宿主菌株不能利用戊糖。在这里，我们设计了利用d -木糖和l -阿拉伯糖的baylyi不动杆菌ADP1。首先利用通量平衡分析对不同戊糖利用途径进行建模，选择最优途径。结合转化和选择的无标记方法促进了Weimberg途径的戊糖分解代谢基因簇整合到A. baylyi基因组中，产生了能够有效利用d -木糖和l -阿拉伯糖作为唯一碳源的菌株，而无需任何额外的工程或适应。对于d -木糖，细胞的生长速度（μ=0.73 h-1）是迄今为止报道的用于戊糖利用的非原生宿主中最高的。l -阿拉伯糖的生长速度为μ=0.40 h-1，也超过了在天然底物葡萄糖上的生长速度（μ=0.37 h-1）。重要的是，戊糖利用与葡萄糖利用同时发生。然后用13C标记木糖进行代谢通量分析，揭示工程菌株d -木糖代谢。为了证明生物生产的潜力，选择l -谷氨酸作为目标化合物。缺失sucAB和gabT以及过表达gdhA使l -谷氨酸产生。在与琥珀酸盐共利用的过程中，该工程菌株的碳产量为34%，在静息细胞的全细胞催化下，碳产量为70%。值得注意的是，从工业相关的半纤维素水解物中直接生产l -谷氨酸得到了证明。本研究为baylyi ADP1的戊糖利用和生物生产建立了一个强大的平台，并强调了代谢优化的潜力。

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引用次数: 0

Engineering a biosensor based high-throughput screening platform for high-yield caffeic acid production in Escherichia coli 设计一个基于生物传感器的高通量筛选平台，用于大肠杆菌高产咖啡酸的生产。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-09-30 DOI: 10.1016/j.ymben.2025.09.010

Daoguang Tian , Zhen Qin , Weilin Liu , Qinggele Caiyin , Weiguo Li , Guang-Rong Zhao , Jianjun Qiao

Caffeic acid (CA) is a valuable phenolic compound with wide applications in pharmaceuticals, food additives, and materials. However, its microbial production faces several challenges, including low heterologous enzyme activity and product toxicity. Here, we report the development of an integrated biosensor-driven high-throughput screening (HTS) platform for the efficient production of CA in Escherichia coli. We first identified and characterized CarR, a novel phenolic acid-responsive transcription factor from Acetobacterium woodii, and engineered it into a p-coumaric acid (p-CA) biosensor. Systematic optimization of the p-CA biosensor, resulting in reduced background, extended dynamic range and increased sensitivity. By coupling this biosensor with fluorescence-activated cell sorting, we established an efficient HTS platform that enabled the rapid selection of an improved FjTAL^G85S mutant with a 6.85-fold enhancement in catalytic activity and a robust p-CA-producing strain (M5) with enhanced tolerance to p-CA and CA. Subsequent bottom-up metabolic engineering in strain CA8 achieved a CA titer of 9.61 g L⁻¹ in a 5-L bioreactor, the highest reported titer to date. Our work not only overcomes key bottlenecks in CA biosynthesis (low tyrosine ammonia-lyase activity, CA and p-CA cytotoxicity) but also provides a powerful tool to accelerate the engineering of microbial cell factories for the production of p-CA and other derived chemicals.

咖啡酸（cafic acid， CA）是一种有价值的酚类化合物，在医药、食品添加剂和材料等方面有着广泛的应用。然而，其微生物生产面临着一些挑战，包括低外源酶活性和产品毒性。在这里，我们报道了一种集成的生物传感器驱动的高通量筛选（HTS）平台的开发，用于在大肠杆菌中高效生产CA。我们首先从woodii乙杆菌中鉴定并表征了一种新的酚酸反应转录因子CarR，并将其设计成对香豆酸（p-CA）生物传感器。系统优化p-CA生物传感器，减少背景，扩大动态范围，提高灵敏度。通过将这种生物传感器与荧光激活的细胞分选相结合，我们建立了一个高效的HTS平台，能够快速选择催化活性提高6.85倍的FjTALG85S突变体和对p-CA和CA耐受性增强的强大的p-CA产生菌株（M5）。随后对菌株CA8进行自下而上的代谢工程，在5-L生物反应器中获得了9.61 g L-1的CA滴度，这是迄今为止报道的最高滴度。我们的工作不仅克服了CA生物合成的关键瓶颈（低酪氨酸解氨酶活性，CA和对CA的细胞毒性），而且为加速微生物细胞工厂的工程生产对CA和其他衍生化学品提供了有力的工具。

