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Not all cytochrome b5s are created equal: How a specific CytB5 boosts forskolin biosynthesis in Saccharomyces cerevisiae 并非所有细胞色素 b5 都是相同的:特定的 CytB5 如何在酿酒酵母中促进福斯克林的生物合成。
IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-11-01 DOI: 10.1016/j.ymben.2024.10.008
Victor Forman , Dan Luo , Sotirios C. Kampranis , Dan Stærk , Birger Lindberg Møller , Irini Pateraki
Cytochrome B5s, or CytB5s, are small heme-binding proteins, ubiquitous across all kingdoms of life that serve mainly as electron donors to enzymes engaged in oxidative reactions. They often function as redox partners of the cytochrome P450s (CYPs), a superfamily of enzymes participating in multiple biochemical processes. In plants, CYPs catalyze key reactions in the biosynthesis of plant specialized metabolites with their activity dependent on electron donation often from cytochrome P450 oxidoreductases (CPRs or PORs). In eukaryotic microsomal CYPs, CytB5s frequently participate in the electron transfer process although their exact role remains understudied, especially in plant systems. In this study, we assess the role of CytB5s in the heterologous biotechnological production of plant specialized metabolites in yeast. For this, we used as a case-study the biosynthesis of forskolin - a bioactive diterpenoid produced exclusively from the plant Coleus forskohlii. The complete biosynthetic pathway for forskolin is known and includes three CYP enzymes. We reconstructed the entire forskolin pathway in the yeast Saccharomyces cerevisiae, and upon co-expression of the three CytB5s - identified in C. forskohlii transcriptomes - alleviation of a CYP-related bottleneck step was noticed only when a specific CytB5, CfCytB5A, was used. Co-expression of CfCytB5A in yeast, in combination with forskolin pathway engineering, resulted in forskolin production at titers of 1.81 g/L in a bioreactor. Our findings demonstrate that CytB5s not only play an important role in plant specialized metabolism but also, they can interact with precision with specific CYPs, indicating that the properties of CytB5s are far from understood. Moreover, our work highlights how CytB5s may act as indispensable components in the sustainable microbial production of plant metabolites, when their biosynthetic pathways involve CYP enzymes.
细胞色素 B5s 或 CytB5s 是一种小型血红素结合蛋白,在生物界无处不在,主要作为电子供体供参与氧化反应的酶使用。它们通常是细胞色素 P450s(CYPs)的氧化还原伙伴,CYPs 是参与多种生化过程的酶超家族。在植物中,CYPs 催化植物特殊代谢物生物合成过程中的关键反应,其活性依赖于细胞色素 P450 氧化还原酶(CPRs 或 PORs)的电子捐赠。在真核微粒体 CYPs 中,CytB5s 经常参与电子传递过程,但它们的确切作用仍未得到充分研究,尤其是在植物系统中。在本研究中,我们评估了 CytB5s 在酵母异源生物技术生产植物特殊代谢物中的作用。为此,我们以福斯可林的生物合成为案例进行了研究,福斯可林是一种生物活性二萜类化合物,专门从植物鞘氨醇中提取。据了解,福斯可林的完整生物合成途径包括三种 CYP 酶。我们在酿酒酵母中重建了整个福斯可林的合成途径,在福斯可林转录组中发现了三种 CytB5,当它们共同表达时,只有在使用特定的 CytB5(CfCytB5A)时,才会发现与 CYP 相关的瓶颈步骤有所缓解。在酵母中联合表达 CfCytB5A 与福斯克林途径工程相结合,可在生物反应器中以 1.81 克/升的滴度生产福斯克林。我们的研究结果表明,CytB5s 不仅在植物特化代谢中发挥着重要作用,而且还能与特定的 CYPs 精确地相互作用,这表明人们对 CytB5s 的特性还知之甚少。此外,我们的工作还突显了当植物代谢物的生物合成途径涉及 CYP 酶时,CytB5s 如何成为可持续微生物生产植物代谢物过程中不可或缺的组成部分。
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引用次数: 0
Adaptive laboratory evolution and metabolic engineering of Cupriavidus necator for improved catabolism of volatile fatty acids 改善挥发性脂肪酸分解代谢的裸冠突炎菌适应性实验室进化和代谢工程
IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-11-01 DOI: 10.1016/j.ymben.2024.10.011
Eric C. Holmes, Alissa C. Bleem, Christopher W. Johnson, Gregg T. Beckham
