Synthetic and Systems Biotechnology最新文献_第2页

A synergistic strategy of metabolic engineering and flocculation recycling for enhanced acetoin production in Zymomonas mobilis 代谢工程和絮凝循环协同策略提高活动单胞菌乙酰素产量

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2026-01-17 DOI: 10.1016/j.synbio.2025.12.014

Weiwei Bao , Qiqun Peng , Hongxiang Yu , Hongwei Yang , Shihui Yang

Acetoin, a valuable platform chemical, faces sustainability challenges in its traditional energy-intensive synthesis. Microbial fermentation using microorganisms such as Zymomonas mobilis offers a promising alternative. To overcome metabolic limitations and process inefficiencies of economic acetoin production, we integrated strategies of metabolic engineering, transcriptomic-guided analysis, flocculation-based cell recycling, and non-food feedstock utilization. A dominant metabolism compromised intermediate (DMCI) chassis of Z. mobilis was constructed by deleting ethanol production and acetoin degradation pathways. Transcriptomics was then employed to identify and knockout latent competing pathway genes of ZMO0318 and ZMO1576 to enhance acetoin production. The engineered strain also tolerated to inhibitors in lignocellulosic hydrolysates, and fed-batch fermentation achieved an acetoin titer of 73 g/L. Furthermore, self-flocculating phenotype was engineered via ZMO1082 modification to enable efficient cell recycling over multiple batches for production cost reduction. This study thus establishes a synergistic strategy to enhance acetoin production, highlighting the role of combining metabolic engineering, omics analyses, and processing engineering for economic biochemical production.

乙托因是一种有价值的平台化学品，在其传统的能源密集型合成中面临着可持续性挑战。微生物发酵利用微生物，如活动单胞菌提供了一个很有前途的选择。为了克服经济乙酰蛋白生产的代谢限制和工艺效率低下，我们综合了代谢工程、转录组引导分析、基于絮凝的细胞回收和非食品原料利用的策略。通过删除产乙醇途径和乙酰胆碱降解途径，构建了一种优势代谢受损中间体（DMCI）底盘。然后利用转录组学技术鉴定并敲除ZMO0318和ZMO1576的潜在竞争通路基因，以促进乙酰素的产生。该工程菌株对木质纤维素水解物中的抑制剂也有耐受性，分批补料发酵的乙酰素滴度达到73 g/L。此外，通过修饰ZMO1082，设计了自絮凝表型，使细胞能够在多批次中高效回收，从而降低生产成本。因此，本研究建立了一种协同策略来提高乙酰丙酮的产量，强调了代谢工程、组学分析和加工工程在经济生化生产中的作用。

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引用次数: 0

Engineering Yarrowia lipolytica for high-level ergosterol production 工程解脂耶氏菌生产高水平麦角甾醇

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2026-01-16 DOI: 10.1016/j.synbio.2026.01.001

Xinyu Liu , Qihang Chen , Wenbao Zhao , Qi Li , Liushuting Xiao , Qian He , Weizhu Zeng , Jingwen Zhou

Ergosterol is the key precursor of steroid drug synthesis. In this experiment, we systematically modified the synthesis of ergosterol. Firstly, we identified key rate-limiting enzymes through systematic screening of the post-squalene pathway. Combinatorial overexpression of IDI1, tHMG1, ERG4, ERG5, ERG27, ERG1 and ERG11 achieved an ergosterol titer of 94.2 mg/L. Molecular dynamics guided mutagenesis of key substrate channel residues, particularly S372V Erg11, enhanced local flexibility and significantly increased ergosterol production. Introduction of the proton-donating mutations S372V-T305H-ERG11 established an artificial proton-dependent pathway, which, together with channel engineering, further increased the titer to 124 mg/L. Lipid droplet engineering and cellular compartmentalization strategies increased the titer to 148.3 mg/L. Ultimately, multi-copy integration of all ergosterol synthesis pathway genes increased the titer to 433.1 mg/L, and fed-batch fermentation in a 5-L bioreactor resulted in a final titer of 4.58 g/L. This study demonstrates a comprehensive hierarchical strategy for high-level sterol production.