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引用次数: 0

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 : 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

A designed hybrid pathway for efficient synthesis of D-pantothenate in E. coli 大肠杆菌高效合成d -泛酸酯的杂交途径设计

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-09-19 DOI: 10.1016/j.ymben.2025.09.007

Chenkai Cao , Jilong Wang , Mengzhen Nie , Jing Zhao , Yuchen Wang , Kechun Zhang

D-Pantothenate (D-PA), a crucial precursor for coenzyme A, is widely used in various industries. Traditional chemical synthesis of D-PA involves toxic inputs, including cyanide, and generates environmental pollution. Total biosynthesis from glucose still has limitation in long reaction time and low titer. To provide a new approach to D-PA, we designed a hybrid pathway. First, we used green chemical method to convert inexpensive glyoxylate and isobutyaldehyde into 2-hydroxy-3-methyl-3-formylbutyric acid (HMFBA). Then we established a biosynthetic pathway to transform HMFBA into D-PA. Specifically, we engineered a malate dehydrogenase to achieve the efficient stereospecific conversion step. Optimization efforts led to a strain producing 116.5 g/L D-PA in 48 h, which is the highest rate and titier reported so far. This new approach offers a potentially economic and high-rate production route of D-PA.

d -泛酸酯（D-PA）是辅酶a的重要前体，广泛应用于各个工业领域。传统的D-PA化学合成涉及有毒物质，包括氰化物，并产生环境污染。葡萄糖全生物合成仍存在反应时间长、效价低的局限性。为了提供一种新的途径，我们设计了一种混合途径。首先，我们采用绿色化学方法将廉价的乙醛酸酯和异丁醛转化为2-羟基-3-甲基-3-甲酰基丁酸（HMFBA）。然后我们建立了将HMFBA转化为D-PA的生物合成途径。具体来说，我们设计了苹果酸脱氢酶来实现高效的立体特异性转化步骤。经过优化，菌株在48小时内产生116.5 g/L D-PA，这是迄今为止报道的最高速率和滴度。该方法为D-PA的经济高效生产提供了一条新的途径。

引用次数: 0

Reusable and modular combinatorial libraries for iterative metabolic engineering of Saccharomyces cerevisiae 用于酿酒酵母迭代代谢工程的可重用和模块化组合库。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-09-19 DOI: 10.1016/j.ymben.2025.09.006

Philip Tinggaard Thomsen, Peter Gockel, Christina Vasileiou, Ingrid Mohr, Marc Cernuda Pastor, Irina Borodina

Efficiently rewiring microbial metabolism for molecule production lies at the core of industrial metabolic engineering. Combinatorial libraries are useful for directing metabolism towards molecule production; however, their construction is labor-intensive, and their use in iterative strain engineering campaigns is often restricted by site-specific genomic integration. Here we present an automation-friendly framework for generating reusable and modular integration-based combinatorial libraries that can be used repeatedly to build high-performing strains. We apply this approach to engineer the production of betacyanins, a commonly used red food colorant extracted from beetroots, in Saccharomyces cerevisiae. Iterative implementation of combinatorial libraries targeting the betacyanin biosynthesis pathway (design space: ∼25,000), precursors (design space: ∼43,000), and cofactors (design space: ∼26,000) consistently improved pigment production by 1.2–5.7-fold per cycle over seven rounds of engineering. Sequencing of high-performing library isolates from each round revealed unique insights into betacyanin and yeast metabolism, e.g. we found strong evidence implicating the S. cerevisiae cytochrome b5 in heterologous red beet pigment production. Altogether, this study demonstrates a framework for combinatorial library engineering well-suited for accelerating the development of high-performing cell factories for industrial fermentation processes.