Bioconversion of high-volume waste streams into value-added products will be an integral component of the growing bioeconomy. Volatile fatty acids (VFAs) (e.g., butyrate, valerate, and hexanoate) are an emerging and promising waste-derived feedstock for microbial carbon upcycling. Cupriavidus necator H16 is a favorable host for conversion of VFAs into various bioproducts due to its diverse carbon metabolism, ease of metabolic engineering, and use at industrial scales. Here, we report that a common strategy to improve product titers in C. necator, deletion of the polyhydroxybutyrate (PHB) biosynthetic operon, results in a significant growth defect on VFA substrates. Using adaptive laboratory evolution, we identify mutations to the regulator gene phaR, the two-component response regulator-histidine kinase pair encoded by H16_A1372/H16_A1373, and the tripartite transporter assembly encoded by H16_A2296-A2298 as causative for improved growth on VFA substrates. Deletion of phaR and H16_A1373 led to significantly reduced NADH abundance accompanied by large changes to expression of genes involved in carbon metabolism, balance of electron carriers, and oxidative stress tolerance that may be responsible for improved growth of these engineered strains. These results provide insight into the role of PHB biosynthesis in carbon and energy metabolism and highlight a key role for the regulator PhaR in global regulatory networks. By combining mutations, we generated platform strains with significant growth improvements on VFAs, which can enable improved conversion of waste-derived VFA substrates to target bioproducts.
将大量废物流生物转化为高附加值产品,将成为不断发展的生物经济不可或缺的组成部分。挥发性脂肪酸(VFAs)(如丁酸盐、戊酸盐和己酸盐)是一种新兴的、前景广阔的废物衍生原料,可用于微生物碳升级再循环。裸冠突铜绿菌 H16 是将 VFAs 转化为各种生物产品的有利宿主,因为它具有多样化的碳代谢、易于进行代谢工程以及可用于工业规模。在这里,我们报告了一种提高 C. necator 产品滴度的常见策略--删除聚羟基丁酸(PHB)生物合成操作子--会导致 VFA 底物上的显著生长缺陷。通过适应性实验室进化,我们确定了调节基因 phaR、由 H16_A1372/H16_A1373 编码的双组分反应调节器-组氨酸激酶对以及由 H16_A2296-A2298 编码的三方转运体组件的突变是改善 VFA 底物生长的原因。缺失 phaR 和 H16_A1373 会导致 NADH 丰度显著降低,同时涉及碳代谢、电子载体平衡和氧化应激耐受性的基因表达也会发生巨大变化,这可能是这些工程菌株生长改善的原因。这些结果让我们深入了解了 PHB 生物合成在碳和能量代谢中的作用,并强调了调控因子 PhaR 在全球调控网络中的关键作用。通过组合突变,我们产生了对 VFAs 有显著生长改善的平台菌株,这能使废物衍生的 VFA 底物更好地转化为目标生物产品。
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引用次数: 0
Coordinated reprogramming of ATP metabolism strongly enhances adipic acid production in Escherichia coli ATP 代谢的协调重编程可大大提高大肠杆菌的己二酸产量。
IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-23 DOI: 10.1016/j.ymben.2024.10.010
Soo Young Moon , Nan Yeong An , Seung Soo Oh , Ju Young Lee
Maintaining a delicate balance of adenosine-5′-triphosphate (ATP) is crucial not only for optimal cellular functions but also for improved metabolite production, indicating the need for careful regulation of ATP demands in metabolic engineering. This study explored the modification of ATP metabolism to enhance adipic acid production in Escherichia coli, focusing on the reverse adipate degradation pathway (RADP), and ATP-consuming cycles were fine-tuned by controlling the overexpression of genes (panK and acs) to balance ATP consumption and adipic acid production. As a result, we successfully achieved a significant increase (19.5-fold) in adipic acid production, reaching 1093.11 mg/L in a shake flask, compared to that in the control strain (wild-type E. coli harboring the RADP). Our transcriptomic analysis indicated that modulation of ATP metabolism, along with a balanced supply of pathway precursors, affects metabolic fluxes, enhancing adipic acid biosynthesis in E. coli. This study suggests the potential of metabolic reprogramming of ATP to meet biosynthetic demands, which may improve the production of adipic acid and other ATP-derived chemicals.