麦角甾醇是合成类固醇药物的关键前体。在本实验中，我们对麦角甾醇的合成进行了系统的修饰。首先，我们通过系统筛选角鲨烯后通路确定了关键的限速酶。IDI1、tHMG1、ERG4、ERG5、ERG27、ERG1和ERG11的组合过表达使麦角甾醇滴度达到94.2 mg/L。分子动力学引导的关键底物通道残基诱变，特别是S372V Erg11，增强了局部柔韧性，显著增加了麦角甾醇的产量。引入供质子突变S372V-T305H-ERG11，建立了人工质子依赖途径，结合通道工程，进一步将滴度提高到124 mg/L。脂滴工程和细胞区隔策略使滴度提高到148.3 mg/L。最终，所有麦角甾醇合成途径基因的多拷贝整合将滴度提高到433.1 mg/L，并在5-L生物反应器中分批补料发酵，最终滴度为4.58 g/L。本研究展示了高水平固醇生产的综合分层策略。

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引用次数: 0

Modifying the upstream open reading frames of cellulase gene enhances cellulase production in Penicillium oxalicum 修改纤维素酶基因上游开放阅读框可提高草青霉的纤维素酶产量

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2026-01-16 DOI: 10.1016/j.synbio.2025.12.016

Shuai Zhao , Xin Shen , Ting-Yu Zhou , Yan-Hao Zhao , Di Tian , Xue-Mei Luo , Zhi-Peng Zhou , Xin-Qing Zhao , Jia-Xun Feng

Cellulase plays an irreplaceable role in biomanufacturing using plant biomass as feedstock. However, improving cellulase production by fungi through manipulation of upstream open reading frames (uORFs) in the 5′-untranslated regions (5′-UTR) of cellulase genes has been less frequently explored. This study aimed to screen uORFs in the 5′-UTR of cellulase genes in Penicillium oxalicum, identify functional uORFs in the 5′-UTR of the eg1 gene which encodes a key endo-β-1,4-glucanase (EG) in P. oxalicum, and enhance fungal cellulase production through uORF modifications. Among the 25 cellulase genes examined in P. oxalicum strain HP7-1, 23 contained uORFs in their 5′-UTR. Seven uORFs were annotated in the 5′-UTR of the eg1 gene. A uORF-green fluorescent protein (GFP) reporter system demonstrated that uORF1 and uORF3 inhibited, while uORF7 enhances, GFP abundance. Overexpression of eg1 containing uORF1 or uORF3 variants where the start codon of the uORF was mutated to AAG in P. oxalicum led to a significant 91.7 % and 62.1 % average increase in carboxymethyl cellulase production after 4 days of induction compared to the start strain ΔPoxKu70. Real-time quantitative reverse transcription-polymerase chain reaction, mRNA stability determination, and in vitro translation experiments collectively revealed that these three uORFs influence the mRNA stability of the downstream mORF, but not translation efficiency. These findings highlight the critical role of uORFs in regulating gene expression during fungal enzyme biosynthesis and offer a valuable alternative strategy for improving enzyme production.

纤维素酶在以植物生物质为原料的生物制造中起着不可替代的作用。然而，真菌通过操纵纤维素酶基因5 ' -非翻译区（5 ' -UTR）的上游开放阅读框（uorf）来提高纤维素酶的产量的研究较少。本研究旨在筛选草青霉纤维素酶基因5′-UTR上的uORF，鉴定草青霉编码关键内端-β-1,4-葡聚糖酶（EG）的eg1基因5′-UTR上的功能性uORF，并通过uORF修饰促进真菌纤维素酶的产生。在草藻菌株HP7-1检测的25个纤维素酶基因中，有23个在其5 ' -UTR中含有uorf。在eg1基因的5 ' -UTR中注释了7个uorf。uorf -绿色荧光蛋白（GFP）报告系统显示，uORF1和uORF3抑制GFP丰度，而uORF7增强GFP丰度。在草藻中过表达含有uORF1或uORF3变异（uORF的起始密码子突变为AAG）的eg1，诱导4天后，与起始菌株ΔPoxKu70相比，羧甲基纤维素酶产量显著增加91.7%和62.1%。实时定量逆转录聚合酶链反应、mRNA稳定性测定和体外翻译实验共同表明，这三种uorf影响下游mORF的mRNA稳定性，但不影响翻译效率。这些发现强调了uORFs在真菌酶生物合成过程中调节基因表达的关键作用，并为改善酶的产生提供了有价值的替代策略。