有效地重组微生物代谢以生产分子是工业代谢工程的核心。组合文库有助于将代谢导向分子生成；然而，它们的构建是劳动密集型的，并且它们在迭代菌株工程活动中的使用经常受到特定位点基因组整合的限制。在这里，我们提出了一个自动化友好的框架，用于生成可重用的和模块化的基于集成的组合库，可以重复使用来构建高性能的菌株。我们将这种方法应用于酿酒酵母中甜菜青素的工程生产，甜菜青素是一种常用的从甜菜根中提取的红色食品着色剂。针对甜菜花青素生物合成途径（设计空间：~ 25,000）、前体（设计空间：~ 43,000）和辅因子（设计空间：~ 26,000）的组合文库的迭代实施在七轮工程中不断提高色素产量，每个周期提高1.2-5.7倍。对每一轮的高效文库分离物进行测序，揭示了甜菜青素和酵母代谢的独特见解，例如，我们发现了强有力的证据，表明酿酒酵母细胞色素b5参与了异源红甜菜色素的生产。总之，本研究展示了一个组合文库工程框架，非常适合于加速工业发酵过程高性能细胞工厂的开发。

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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 : 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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引用次数: 0

Multi-layered transcriptional and post-translational fine-tuning of metabolic pathways for overproduction of L-valine l -缬氨酸过量产生的代谢途径的多层转录和翻译后微调。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-09-18 DOI: 10.1016/j.ymben.2025.09.005

Xutao Lang , Wenwen Yu , Xuewen Zhu , Xianhao Xu , Yanfeng Liu , Jianghua Li , Guocheng Du , Jian Chen , Xueqin Lv , Long Liu

L-valine is an essential amino acid widely used in the food, pharmaceutical, and animal feed industries. Currently, engineering microbial cell factories to produce L-valine from low-cost feedstocks has emerged as a leading strategy. However, there is still a lack of an L-valine-producing strain that simultaneously exhibits high titer, high yield, and high productivity. The metabolic engineering strategies reported for L-valine biosynthesis primarily rely on conventional unidimensional, transcriptional-level modifications, which limit fine-tuning and do not provide comprehensive, multi-layered regulation. In this study, we constructed an Escherichia coli hyperproducer of L-valine by multiplexed transcriptional and post-translational fine-tuning of metabolic pathways. Initially, the transcriptional repression in L-valine synthetic pathway was eliminated by using promoter engineering strategy. Then, the “push-pull-inhibit” and transport engineering strategies were used to improve L-valine accumulation, achieving a flask fermentation titer of 21.6 g/L and a productivity of 0.45 g/L/h. Subsequently, we rationally designed membraneless organelles (MLOs) to enable spatial regulation of metabolic biosynthesis, which enhanced the targeted recruitment of dihydroxy-acid dehydratase and branched-chain amino acid aminotransferase. This spatial reorganization led to a 95.6 % increase in productivity, reaching 0.88 g/L/h. Finally, the best-performing strain produced 90.6 g/L L-valine in a 3-L bioreactor at 28 h, with a yield of 0.48 g/g glucose and a productivity of 3.24 g/L/h. To the best of our knowledge, this represents the highest L-valine productivity achieved to date. Our strategy provides a practical and effective approach for advancing microbial amino acid biosynthesis by multi-layered transcriptional and post-translational regulation of metabolic pathways.

l -缬氨酸是一种必需氨基酸，广泛应用于食品、制药和动物饲料行业。目前，工程微生物细胞工厂以低成本原料生产l -缬氨酸已成为一种主要策略。然而，仍然缺乏一种l -缬氨酸生产菌株，同时表现出高滴度，高产率和高生产力。据报道，l-缬氨酸生物合成的代谢工程策略主要依赖于传统的一维转录水平修饰，这限制了微调，不能提供全面的、多层次的调控。在这项研究中，我们通过多重转录和翻译后的代谢途径微调构建了大肠杆菌l -缬氨酸高产菌。最初，利用启动子工程策略消除了l -缬氨酸合成途径中的转录抑制。然后，采用“推-拉-抑制”和运输工程策略提高L-valine积累，达到了21.6 g/L的烧瓶发酵滴度和0.45 g/L/h的产率。随后，我们合理设计无膜细胞器（MLOs），实现代谢生物合成的空间调控，增强了二羟基酸脱水酶和支链氨基酸转氨酶的靶向募集。这种空间重组使生产力提高了95.6%，达到0.88 g/L/h。结果表明，最佳菌株在3-L生物反应器中28h产L-valine 90.6 g/L，产糖量为0.48 g/g，产率为3.24 g/L/h。据我们所知，这代表了迄今为止最高的l -缬氨酸生产力。我们的策略提供了一种实用有效的方法，通过多层转录和翻译后代谢途径的调节来推进微生物氨基酸的生物合成。