保持腺苷-5'-三磷酸(ATP)的微妙平衡不仅对优化细胞功能至关重要,而且对提高代谢产物的产量也至关重要,这表明在代谢工程中需要仔细调节 ATP 需求。本研究以反向己二酸降解途径(RADP)为重点,探索了如何改变 ATP 代谢以提高大肠杆菌的己二酸产量,并通过控制基因(panK 和 acs)的过量表达对 ATP 消耗循环进行了微调,以平衡 ATP 消耗和己二酸产量。结果,与对照菌株(携带 RADP 的野生型大肠杆菌)相比,我们成功地使己二酸产量大幅增加(19.5 倍),在摇瓶中达到 1,093.11 mg/L。我们的转录组分析表明,调节 ATP 代谢以及途径前体的平衡供应会影响代谢通量,从而增强大肠杆菌的己二酸生物合成。这项研究表明,ATP 的代谢重编程有可能满足生物合成的需求,从而提高己二酸和其他 ATP 衍生化学物质的产量。
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引用次数: 0
Evolution-assisted engineering of E. coli enables growth on formic acid at ambient CO2 via the Serine Threonine Cycle 大肠杆菌的进化辅助工程可通过丝氨酸苏氨酸循环在环境二氧化碳条件下利用甲酸进行生长。
IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-22 DOI: 10.1016/j.ymben.2024.10.007
Sebastian Wenk , Vittorio Rainaldi , Karin Schann , Hai He , Madeleine Bouzon , Volker Döring , Steffen N. Lindner , Arren Bar-Even
Atmospheric CO2 poses a major threat to life on Earth by causing global warming and climate change. On the other hand, it can be considered as a resource that is scalable enough to establish a circular carbon economy. Accordingly, technologies to capture and convert CO2 into reduced one-carbon (C1) compounds (e.g. formic acid) are developing and improving fast. Driven by the idea of creating sustainable bioproduction platforms, natural and synthetic C1-utilization pathways are engineered into industrially relevant microbes. The realization of synthetic C1-assimilation cycles in living organisms is a promising but challenging endeavour. Here, we engineer the Serine Threonine Cycle, a synthetic C1-assimilation cycle in Escherichia coli to achieve growth on formic acid. Our stepwise engineering approach in tailored selection strains combined with adaptive laboratory evolution experiments enabled formatotrophic growth of the organism. Whole genome sequencing and reverse engineering allowed us to determine the key mutations linked to pathway activity. The Serine Threonine Cycle strains created in this work use formic acid as sole carbon and energy source and can grow at ambient CO2 cultivation conditions. This work sets an example for the engineering of complex C1-assimilation cycles in heterotrophic microbes.
大气中的二氧化碳会导致全球变暖和气候变化,对地球上的生命构成重大威胁。另一方面,它也可以被视为一种资源,其规模足以建立循环碳经济。因此,捕获二氧化碳并将其转化为还原一碳(C1)化合物(如甲酸)的技术正在快速发展和改进。在创建可持续生物生产平台这一理念的推动下,天然和合成的 C1 利用途径被设计到与工业相关的微生物中。在生物体内实现合成 C1 同化循环是一项前景广阔但极具挑战性的工作。在这里,我们在大肠杆菌中设计了合成 C1 同化循环的丝氨酸苏氨酸循环,以实现在甲酸上的生长。我们在量身定制的选择菌株中采用分步工程方法,并结合适应性实验室进化实验,实现了该生物的格式营养生长。通过全基因组测序和逆向工程,我们确定了与途径活性相关的关键突变。在这项工作中创建的丝氨酸苏氨酸循环菌株使用甲酸作为碳源和能源,可以在环境二氧化碳培养条件下生长。这项工作为在异养微生物中设计复杂的 C1 同化循环树立了榜样。
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引用次数: 0
AI-based automated construction of high-precision Geobacillus thermoglucosidasius enzyme constraint model. 基于人工智能的高精度热葡聚糖地衣芽孢杆菌酶约束模型的自动构建。
IF 8.4 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-18 DOI: 10.1016/j.ymben.2024.10.006
Minghao Zhang,Haijiao Shi,Xiaohong Wang,Yanan Zhu,Zilong Li,Linna Tu,Yu Zheng,Menglei Xia,Weishan Wang,Min Wang
