Synthetic and Systems Biotechnology最新文献_第9页

The coupling effect of Penicillium baileys W2 in the Aspergillus flavus inhibition and peanut growth promotion 贝利青霉W2抑制黄曲霉与促进花生生长的耦合效应

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-04 DOI: 10.1016/j.synbio.2025.09.003

Qianqian Wang , Shihua Shan , Quanxi Sun , Xiaobo Zhao , Cuiling Yuan , Yifei Mou , Juan Wang , Caixia Yan , Qi Wang , Qingchen Rui , Chunjuan Li

Aspergillus flavus is a significant plant pathogen, and peanut crops are particularly vulnerable to aflatoxin contamination. This vulnerability underscores the need for more effective control methods. In this study, the strain Penicillium baileys W2 was isolated from the rhizosphere soils of healthy peanut seedlings. The fermentation extract exhibited concentration-dependent inhibition of pathogenic A. flavus growth, with a minimum inhibitory concentration (MIC) of 55 % and a minimum fungicidal concentration (MFC) of 60 %. Physiological data and transcriptome analysis demonstrated that the W2 fermentation supernatant inhibited A. flavus growth by disrupting membrane permeability. Metabolomics analysis identified active compounds, including propylparaben, taxifolin, and phloretin, which exhibited significant antagonistic effects against A. flavus. Additionally, we evaluated the impact of the W2 fermentation broth on peanut growth promotion and on rhizosphere microbial community structure using metagenomic sequencing. The reduction of harmful soil microorganisms contributed to the maintenance of soil health, whereas the increased abundance of beneficial microorganisms enhanced peanut seedling growth by facilitating soil nutrient cycling. These findings indicate that the development and application of P. baileys strain W2 or its fermentation extract aligns with sustainable agricultural principles and offers a promising biological control approach.

黄曲霉是一种重要的植物病原菌，花生作物特别容易受到黄曲霉毒素的污染。这一脆弱性强调需要更有效的控制方法。本研究从健康花生幼苗根际土壤中分离出百利青霉W2菌株。发酵提取物对致病性黄曲霉生长的抑制呈浓度依赖性，最低抑制浓度（MIC）为55%，最低杀菌浓度（MFC）为60%。生理数据和转录组分析表明，W2发酵上清通过破坏膜通透性来抑制黄曲霉的生长。代谢组学分析鉴定出活性化合物，包括对羟基苯甲酸丙酯、杉木素和根皮素，对黄曲霉具有显著的拮抗作用。此外，我们还利用宏基因组测序技术评估了W2发酵液对花生生长的促进作用和对根际微生物群落结构的影响。有害微生物的减少有助于维持土壤健康，而有益微生物丰度的增加通过促进土壤养分循环来促进花生幼苗的生长。这些结果表明，开发和应用白僵菌菌株W2或其发酵提取物符合可持续农业原则，是一种有前景的生物防治方法。

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引用次数: 0

Enhanced vitamin B6 production in engineered Escherichia coli via restricted mix-carbon feeding and pressure-controlled fermentation 通过限制混合碳饲养和压力控制发酵提高工程大肠杆菌维生素B6的产量

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-04 DOI: 10.1016/j.synbio.2025.09.004

Long Chen , Miao-Miao Xia , Hui-Na Dong , Hao-Ran Ma , Xu-Yang Huang , Gong-Jin Shen , Zhao-Xia Jin , Lin-Xia Liu , Da-Wei Zhang

Pyridoxine (PN), a major commercial form of vitamin B₆, is mainly produced via chemical synthesis, raising environmental concerns. Microbial fermentation offers a greener alternative, but low biomass and titer limit industrial application. In this study, we developed an integrated strategy combining DO-stat restricted mix-carbon feeding with two-stage pressure-controlled fermentation conditions, and medium optimization to enhance cell growth and PN production in Escherichia coli. DO-stat feeding enabled dynamic regulation of oxygen and carbon sources, while a two-stage pressure-controlled (0.1 bar for 0–24 h, 0.2 bar thereafter) improved both growth and PN biosynthesis. Meanwhile, a novel medium (CRS-67), optimized via Taguchi design, increased shake flask PN titer by 275 mg/L, 75.5 % over CS medium. Under systematic fermentation optimization in a 5-L bioreactor, OD₆₀₀ reached 142.8 and PN titer reached 3.33 g/L—the highest reported level to date. This study presents a robust and scalable process for microbial PN production and offers insights for industrial biomanufacturing of vitamin B₆.