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引用次数: 0

The functional analysis of NirBD in microorganisms and the efficacies of NirBD on the soil nitrogen storage and N2O emission NirBD在微生物中的功能分析及对土壤氮储存和N2O排放的影响

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2026-01-15 DOI: 10.1016/j.synbio.2025.12.017

Yidan Peng , Tengxia He , Qimin Zhou , Mengyuan Yin , Chengtao Jin

The siroheme-containing nitrite reductase (NirBD), which is encoded by the nirBD gene, is a functional enzyme in the nitrogen cycle. The NirBD enzyme can regulate N₂O release through the two distinct pathways of assimilatory nitrate/nitrite reduction to biomass and dissimilatory nitrate/nitrite reduction to ammonium. Therefore, a thorough comprehension of the function of NirBD in microorganisms can enable us to better understanding the contributions for nitrogen retention and N₂O emission. However, the knowledge of the functions and expression mechanisms of nirBD gene across different microorganisms remains limited. This review synthesized the current research on the phylogenetic distribution and catalytic versatility of NirBD in fungi, bacteria, and actinomycetes. The contributions of NirBD for nitrogen retention and N₂O emission were extensively discussed under anaerobic and aerobic conditions. The expression mechanism of NirBD was demonstrated. The factors that affect the expression amount of the NirBD enzyme in microorganisms were clarified systematically. This review not only elucidated the unique role of NirBD in the nitrogen metabolism but also provided a critical theoretical foundation for developing future strategies to enhance soil nitrogen fertility and mitigate N₂O emissions.

含siroheme亚硝酸盐还原酶（NirBD）是一种氮循环中的功能性酶，由NirBD基因编码。NirBD酶可通过同化性硝酸盐/亚硝酸盐还原为生物质和异化性硝酸盐/亚硝酸盐还原为铵两种不同的途径调节N2O的释放。因此，深入了解微生物中NirBD的功能有助于我们更好地了解微生物对氮保留和N2O排放的贡献。然而，对nirBD基因在不同微生物中的功能和表达机制的了解仍然有限。本文综述了NirBD在真菌、细菌和放线菌中的系统发育分布和催化通用性的研究进展。在厌氧和好氧条件下，广泛讨论了NirBD对氮保留和N2O排放的贡献。证实了NirBD的表达机制。系统地阐明了微生物中影响NirBD酶表达量的因素。该综述不仅阐明了NirBD在氮素代谢中的独特作用，也为未来制定提高土壤氮肥肥力和减少N2O排放的策略提供了重要的理论基础。

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引用次数: 0

De novo synthesis of L-2-aminobutyric acid in Escherichia coli based on multi-layered metabolic engineering strategies 基于多层代谢工程策略的大肠杆菌从头合成l -2-氨基丁酸

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2026-01-15 DOI: 10.1016/j.synbio.2026.01.004

Zhenqiang Zhao , Yizheng Liu , Rongshuai Zhu , Fengyu Yang , Zhifei Liu , Jiajia You , Xuewei Pan , Jianming Yang , Zhiming Rao