Geobacillus thermoglucosidasius NCIMB 11955 possesses advantages, such as high-temperature tolerance, rapid growth rate, and low contamination risk. Additionally, it features efficient gene editing tools, making it one of the most promising next-generation cell factories. However, as a non-model microorganism, a lack of metabolic information significantly hampers the construction of high-precision metabolic flux models. Here, we propose a BioIntelliModel (BIM) strategy based on artificial intelligence technology for the automated construction of enzyme-constrained models. 1) . BIM utilises the Contrastive Learning Enabled Enzyme Annotation (CLEAN) prediction tool to analyse the entire genome sequence of G. thermoglucosidasius NCIMB 11955, uncovering potential functional proteins in non-model strains. 2). The MetaPatchM module of BIM automates the repair of the metabolic network model. 3). The Tianjin University of Science and Technology-kcat (TUST-kcat) module predicts the kcat values of enzymes within the model. 4). The Enzyme-insert procedure constructs an enzyme-constrained model and performs a global scan to address overconstraint issues. Enzymatic data were automatically integrated into the metabolic flux model, creating an enzyme-constrained model, ec_G-ther11955. To validate model accuracy, we used both the p-thermo and ec_G-ther11955 models to predict riboflavin production strategies. The ec_G-ther11955 model demonstrated significantly higher accuracy. To further verify its efficacy, we employed ec_G-ther11955 to guide the rational design of L-valine-producing strains. Using the Optimisation Procedure for Identifying All Genetic Manipulations Leading to Targeted Overproductions (OptForce), Predictive Knockout Targeting (PKT), and Flux Scanning based on Enforced Objective Flux (FSEOF) algorithms, we identified 24 knockout and overexpression targets, achieving an accuracy rate of 87.5%. Ultimately, this led to an increase of 664.04% in L-valine titre. This study provides a novel strategy for rapidly constructing non-model strain models and demonstrates the tremendous potential of artificial intelligence in metabolic engineering.
Geobacillus thermoglucosidasius NCIMB 11955 具有耐高温、生长速度快、污染风险低等优点。此外,它还具有高效的基因编辑工具,是最有前途的下一代细胞工厂之一。然而,作为一种非模型微生物,代谢信息的缺乏极大地阻碍了高精度代谢通量模型的构建。在此,我们提出了一种基于人工智能技术的生物智能模型(BIM)策略,用于自动构建酶约束模型。1) .BIM 利用对比学习酶注释(CLEAN)预测工具来分析热葡糖球菌 NCIMB 11955 的整个基因组序列,从而发现非模型菌株中潜在的功能蛋白。2).BIM 的 MetaPatchM 模块可自动修复代谢网络模型。3).天津科技大学-kcat(TUST-kcat)模块可预测模型中酶的 kcat 值。4).4). Enzyme-insert 程序构建酶约束模型,并进行全局扫描以解决过度约束问题。酶数据被自动整合到代谢通量模型中,创建了一个酶约束模型 ec_G-ther11955。为了验证模型的准确性,我们使用 p-thermo 和 ec_G-ther11955 模型预测核黄素生产策略。ec_G-ther11955模型的准确性明显更高。为了进一步验证其有效性,我们使用 ec_G-ther11955 来指导合理设计生产 L-缬氨酸的菌株。利用识别导致定向过量生产的所有遗传操作的优化程序(OptForce)、预测性敲除靶标(PKT)和基于强制目标通量的通量扫描(FSEOF)算法,我们识别出了 24 个敲除和过表达靶标,准确率达到 87.5%。最终,L-缬氨酸滴度提高了 664.04%。这项研究为快速构建非模型菌株模型提供了一种新策略,并展示了人工智能在代谢工程中的巨大潜力。
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引用次数: 0
AI-based automated construction of high-precision Geobacillus thermoglucosidasius enzyme constraint model 基于人工智能的高精度热葡聚糖地衣芽孢杆菌酶约束模型的自动构建。
IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-18 DOI: 10.1016/j.ymben.2024.10.006
Minghao Zhang , Haijiao Shi , Xiaohong Wang , Yanan Zhu , Zilong Li , Linna Tu , Yu Zheng , Menglei Xia , Weishan Wang , Min Wang
Geobacillus thermoglucosidasius NCIMB 11955 possesses advantages, such as high-temperature tolerance, rapid growth rate, and low contamination risk. Additionally, it features efficient gene editing tools, making it one of the most promising next-generation cell factories. However, as a non-model microorganism, a lack of metabolic information significantly hampers the construction of high-precision metabolic flux models. Here, we propose a BioIntelliModel (BIM) strategy based on artificial intelligence technology for the automated construction of enzyme-constrained models. 