吡哆醇（Pyridoxine， PN）是维生素B6的一种主要商业形式，主要通过化学合成生产，引起了环境问题。微生物发酵提供了一个更环保的选择，但低生物量和滴度限制工业应用。在本研究中，我们开发了一种综合策略，将DO-stat限制性混合碳饲养与两阶段压力控制发酵条件相结合，并对培养基进行优化，以提高大肠杆菌的细胞生长和PN产量。DO-stat喂养可以动态调节氧和碳源，而两级压力控制（0-24小时0.1 bar，之后0.2 bar）促进了生长和PN的生物合成。同时，经田口设计优化的新型培养基CRS-67可使摇瓶PN效价提高275 mg/L，比CS培养基提高75.5%。在5-L的生物反应器中进行系统发酵优化，OD600达到142.8，PN滴度达到3.33 g/ l，是迄今为止报道的最高水平。本研究提出了一个强大的、可扩展的微生物PN生产过程，并为维生素B6的工业生物制造提供了见解。

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引用次数: 0

Construction and analysis of a cell factory for terpenoid biosynthesis in Pichia pastoris via metabolic engineering and metabolomics 基于代谢工程和代谢组学的毕赤酵母萜类生物合成细胞工厂的构建与分析

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-03 DOI: 10.1016/j.synbio.2025.08.015

Chenfan Sun , Cuifang Ye , Xiaoqian Li , Jiabin Xu , Huiru Yu , Jucan Gao , Chengran Guan , Jintao Cheng

Terpenoids are widely distributed in nature and have various applications in health products, pharmaceuticals, and fragrances. Despite the tremendous potential of terpenoids, traditional production methods such as plant extraction and chemical synthesis face challenges in meeting current market demands. With the developments in synthetic biology and metabolic engineering, it has become feasible to construct efficient microbial cell factories for large-scale production for terpenoids. In this work, using the yeast Pichia pastoris as the host cell, a "plug-and-play" cell factory for universal terpenoid production was constructed by enhancing the expression of the MVA pathway for common precursor synthesis and reducing branch pathway diversion strategies. We have successfully and efficiently synthesized β-elemene, β-farnesene, (+)-valencene, (−)-α-bisabolol by this cell factory. Furthermore, by analyzing metabolites in different engineered strains in terms of system biology, it was discovered that an increase in key protein copy numbers enhanced the synthesis of arginine and other metabolic pathways. The robustness of the strains and the tightly regulated metabolic network constrain rational metabolic engineering transformations. These data provide important clues for the modification and optimization of production strains.

萜类化合物广泛存在于自然界中，在保健品、药品和香料中有多种应用。尽管萜类化合物潜力巨大，但传统的植物提取和化学合成等生产方法在满足当前市场需求方面面临挑战。随着合成生物学和代谢工程学的发展，构建高效的微生物细胞工厂大规模生产萜类化合物已成为可能。本研究以毕氏酵母为宿主细胞，通过增强MVA通路的表达，减少分支通路的转移策略，构建了一个“即插即用”的通用萜类化合物生产细胞工厂。通过该细胞工厂，我们成功地合成了β-榄香烯、β-法尼烯、(+)-价烯、(−)-α-双abolol。此外，通过对不同工程菌株的代谢产物进行系统生物学分析，发现关键蛋白拷贝数的增加促进了精氨酸和其他代谢途径的合成。菌株的鲁棒性和严格调控的代谢网络约束了合理的代谢工程转化。这些数据为生产菌株的改良和优化提供了重要线索。

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引用次数: 0

A near-infrared ratio fluorescent probe achieves mitochondrial hydrogen polysulfide imaging for monitoring ferroptosis in arthritis 近红外比例荧光探针实现线粒体多硫化氢成像监测关节炎中的铁下垂

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-02 DOI: 10.1016/j.synbio.2025.08.016