L-2-Aminobutyric acid (L-2-ABA) is a non-proteinogenic amino acid and an important chiral intermediate widely used in pharmaceuticals and fine chemicals. However, its fermentative production is limited by intermediate toxicity and imbalanced metabolic flux. In this study, Escherichia coli was systematically engineered for efficient de novo synthesis of L-2-ABA using a multi-layer metabolic engineering strategy. A quorum-sensing–based dynamic control circuit was introduced to decouple cell growth from 2-oxobutyric acid formation, thereby alleviating precursor toxicity and improving flux coordination. Combined with optimization of the L-2-ABA conversion pathway, model-guided carbon flux redistribution, cofactor regeneration, and tuning of global transcriptional regulation, a high-performance production strain was obtained without the need for antibiotics or inducers. The final engineered strain ABA40 achieved 45.3 g/L L-2-ABA with a yield of 0.31 g/g glucose in a 72 h fed-batch fermentation. This study demonstrates the effectiveness of dynamic and integrated metabolic engineering strategies for the biosynthesis of non-natural amino acids.

l -2-氨基丁酸（L-2-ABA）是一种非蛋白质原性氨基酸，是一种重要的手性中间体，广泛应用于医药和精细化工领域。然而，其发酵生产受到中间毒性和代谢通量不平衡的限制。在这项研究中，利用多层代谢工程策略，系统地改造大肠杆菌，使其高效地从头合成L-2-ABA。引入基于群体感应的动态控制电路，将细胞生长与2-氧丁酸形成解耦，从而减轻前体毒性并改善通量协调。通过对L-2-ABA转化途径的优化、模型引导的碳通量再分配、辅因子再生和全局转录调控的调整，获得了无需抗生素和诱导剂的高效生产菌株。最终工程菌株ABA40在补料批发酵72 h后，L-2- aba产量为45.3 g/L，葡萄糖产量为0.31 g/g。该研究证明了动态和综合代谢工程策略在非天然氨基酸生物合成中的有效性。

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引用次数: 0

Enhanced AAV production via rational design of a novel pHelper vector integrated with HSV-1 helper genes 通过合理设计整合HSV-1辅助基因的新型辅助载体提高AAV的产生

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2026-01-15 DOI: 10.1016/j.synbio.2025.12.018

He Ren , Jianqi Nie , Zichuan Song , Wanting Mo , Yankun Yang , Zhonghu Bai

Adeno-associated virus (AAV) vectors are widely used in gene therapy owing to their safety, stability, and broad tissue tropism. However, current plasmid-based AAV manufacturing platforms suffer from low yield and high manufacturing cost, limiting their scalability for clinical and commercial applications. Rational engineering of pHelper vector offers an effective strategy to enhance AAV production. In this study, we engineered a novel helper vector (UL12-ICP22-miniHelper) by integrating UL12 and ICP22 genes from herpes simplex virus type 1 (HSV-1) into a size-reduced pHelper backbone (mini-pHelper) with partial deletion of E2a and E4 regions. In a triple-plasmid transfection system, UL12-ICP22-miniHelper increased AAV5 vector yield from 1.35 × 10¹¹ to 2.85 × 10¹¹ vg/mL (2.11-fold) without altering the proportion of full capsids. Enhanced productivity was also observed across multiple serotypes, with increases of 2.24-fold for AAV1, 1.54-fold for AAV2, 1.88-fold for AAV6, and 2.03-fold for AAV9, while maintaining transduction efficiency. Mechanistic analysis indicated that the improved productivity was associated with elevated viral genome replication and increased Rep/Cap protein expression. Collectively, these results demonstrate that the novel UL12-ICP22-miniHelper provides a broadly applicable and cost-effective strategy for improving AAV vector manufacturing in both clinical and industrial applications.