1). BIM utilises the Contrastive Learning Enabled Enzyme Annotation (CLEAN) prediction tool to analyse the entire genome sequence of G. thermoglucosidasius NCIMB 11955, uncovering potential functional proteins in non-model strains. 2). The MetaPatchM module of BIM automates the repair of the metabolic network model. 3). The Tianjin University of Science and Technology-kcat (TUST-kcat) module predicts the kcat values of enzymes within the model. 4). The Enzyme-insert procedure constructs an enzyme-constrained model and performs a global scan to address overconstraint issues. Enzymatic data were automatically integrated into the metabolic flux model, creating an enzyme-constrained model, ec_G-ther11955. To validate model accuracy, we used both the p-thermo and ec_G-ther11955 models to predict riboflavin production strategies. The ec_G-ther11955 model demonstrated significantly higher accuracy. To further verify its efficacy, we employed ec_G-ther11955 to guide the rational design of L-valine-producing strains. Using the Optimisation Procedure for Identifying All Genetic Manipulations Leading to Targeted Overproductions (OptForce), Predictive Knockout Targeting (PKT), and Flux Scanning based on Enforced Objective Flux (FSEOF) algorithms, we identified 24 knockout and overexpression targets, achieving an accuracy rate of 87.5%. Ultimately, this led to an increase of 664.04% in L-valine titre. This study provides a novel strategy for rapidly constructing non-model strain models and demonstrates the tremendous potential of artificial intelligence in metabolic engineering.
Geobacillus thermoglucosidasius NCIMB 11955 具有耐高温、生长速度快、污染风险低等优点。此外,它还具有高效的基因编辑工具,是最有前途的下一代细胞工厂之一。然而,作为一种非模型微生物,代谢信息的缺乏极大地阻碍了高精度代谢通量模型的构建。在此,我们提出了一种基于人工智能技术的生物智能模型(BIM)策略,用于自动构建酶约束模型。1) .BIM 利用对比学习酶注释(CLEAN)预测工具来分析热葡糖球菌 NCIMB 11955 的整个基因组序列,从而发现非模型菌株中潜在的功能蛋白。2).BIM 的 MetaPatchM 模块可自动修复代谢网络模型。3).天津科技大学-kcat(TUST-kcat)模块可预测模型中酶的 kcat 值。4).4). Enzyme-insert 程序构建酶约束模型,并进行全局扫描以解决过度约束问题。酶数据被自动整合到代谢通量模型中,创建了一个酶约束模型 ec_G-ther11955。为了验证模型的准确性,我们使用 p-thermo 和 ec_G-ther11955 模型预测核黄素生产策略。ec_G-ther11955模型的准确性明显更高。为了进一步验证其有效性,我们使用 ec_G-ther11955 来指导合理设计生产 L-缬氨酸的菌株。利用识别导致定向过量生产的所有遗传操作的优化程序(OptForce)、预测性敲除靶标(PKT)和基于强制目标通量的通量扫描(FSEOF)算法,我们识别出了 24 个敲除和过表达靶标,准确率达到 87.5%。最终,L-缬氨酸滴度提高了 664.04%。这项研究为快速构建非模型菌株模型提供了一种新策略,并展示了人工智能在代谢工程中的巨大潜力。
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引用次数: 0
Butyrate as a growth factor of Clostridium acetobutylicum 作为乙酰丁酸梭菌生长因子的丁酸盐。
IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-15 DOI: 10.1016/j.ymben.2024.10.005
Hyeongmin Seo, Sofia H. Capece, John D. Hill, Jonathan K. Otten, Eleftherios T. Papoutsakis
The butyrate biosynthetic pathway not only contributes to electron management and energy generation in butyrate forming bacteria, but also confers evolutionary advantages to the host by inhibiting the growth of surrounding butyrate-sensitive microbes. While high butyrate levels induce toxic stress, effects of non-toxic levels on cell growth, health, metabolism, and sporulation remain unclear. Here, we show that butyrate stimulates cellular processes of Clostridium acetobutylicum, a model butyrate forming Firmicute. First, we deleted the 3-hydroxybutyryl-CoA dehydrogenase gene (hbd) from the C. acetobutylicum chromosome to eliminate the butyrate synthetic pathway and thus butyrate formation. A xylose inducible Cas9 cassette was chromosomally integrated and utilized for the one-step markerless gene deletions. Non-toxic butyrate