Ting Cao , Ziwen Xiao , Wenhua Dong , Hong Ma , Deyan Gong , Zhefeng Fan

Reactive sulfur (RSS) is a type of sulfur-containing molecule widely present in biological systems. Hydrogen polysulfide (H₂S_n, n > 1), as a member of the active sulfur family, plays an indispensable role in many physiological and pathological processes. Ferroptosis is a special cell death mode driven by iron-dependent lipid peroxidation, which is involved in the occurrence and development of various human diseases. Ferroptosis is manifested by increased lipid peroxidation and elevated levels of reactive oxygen species (ROS), which further lead to an increase in H₂S_n content in cells. Emerging research suggests a close association between ferroptosis and arthritis related diseases. This work successfully constructed a mitochondrial-targeted ratiometric near-infrared fluorescent probe for the specific detection of H₂S_n. The experimental results show that the probe (Cy-S₄) has a large Stokes shift (∼218 nm), excellent optical properties, extremely fast response time (8 s), high sensitivity (DL = 0.23 μM), and strong specificity. This probe has been successfully applied to tracking the content of H₂S_n in ferroptosis process and fluorescence imaging of H₂S_n in inflammatory cell mitochondria. Pathological section data confirmed that the probe has good in vivo imaging ability, and more importantly, in vivo arthritis imaging experiments showed that the expression of H₂S_n plays an important role in ferroptosis. These experimental results will provide a reliable monitoring tool for the treatment and prevention of arthritis, enriching the theoretical research related to this disease.

活性硫（RSS）是一类广泛存在于生物系统中的含硫分子。多硫化氢（H2Sn, n > 1）作为活性硫家族的一员，在许多生理病理过程中起着不可缺少的作用。铁下垂是一种由铁依赖性脂质过氧化作用驱动的特殊细胞死亡模式，参与了人类多种疾病的发生和发展。铁下垂表现为脂质过氧化和活性氧（ROS）水平升高，这进一步导致细胞中H2Sn含量增加。新兴研究表明，铁下垂与关节炎相关疾病密切相关。本工作成功构建了一种线粒体靶向比例荧光探针，用于特异检测H2Sn。实验结果表明，该探针（Cy-S4）具有较大的Stokes位移（~ 218 nm）、优异的光学性能、极快的响应时间（8 s）、高灵敏度（DL = 0.23 μM）和强特异性。该探针已成功应用于铁下垂过程中H2Sn含量的跟踪和炎症细胞线粒体中H2Sn的荧光成像。病理切片数据证实该探针具有良好的体内成像能力，更重要的是，体内关节炎成像实验表明H2Sn的表达在铁下垂中起重要作用。这些实验结果将为关节炎的治疗和预防提供可靠的监测工具，丰富该疾病的相关理论研究。

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引用次数: 0

Combining multiplex metabolic engineering with adaptive evolution strategies for high-level succinic acid production in Yarrowia lipolytica 结合多元代谢工程和适应性进化策略的多脂耶氏菌高产琥珀酸的研究

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-08-30 DOI: 10.1016/j.synbio.2025.08.011

Tao Sun , Mei-Li Sun , Lu Lin , Jian Gao , Rodrigo Ledesma-Amaro , Kaifeng Wang , Xiao-Jun Ji

Succinic acid, an essential platform chemical with extensive utility in biodegradable materials, pharmaceuticals, and the food industry, faces challenges of high energy consumption and environmental pollution in traditional chemical synthesis. Here, we employed multiplex metabolic engineering and adaptive laboratory evolution to enhance succinic acid biosynthesis in Yarrowia lipolytica. By attenuating succinate dehydrogenase (Sdh) activity, mitigating by-product accumulation, and enhancing the succinate synthesis pathway, engineered strains showed efficient succinic acid production from glycerol. The titer reached 130.99 g/L under unregulated pH conditions, translating to a yield of 0.35 g/g and a productivity of 0.70 g/(L·h). Subsequently, transporter engineering and adaptive evolution strategies were applied to enhance glucose utilization for succinic acid synthesis, yielding an evolved strain that eliminated the growth lag phase and produced 106.68 g/L succinic acid from glucose, which translated to a yield of 0.32 g/g and a productivity of 0.64 g/(L·h). Additionally, transcriptomic analysis and inverse metabolic engineering revealed that 4-hydroxyphenylpyruvate dioxygenase (4-Hppd) in the tyrosine degradation pathway partially restored the growth of Sdh-deficient strains on glucose, offering new insights for subsequent succinic acid biomanufacturing using Y. lipolytica.