腺相关病毒（AAV）载体因其安全性、稳定性和广泛的组织亲和性而广泛应用于基因治疗。然而，目前基于质粒的AAV制造平台存在产量低、制造成本高的问题，限制了其临床和商业应用的可扩展性。载体的合理工程设计是提高AAV产量的有效策略。在这项研究中，我们将来自1型单纯疱疹病毒（HSV-1）的UL12和ICP22基因整合到一个缩小的、E2a和E4区域部分缺失的helper主干（mini- helper）中，设计了一个新的辅助载体UL12-ICP22- minihelper。在三质粒转染系统中，UL12-ICP22-miniHelper在不改变完整衣壳比例的情况下，将AAV5载体产量从1.35 × 1011提高到2.85 × 1011 vg/mL（2.11倍）。在保持转导效率的情况下，多种血清型的效率也有所提高，其中AAV1提高了2.24倍，AAV2提高了1.54倍，AAV6提高了1.88倍，AAV9提高了2.03倍。机制分析表明，产量的提高与病毒基因组复制的增加和Rep/Cap蛋白表达的增加有关。总之，这些结果表明，新型UL12-ICP22-miniHelper为改善临床和工业应用中的AAV载体制造提供了一种广泛适用且具有成本效益的策略。

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引用次数: 0

RECC: A Red/ET–CRISPR/Cas9-based system enabling genome mining of marine Pseudoalteromonas for novel natural products RECC：一个基于Red/ ET-CRISPR / cas9的系统，可以对海洋假交替单胞菌进行基因组挖掘，以获得新的天然产物

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2026-01-10 DOI: 10.1016/j.synbio.2025.12.015

Zong-jie Wang , Haibo Zhou , Youming Zhang , Fu Yan , Liujie Huo , Xiaotong Wang

Marine microorganisms possess vast biosynthetic potential, yet most of their biosynthetic gene clusters (BGCs) remain transcriptionally silent under laboratory conditions. Genetic intractability has been a major barrier to activating these cryptic pathways. Here, we present RECC, an integrated Red/ET–CRISPR/Cas9 system that enables seamless, marker-free genome editing in marine bacteria. RECC couples Red/ET recombineering with CRISPR/Cas9-mediated cleavage, allowing the incorporation of homology arms and protospacers into a single construct through one-step Gibson assembly, thereby substantially simplifying the engineering process. Using Pseudoalteromonas flavipulchra DSM 14401 as a model, we employed RECC to replace the native promoter of a silent nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) hybrid gene cluster with a strong constitutive promoter. This targeted activation led to the production of a series of previously unknown cyclolipopeptides, designated flavipulchrins. Structural characterization and bioinformatic analysis revealed a plausible biosynthetic pathway for these metabolites. Collectively, RECC provides a robust and generalizable genome-editing platform that facilitates the systematic exploration of biosynthetic potential in genetically recalcitrant marine microorganisms.

海洋微生物具有巨大的生物合成潜力，但它们的大多数生物合成基因簇（BGCs）在实验室条件下仍保持转录沉默。遗传上的难缠性一直是激活这些隐秘途径的主要障碍。在这里，我们提出了RECC，这是一个集成的Red/ ET-CRISPR /Cas9系统，可以在海洋细菌中进行无缝，无标记的基因组编辑。RECC将Red/ET重组与CRISPR/ cas9介导的裂解结合在一起，通过一步Gibson组装将同源臂和原间隔物整合到一个构建体中，从而大大简化了工程过程。以黄假互变单胞菌（Pseudoalteromonas flavipulchra） DSM 14401为模型，采用RECC将沉默的非核糖体肽合成酶-聚酮合成酶（nrpps - pks）杂交基因簇的原生启动子替换为强组成启动子。这种靶向激活导致产生一系列以前未知的环脂肽，称为黄嘌呤。结构表征和生物信息学分析揭示了这些代谢物的合理生物合成途径。总的来说，RECC提供了一个强大的、通用的基因组编辑平台，有助于系统地探索遗传顽固性海洋微生物的生物合成潜力。

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引用次数: 0

Multi-gene Co-expression systems in E. coli: From single-vector designs to programmable expression platforms 大肠杆菌的多基因共表达系统：从单载体设计到可编程表达平台

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2026-01-10 DOI: 10.1016/j.synbio.2025.12.008

Rui Liu , Lu-Wei Wang , Zi-Han Gao , Xiao-Tong Sun , Shu-Ran Lv , Huan Liu , Sa-ouk Kang , Bo Sun