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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 : 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

Systematic rewiring of Bacillus subtilis for efficient de novo biosynthesis of the neuroprotectant cytidine-5′-diphosphocholine 枯草芽孢杆菌系统重新布线，有效地重新合成神经保护剂胞苷-5'-二磷酸胆碱。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-09-11 DOI: 10.1016/j.ymben.2025.09.004

Shaomei Yang , Xu Feng , Hao Wei , Yanshuang Wang , Shouying Fu , Xiuzhen Gao , Qinyuan Ma

Cytidine-5′-diphosphocholine (CDP-choline) is a crucial neuroprotective agent. Current industrial production relies on chemical and enzymatic methods that face inherent sustainability challenges and share a dependence on the costly precursor, cytidine monophosphate (CMP). Here, we report the systems metabolic engineering of Bacillus subtilis for the efficient, de novo biosynthesis of CDP-choline from glucose, completely obviating the need for CMP. A synthetic pathway was first established by introducing heterologous choline kinase and phosphocholine cytidylyltransferase. Subsequently, a multi-module engineering strategy was implemented, focusing on enhancing precursor supply and redirecting carbon metabolism. This involved systematically optimizing choline uptake by overexpressing the transporter OpuD and deleting the transcriptional repressor opcR, fortifying the cytidine triphosphate (CTP) pool by overexpressing feedback-resistant CTP synthase gene pyrG^E156K, and deleting the transcriptional repressor pyrR along with other pyrimidine nucleotide consumption genes, and channeling carbon flux towards the TCA cycle by reducing pyruvate and malate consumption. The final engineered strain achieved a titer of 4.79 ± 0.24 g/L CDP-choline in a 5 L fed-batch bioreactor, with a high specific yield of 149.0 ± 5.8 mg/g DCW. Notably, the process exhibited a highly advantageous intracellular accumulation of 92.7 %, which simplifies downstream purification. This study represents the first successful demonstration of CDP-choline production from simple sugars in a microbial host, establishing a robust and economically competitive platform for its industrial manufacture.

胞苷-5′-二磷酸胆碱（cdp -胆碱）是一种重要的神经保护剂。目前的工业生产依赖于化学和酶的方法，这些方法面临着固有的可持续性挑战，并且依赖于昂贵的前体，单磷酸胞苷（CMP）。在这里，我们报道了枯草芽孢杆菌的系统代谢工程，以有效地从葡萄糖中重新合成cdp -胆碱，完全消除了对CMP的需要。通过引入外源胆碱激酶和磷酸胆碱胞基转移酶，初步建立了一条合成途径。随后，实施了多模块工程策略，重点是增加前体供应和重定向碳代谢。这包括通过过表达转运体OpuD和删除转录抑制因子opcR来系统地优化胆碱摄取，通过过表达反馈抗性CTP合成酶基因pyrGE156K来强化三磷酸胞苷（CTP）库，并删除转录抑制因子pyrR和其他嘧啶核苷酸消耗基因，并通过减少丙酮酸和苹果酸消耗来引导碳通量进入TCA循环。在5 L补料间歇式生物反应器中，最终工程菌株的滴度为4.79±0.24 g/L，比产率为149.0±5.8 mg/g DCW。值得注意的是，该工艺具有92.7%的胞内富集率，简化了下游纯化。这项研究首次成功地展示了在微生物宿主中由单糖生产cdp -胆碱，为其工业生产建立了一个强大的、具有经济竞争力的平台。

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