琥珀酸是生物降解材料、制药、食品等行业中广泛应用的重要平台化学品，传统的化学合成面临着高能耗和环境污染的挑战。在这里，我们采用多重代谢工程和适应性实验室进化来增强脂耶氏菌的琥珀酸生物合成。通过降低琥珀酸脱氢酶（Sdh）活性，减轻副产物积累，增强琥珀酸合成途径，工程菌株显示出从甘油高效生产琥珀酸的能力。在不调节pH条件下，滴度达到130.99 g/L，产率为0.35 g/g，产率为0.70 g/（L·h）。随后，利用转运体工程和适应性进化策略提高葡萄糖对琥珀酸合成的利用，获得了一种进化菌株，该菌株消除了生长滞后期，从葡萄糖中产生106.68 g/L琥珀酸，产率为0.32 g/g，生产力为0.64 g/（L·h）。此外，转录组学分析和逆向代谢工程显示，酪氨酸降解途径中的4-羟基苯基丙酮酸双加氧酶（4-Hppd）部分恢复了sdh缺陷菌株对葡萄糖的生长，为后续利用聚脂Y. lipolytica生物制造琥珀酸提供了新的见解。

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引用次数: 0

Development of a novel transformation system using zhongshengmycin in Aspergillus oryzae and Aspergillus niger 中生霉素在米曲霉和黑曲霉中转化体系的建立

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-08-29 DOI: 10.1016/j.synbio.2025.08.014

Futi Bi , Qichen Huang , Baoxiang Pan , Bin Wang , Li Pan

Filamentous fungi such as Aspergillus oryzae and Aspergillus niger serve as valuable microbial cell factories with diverse applications. However, genetic manipulation in these fungi is often hindered by the lack of efficient resistance-based transformation systems. To address this challenge, we developed a novel transformation system for both A. oryzae and A. niger based on zhongshengmycin, a cost-effective streptothricin-class antibiotic, used in combination with the resistance gene encoding nourseothricin acetyltransferase (NAT). Our tests revealed that sensitization with SDS and EDTA-Na₂ disrupts cell membrane integrity, thereby enhancing the susceptibility of Aspergillus to zhongshengmycin. Through an attB/attP-mediated integration system, we achieved heterologous expression of β-glucuronidase in A. oryzae with a selection efficiency of 85.71 % (6/7). Additionally, recombination of megfp-sed5 in A. niger achieved a transformation efficiency of 83.33 % (10/12), comparable to that of the PyrG system, thus establishing a versatile platform for fungal biotechnology. This system enables precise genomic integration for metabolic rewiring, offering new strategies for the synthetic biology-driven improvement of industrial filamentous fungi.

丝状真菌如米曲霉和黑曲霉是具有广泛应用价值的微生物细胞工厂。然而，由于缺乏有效的基于抗性的转化系统，这些真菌的遗传操作常常受到阻碍。为了解决这一挑战，我们开发了一种基于中胜霉素（一种具有成本效益的链霉素类抗生素）和耐药基因编码的壬基乙酰转移酶（NAT）联合使用的新型米霉和黑霉转化系统。我们的实验表明，SDS和EDTA-Na2的致敏破坏了细胞膜的完整性，从而增强了曲霉对中生霉素的敏感性。通过atb / attp介导的整合系统，我们实现了β-葡萄糖醛酸酶在水稻芽孢杆菌中的异源表达，选择效率为85.71%（6/7）。此外，megfp-sed5在黑曲霉中的重组转化效率达到83.33%(10/12)，与PyrG系统相当，从而为真菌生物技术建立了一个多功能的平台。该系统为代谢重布线提供了精确的基因组整合，为工业丝状真菌的合成生物学驱动改进提供了新的策略。

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引用次数: 0

Hfq modulates system-wide regulatory networks controlling biocontrol property in Lysobacter enzymogenes Hfq调节溶菌酶原生物防治特性的全系统调控网络

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-08-29 DOI: 10.1016/j.synbio.2025.08.013

Xinyi Cheng , Wenhui Chen , Yangyang Zhao , Yancun Zhao , Fengquan Liu , Gaoge Xu