levels significantly affected growth, health, and sporulation of C. acetobutylicum. After deleting spo0A, the gene encoding the master regulator of sporulation, Spo0A, and conducting butyrate addition experiments, we conclude that butyrate affects cellular metabolism through both Spo0A-dependent and independent mechanisms. We also deleted the hbd gene from the chromosome of the asporogenous C. acetobutylicum M5 strain lacking the pSOL1 plasmid to examine the potential involvement of pSOL1 genes on the observed butyrate effects. Addition of crotonate, the precursor of butyrate biosynthesis, to the hbd deficient M5 strain was used to probe the role of butyrate biosynthesis pathway in electron and metabolic fluxes. Finally, we found that butyrate addition can enhance the growth of the non-butyrate forming Clostridium saccharolyticum. Our data suggest that butyrate functions as a stimulator of cellular processes, like a growth factor, in C. acetobutylicum and potentially evolutionarily related Clostridium organisms.
丁酸盐生物合成途径不仅有助于丁酸盐形成细菌的电子管理和能量生成,还能抑制周围对丁酸盐敏感的微生物的生长,从而为宿主带来进化优势。虽然高水平的丁酸盐会引起毒性应激,但无毒水平的丁酸盐对细胞生长、健康、新陈代谢和孢子的影响仍不清楚。在这里,我们发现丁酸盐能刺激乙酰丁酸梭菌(一种典型的丁酸盐形成固缩菌)的细胞过程。首先,我们从乙酰丁酸梭菌染色体中删除了 3-hydroxybutyryl-CoA dehydrogenase 基因(hbd),以消除丁酸合成途径,从而消除丁酸的形成。在染色体上整合了木糖诱导型 Cas9 基因盒,并利用该基因盒进行一步无标记基因删除。无毒的丁酸盐浓度明显影响了乙酰丁酸杆菌的生长、健康和孢子发育。在删除 spo0A(编码孢子形成主调节因子 Spo0A 的基因)并进行丁酸盐添加实验后,我们得出结论:丁酸盐通过依赖 Spo0A 和独立机制影响细胞代谢。我们还从缺乏 pSOL1 质粒的无孢子 C. acetobutylicum M5 菌株的染色体中删除了 hbd 基因,以研究 pSOL1 基因对所观察到的丁酸盐效应的潜在参与。在缺乏 hbd 的 M5 菌株中添加巴豆酸盐(丁酸盐生物合成的前体),以探究丁酸盐生物合成途径在电子和代谢通量中的作用。最后,我们发现丁酸盐的添加可以促进不形成丁酸盐的糖化梭菌的生长。我们的数据表明,丁酸盐在乙酰丁酸梭菌和可能与之有进化关系的梭菌中起着刺激细胞过程的作用,就像生长因子一样。
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引用次数: 0
Growth-coupled production of L-isoleucine in Escherichia coli via metabolic engineering 通过代谢工程在大肠杆菌中生长耦合生产 L-异亮氨酸。
IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-14 DOI: 10.1016/j.ymben.2024.10.004
Nan Lu , Minhua Wei , Xuejing Yang , Yingzi Li , Hao Sun , Qianyu Yan , Haibin Zhang , Jilong He , Jie Ma , Menglei Xia , Chenglin Zhang
L-isoleucine, an essential amino acid, is widely used in the pharmaceutical and food industries. However, the current production efficiency is insufficient to meet the increasing demands. In this study, we aimed to develop an efficient L-isoleucine-producing strain of Escherichia coli. First, accumulation of L-isoleucine was achieved by employing feedback-resistant enzymes. Next, a growth-coupled L-isoleucine synthetic pathway was established by introducing the metA-metB-based α-ketobutyrate-generating bypass, which significantly increased L-isoleucine production to 7.4 g/L. Upon employing an activity-improved cystathionine γ-synthase mutant obtained from adaptive laboratory evolution, L-isoleucine production further increased to 8.5 g/L. Subsequently, the redox flux was improved by bypassing the NADPH-dependent aspartate aminotransferase pathway and employing the NADH-dependent pathway and transhydrogenase. Finally, L-isoleucine efflux was enhanced by modifying the transport system. After fed-batch fermentation for 48 h, the resultant strain, ISO-12, reached an L-isoleucine production titer of 51.5 g/L and yield of 0.29 g/g glucose. The strains developed in this study achieved a higher L-isoleucine production efficiency than those reported previously. These strategies will aid in the development of cell factories that produce L-isoleucine and related products.