Lysobacter enzymogenes is a beneficial soil bacterium renowned for its potent biocontrol properties, primarily attributed to antimicrobial secondary metabolites such as HSAF and WAP-8294A2, as well as specialized secretion systems. In this study, we investigate the post-transcriptional regulatory roles of the RNA chaperone Hfq in L. enzymogenes OH11 using an integrated multi-omics approach, combining transcriptomic, proteomic, and RNA-binding data. Our comprehensive analysis reveals that Hfq systemically regulates central metabolism and coordinates biocontrol-associated processes, including antimicrobial biosynthesis and secretion systems. Notably, Hfq extensively modulates multiple regulatory pathways involved in HSAF biosynthesis, a well-studied compound with potential applications in combating fungal and oomycete diseases. Additionally, we identify a set of Hfq-associated small RNAs. Through target prediction, we inferred that many of these sRNAs likely influence cellular functions and stress responses, reinforcing Hfq's role as a global regulator of bacterial physiology. These findings provide a systems-level understanding of Hfq's regulatory mechanisms in L. enzymogenes, offering valuable insights for optimizing its biocontrol potential.

溶酶杆菌是一种有益的土壤细菌，以其强大的生物防治特性而闻名，主要归因于抗菌次级代谢物，如HSAF和WAP-8294A2，以及专门的分泌系统。在这项研究中，我们使用综合多组学方法，结合转录组学、蛋白质组学和RNA结合数据，研究了RNA伴侣Hfq在L.酶基因OH11中的转录后调控作用。我们的综合分析表明，Hfq系统地调节中枢代谢和协调生物防治相关过程，包括抗菌生物合成和分泌系统。值得注意的是，Hfq广泛调节与HSAF生物合成有关的多种调控途径，HSAF是一种被充分研究的化合物，在对抗真菌和卵霉菌疾病方面具有潜在的应用前景。此外，我们还鉴定了一组与hfq相关的小rna。通过目标预测，我们推断许多这些sRNAs可能影响细胞功能和应激反应，从而加强了Hfq作为细菌生理全局调节剂的作用。这些发现提供了对Hfq在L. enzymatic genes中的调控机制的系统级理解，为优化其生物防治潜力提供了有价值的见解。

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引用次数: 0

Biofilm-based continuous secretion of human lysozyme through cell-surface display of FimH on Saccharomyces cerevisiae 基于生物膜的人溶菌酶的连续分泌，通过在酿酒酵母上的细胞表面显示FimH

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-08-27 DOI: 10.1016/j.synbio.2025.08.012

Zhenyu Wang , Bangguo Fu , Huifang Zhang , Mengting Li , Xiwei Peng , Huanqing Niu , Yong Chen , Chenjie Zhu , Jia Liu , Dong Liu , Hanjie Ying

Biofilms enhance microbial tolerance to harsh environments while preserving cellular activity over prolonged periods. Although biofilm-based continuous fermentation is widely applied for production of small-molecule chemicals, its application to macromolecular protein production has been rarely reported. Here, FimH, an adhesin from E. coli fimbriae that is employed for mannose-specific adherence and promotes biofilm formation, was displayed on the surface of S. cerevisiae using the anchoring proteins Sag1C, Sed1, Cwp2 and Ccw12, resulting in an 80–150 % enhancement in biofilm formation. Among them, Sed1 was the most effective, followed by Sag1C. A biofilm-based fermentation system for continuous secretion of human lysozyme (hLYZ) was established via cell-surface display of FimH in S. cerevisiae, achieving stable operation for over 350 h. The engineered strain BY4742-Sag1C-hlyz achieved an average extracellular hLYZ activity of 113.1 U/mL, significantly higher than the control BY4742-hlyz (41.6 U/mL), owing to enhanced cell adhesion that increased the total cell number in biofilm-based fermentation. Its productivity reached 2.36 U/mL/h, representing a 77.4 % increase compared with free-cell fermentation of BY4742-hlyz (1.33 U/mL/h). In conclusion, this study first achieved heterologous expression of a bacterial biofilm-forming gene in S. cerevisiae, enhancing biofilm formation and providing a reference for future expression of bacterial biofilm-related genes in yeast. Furthermore, biofilm-based fermentation enabled continuous secretion of hLYZ, highlighting a promising strategy for continuous production of recombinant proteins.