L- 异亮氨酸是一种必需氨基酸,被广泛应用于制药和食品行业。然而,目前的生产效率不足以满足日益增长的需求。在本研究中,我们旨在培育一株高效的大肠杆菌 L-异亮氨酸生产菌株。首先,通过使用抗反馈酶实现了 L-异亮氨酸的积累。接着,通过引入基于 metA-metB 的α-酮丁酸生成旁路,建立了生长耦合的 L-异亮氨酸合成途径,将 L-异亮氨酸产量显著提高到 7.4 克/升。在使用从适应性实验室进化中获得的活性提高的胱硫醚γ-合成酶突变体后,L-异亮氨酸的产量进一步提高到 8.5 克/升。随后,通过绕过依赖 NADPH 的天冬氨酸氨基转移酶途径,采用依赖 NADH 的途径和反式氢化酶,提高了氧化还原通量。最后,通过改造转运系统提高了 L-异亮氨酸的外流。经过 48 小时的饲料批量发酵后,产生的菌株 ISO-12 的 L-异亮氨酸生产滴度达到 51.5 克/升,葡萄糖产量为 0.29 克/克。与之前报道的菌株相比,本研究开发的菌株实现了更高的 L-异亮氨酸生产效率。这些策略将有助于开发生产 L-异亮氨酸及相关产品的细胞工厂。
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引用次数: 0
Improving the growth and intestinal colonization of Escherichia coli Nissle 1917 by strengthening its oligopeptides importation ability 通过加强大肠杆菌 Nissle 1917 的寡肽导入能力,改善其生长和肠道定植。
IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-09 DOI: 10.1016/j.ymben.2024.10.002
Ruxue Sun , Peijun Yu , Liying Guo , Yufei Huang , Yanhong Nie , Yunpeng Yang
Escherichia coli Nissle 1917 (EcN), the probiotic featured with well-established safety in different host, is emerging as a favored chassis for the construction of engineered probiotics for disease treatment. However, limited by the low intestinal colonization ability of EcN, repeated administration is required to maximize the health benefits of the EcN-derived engineered probiotics. Here, using fecal metabolites as “metabolites pool”, we developed a metabolomic strategy to characterize the comprehensive metabolic profile of EcN. Compared with Prevotella copri DSM 18205 (P. copri), one of the dominant microbes in gut flora, EcN exhibited minor growth advantage under the fecal metabolites-containing condition for its lower metabolic capability towards fecal metabolites. Further study indicated that EcN lacked the ability to import the oligopeptides containing more than two amino acids. The shortage of oligopeptides-derived amino acids might limit the growth of EcN by restricting its purine metabolism. Assisted with the bioinformatic and qRT-PCR analyses, we identified a tripeptides-specific importer Pc-OPT in P. copri, which was mainly distributed in genera Prevotella and Bacteroides. Overexpression of Pc-OPT improved the tripeptides importation of EcN and promoted its growth and intestinal colonization. Notably, 16S rRNA gene amplicon sequencing results indicated that strengthening the oligopeptides importation ability of EcN might promote its intestinal colonization by adjusting the gut microbial composition. Our study reveals that the growth and intestinal colonization of EcN is limited by its insufficient oligopeptides importation and paves road for promoting the efficacy of the EcN-derived synthetic probiotics by improving their intestinal colonization ability.
大肠杆菌 Nissle 1917(EcN)是一种益生菌,在不同宿主体内具有公认的安全性,正在成为构建用于疾病治疗的工程益生菌的首选底盘。然而,受限于 EcN 较低的肠道定植能力,需要重复给药才能最大限度地发挥 EcN 衍生的工程益生菌的健康益处。在此,我们利用粪便代谢物作为 "代谢物池",开发了一种代谢组学策略,以描述 EcN 的综合代谢特征。与肠道菌群中的优势微生物之一 Copri Prevotella DSM 18205(P. copri)相比,EcN 在含有粪便代谢物的条件下表现出较小的生长优势,因为它对粪便代谢物的代谢能力较低。进一步的研究表明,EcN 缺乏导入含有两个以上氨基酸的寡肽的能力。寡肽衍生氨基酸的缺乏可能限制了 EcN 的嘌呤代谢,从而限制了其生长。在生物信息学和 qRT-PCR 分析的帮助下,我们在 P. copri 中发现了一种三肽特异性导入器 Pc-OPT,它主要分布在 Prevotella 和 Bacteroides 属中。过表达 Pc-OPT 可提高 EcN 的三肽导入能力,促进其生长和肠道定植。值得注意的是,16S rRNA基因扩增片段测序结果表明,加强EcN的寡肽导入能力可能会通过调整肠道微生物组成促进其肠道定植。我们的研究揭示了 EcN 的生长和肠道定植受限于其寡肽导入能力不足,并为通过提高其肠道定植能力来促进源自 EcN 的合成益生菌的功效铺平了道路。
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引用次数: 0
Engineering a novel pathway for efficient biosynthesis of salicin in Escherichia coli 在大肠杆菌中设计一条高效生物合成水杨素的新途径。
IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-09 DOI: 10.1016/j.ymben.2024.10.003
Jingyan Wang, Qianjing Zhao, Xin Chen, Yichen Lu, Xinxiao Sun, Qipeng Yuan, Jia Wang, Xiaolin Shen
Salicin is a natural glycoside compound widely used to treat fever, inflammation, and analgesia. Currently, salicin is primarily extracted from willow bark, which is not only cumbersome in terms of extraction and separate steps, but also subject to seasonal and geographic limitations. In this study, a highly efficient biosynthetic pathway for salicin synthesis was designed and constructed in E. coli. The most important precursor in the synthetic pathway of salicin designed in this study is salicyl alcohol. Building on a previously constructed biosynthetic salicylic acid metabolic pathway, the production of salicyl alcohol in shake flask fermentation reached 1.7 g/L by increasing the supply of shikimic acid pathway precursor PEP and salicyl alcohol precursor chorismate. According to the principle of substrate similarity, this study identified the key enzyme OsSGT1 from Oryza sativa, which uses E. coli endogenous UDP-glucose as a glycosyl donor to glycosylate salicyl alcohol into salicin. By redefining the optimal substrate of OsSGT1, and balancing metabolic flux along with increasing the supply of UDP-glucose, salicin production in shake flasks reached 4 g/L. Finally, culturing the high-yield strain in a 3-L fermenter resulted in the synthesis of 14.62 g/L of salicin. To the best of our knowledge, this achievement marks the highest salicin production through microbial fermentation to date.
水杨素是一种天然苷类化合物,广泛用于治疗发烧、炎症和镇痛。目前,水杨素主要从柳树皮中提取,不仅提取和分离步骤繁琐,而且受到季节和地域的限制。本研究在大肠杆菌中设计并构建了一条高效合成水杨素的生物合成途径。本研究设计的水杨素合成途径中最重要的前体是水杨醇。在之前构建的水杨酸生物合成代谢途径的基础上,通过增加莽草酸途径前体 PEP 和水杨醇前体胆氨酸的供应,摇瓶发酵中水杨醇的产量达到了 1.7 g/L。根据底物相似性原理,本研究确定了大麦中的关键酶 OsSGT1,它以大肠杆菌内源 UDP-葡萄糖为糖基供体,将水杨醇糖基化为水杨素。通过重新确定 OsSGT1 的最佳底物、平衡代谢通量以及增加 UDP 葡萄糖的供应,摇瓶中的水杨素产量达到了 4 克/升。最后,在 3 升发酵罐中培养高产菌株,合成了 14.62 克/升的水杨素。据我们所知,这是迄今为止通过微生物发酵生产水杨素的最高产量。
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Metabolic engineering
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