生物膜增强微生物对恶劣环境的耐受性，同时长时间保持细胞活性。尽管基于生物膜的连续发酵广泛应用于小分子化学品的生产，但其在大分子蛋白质生产中的应用却很少报道。本文利用锚定蛋白Sag1C、Sed1、Cwp2和Ccw12，将来自大肠杆菌菌毛的粘附素FimH展示在酿酒酵母表面，用于甘露糖特异性粘附并促进生物膜的形成，从而使生物膜的形成增强80 - 150%。其中Sed1最有效，Sag1C次之。通过在酿酒酵母细胞表面展示溶菌酶（FimH），建立了基于生物膜的连续分泌人溶菌酶（hLYZ）的发酵体系，稳定运行350 h以上。工程菌株BY4742-Sag1C-hlyz的平均胞外hLYZ活性为113.1 U/mL，显著高于对照BY4742-hlyz (41.6 U/mL)，这是由于在生物膜发酵过程中增强了细胞黏附，增加了细胞总数。其产率达到2.36 U/mL/h，比BY4742-hlyz的自由细胞发酵（1.33 U/mL/h）提高77.4%。总之，本研究首次在酿酒酵母中实现了细菌生物膜形成基因的异源表达，促进了细菌生物膜的形成，为今后在酵母中表达细菌生物膜相关基因提供了参考。此外，基于生物膜的发酵使hLYZ能够连续分泌，这凸显了重组蛋白连续生产的前景。

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引用次数: 0

Combination of enzyme engineering and quorum sensing system for efficient de novo biosynthesis of β-arbutin in Escherichia coli 结合酶工程和群体感应系统在大肠杆菌中高效从头合成β-熊果苷

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-08-22 DOI: 10.1016/j.synbio.2025.08.010

Zhuoyuan Liu , Dongming Liu , Lihao Deng , Kai LingHu , Xiaoyu Shan , Jingwen Zhou , Weizhu Zeng

β-Arbutin is a plant-derived glycoside with various biological activities, which has been applied in pharmaceuticals and whitening cosmetics. Uneven carbon flux distribution and the low catalytic efficiency of arbutin synthase limit the biosynthesis of β-arbutin in microorganisms. In addition, commonly used high-cost, or toxic, inducible expression agents are not practical to use in industrial production. In this study, a biosynthetic pathway for β-arbutin was constructed in Escherichia coli. The competition and degradation pathways were knocked out, and the key pathway enzymes were identified and overexpressed, to increase β-arbutin production. Furthermore, by combining modifications to the phosphoketolase pathway and the acetic acid pathway, the carbon flux was redirected to the shikimic acid pathway. The substrate access channel of the arbutin synthase active site was modified by protein engineering, resulting in the R241H mutant, which increased β-arbutin production by 16.7 %, compared with wild type (WT). Finally, the luxR-based quorum sensing (QS) system was used to regulate the allocation of metabolic fluxes between cell growth and product synthesis, achieving a self-induced regulation of E. coli, without requiring exogenous inducers. The final engineered strain produced 81.9 g/L of β-arbutin from a fed-batch fermentation in a 5 L fermenter and the yield from glucose reached 0.29 g/g. This study presents a systematic strategy that can provide a reference for the construction of β-arbutin microbial cell factories.

β-熊果苷是一种具有多种生物活性的植物源性糖苷，在医药、美白化妆品等领域有着广泛的应用。碳通量分布不均和熊果苷合酶催化效率低限制了微生物对β-熊果苷的生物合成。此外，常用的高成本或有毒的诱导表达剂不适合用于工业生产。本研究在大肠杆菌中构建了β-熊果苷的生物合成途径。敲除竞争和降解途径，鉴定并过表达关键途径酶，增加β-熊果苷的产量。此外，通过结合磷酸酮醇酶途径和乙酸途径的修饰，将碳通量重定向到莽草酸途径。通过蛋白工程修饰熊果苷合成酶活性位点的底物通路，得到的R241H突变体β-熊果苷产量较野生型（WT）提高16.7%。最后，利用基于luxr的群体感应（quorum sensing， QS）系统调节代谢通量在细胞生长和产物合成之间的分配，实现了大肠杆菌不需要外源诱导剂的自我诱导调节。最终工程菌株在5 L的发酵罐中分批补料发酵，β-熊果苷产量为81.9 g/L，葡萄糖产量为0.29 g/g。本研究为β-熊果苷微生物细胞工厂的建设提供了系统的策略参考。

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引用次数: 0

Topoisomerase II-Like variants enhance acid tolerance and low-pH l-malic acid biosynthesis in metabolic engineered Aspergillus nidulans 拓扑异构酶ii样变异体增强代谢工程曲霉的耐酸性和低ph的l-苹果酸生物合成

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-08-22 DOI: 10.1016/j.synbio.2025.08.008

Lingling Pei, Yan Dong, Ziqing Chen, Chi Zhang, Ling Lu

l-malic acid is a versatile compound having extensive applications across food, pharmaceutical, medical, and chemical industries while its production from microbial fermentation offers sustainable advantages including renewable feedstocks and environmental friendliness. However, a major challenge faced by microbial l-malic acid production is acid-induced pH drop during the fermentation process, requiring excessive neutralizers (e.g., CaCO₃) that increase costs and environmental burdens greatly. In this study, we firstly made an engineered Aspergillus nidulans strain as a production chassis through metabolic engineering by overexpression of fructose-6-phosphate kinase gene (pfkA) combined with deletion of pyruvate decarboxylase gene (pdcA) so that the titer of l-malic acid was increased by 20.3 %. Secondly, overexpression of glucose transporter gene (mstE) further boosted production to the highest reported shake-flask titer (44.3 g/L) for A. nidulans. Moreover, acid-tolerant isolates capable of growth at pH 2.8 has been successfully obtained by adaptive laboratory evolution in aforementioned engineered strains. They achieved 75 % neutralizer reduction (just adding 10 g/L CaCO₃) while maintaining original titers. Whole-genome sequencing comparison identified mutations in gene (ANIA_04699) encoding a protein with a topoisomerase II associated PAT I domain happened in acid-tolerance isolates. Functional validation via engineered point-mutants confirmed mutations of the gene (ANIA_04699) as a key acid-tolerance determinant, contributes a major role enabling CaCO3 reduction without titer loss. Meanwhile, phylogenetic studies showed there are about 71–75 % homology across selected Aspergillus spp. suggesting high conservation and broad applicability. Function prediction analysis based on previous verified PAT I domains imply that mutations of this topoisomerase II-Like protein might be tolerant to DNA damage induced by low pH environment. This work establishes A. nidulans as a platform for low-pH organic acid production and identifies topoisomerase II variants as new targeted candidates for acid tolerance engineering.

l-苹果酸是一种用途广泛的化合物，在食品、制药、医疗和化学工业中有着广泛的应用，而其微生物发酵生产具有可持续的优势，包括可再生原料和环境友好性。然而，微生物l-苹果酸生产面临的一个主要挑战是在发酵过程中酸引起的pH值下降，需要过多的中和剂（例如CaCO3），这大大增加了成本和环境负担。本研究首先通过代谢工程的方法，通过过表达果糖-6-磷酸激酶基因（pfkA）结合丙酮酸脱羧酶基因（pdcA）的缺失，构建了一株工程菌作为生产基质，使l-苹果酸滴度提高了20.3%。其次，葡萄糖转运蛋白基因（mstE）的过表达进一步提高了假木兰花的产量，达到了所报道的最高摇瓶滴度（44.3 g/L）。此外，通过上述工程菌株的适应性实验室进化，成功获得了能够在pH 2.8下生长的耐酸菌株。在保持原始滴度的情况下，他们实现了75%的中和剂还原（仅添加10 g/L CaCO3）。全基因组测序比较发现，在耐酸菌株中，编码拓扑异构酶II相关的PAT I结构域的蛋白的基因（ANIA_04699）发生了突变。通过工程点突变的功能验证证实了该基因（ANIA_04699）的突变是一个关键的耐酸决定因素，在不损失滴度的情况下促进CaCO3的还原。同时，系统发育研究表明，所选曲霉属的同源性约为71 ~ 75%，具有较高的保守性和广泛的适用性。基于先前验证的PAT I结构域的功能预测分析表明，这种拓扑异构酶ii样蛋白的突变可能耐受低pH环境诱导的DNA损伤。本研究确定了a . nidulans作为低ph有机酸生产的平台，并确定了拓扑异构酶II变体作为耐酸工程的新靶标候选者。

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