Escherichia coli (E. coli) has long served as a versatile workhorse for recombinant protein production. As synthetic biology expands the demand for coordinated expression of multiple genes, co-expression systems in E. coli have evolved from basic dual-gene constructs to programmable, polygenic expression platforms. This review critically examines the major strategies enabling multigene co-expression in E. coli, including internal ribosome entry sites (IRES), 2A self-cleaving peptides, dual-promoter cassettes, multicistronic operons, and multi-plasmid configurations. We highlight the mechanistic principles, design trade-offs, and regulatory bottlenecks associated with each approach, such as translational imbalance, inclusion body formation, and plasmid compatibility. Real-world applications in metabolic engineering, complex protein assembly, and biomanufacturing are analyzed to demonstrate the functional advantages of these systems. Finally, we explore emerging programmable toolkits that integrate modular architecture, expression modeling, and AI-assisted design, paving the way for next-generation synthetic expression control in microbial chassis. This review offers a comprehensive and strategic roadmap for researchers engineering multi-gene systems in E. coli and beyond.

大肠杆菌（E. coli）长期以来一直是重组蛋白生产的全能主力。随着合成生物学对多基因协同表达的需求不断扩大，大肠杆菌的共表达系统已经从基本的双基因结构发展到可编程的多基因表达平台。本文综述了大肠杆菌中实现多基因共表达的主要策略，包括内部核糖体进入位点（IRES）、2A自切割肽、双启动子磁带、多顺反子操纵子和多质粒结构。我们强调了与每种方法相关的机制原则、设计权衡和监管瓶颈，如翻译不平衡、包涵体形成和质粒相容性。分析了这些系统在代谢工程、复杂蛋白质组装和生物制造中的实际应用，以展示这些系统的功能优势。最后，我们探索了集成模块化架构、表达建模和人工智能辅助设计的新兴可编程工具包，为下一代微生物底盘合成表达控制铺平了道路。这篇综述为研究人员在大肠杆菌和其他基因系统中设计多基因系统提供了一个全面和战略性的路线图。

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引用次数: 0

Integrated amplification of NADPH-regenerating modules enhances cytidine biosynthesis in Escherichia coli nadph再生模块的整合扩增增强了大肠杆菌胞苷的生物合成

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2026-01-10 DOI: 10.1016/j.synbio.2025.12.009

Lu Liu , Xiangjun Zhang , Tengteng Zhu , Tong Ye , Zongqian Li , Wei Ding , Huiyan Liu , Haitian Fang

In Escherichia coli, cofactor imbalance serves as a crucial limiting factor in cytidine biosynthesis, with nicotinamide adenine dinucleotide phosphate (NADPH) insufficiency representing the principal metabolic barrier. To overcome this limitation, an integrated engineering strategy targeting the enhancement of NADPH metabolism was implemented. Via CRISPR-Cas9-mediated multiplex genomic editing and strong constitutive promoter replacement, three NADPH-regenerating modules were concurrently enhanced: the membrane-bound transhydrogenase (pntAB), the oxidative pentose phosphate pathway (zwf-encoded glucose-6-phosphate dehydrogenase), and the decarboxylation shunt (gnd-encoded 6-phosphogluconate dehydrogenase). After 54-hour fermentation in 500 mL shake flasks, the cytidine titer of the engineered strain NXBG-20 reached 7.83 g/L, representing a 9.10-fold increase compared to the start strain. Systematic multi-omics profiling revealed that the metabolic network had undergone substantial alterations. These alterations were characterized by the redirection of glycolytic flux towards nucleotide precursor substances and the enhancement of ribose-5-phosphate biosynthesis. This engineering approach not only establishes a novel microbial platform for cytidine bioproduction but also provides mechanistic insights into cofactor-driven metabolic flux control.

在大肠杆菌中，辅助因子失衡是胞苷生物合成的关键限制因素，而烟酰胺腺嘌呤二核苷酸磷酸（NADPH）不足是主要的代谢屏障。为了克服这一限制，我们实施了一种以增强NADPH代谢为目标的综合工程策略。通过crispr - cas9介导的多重基因组编辑和强组成型启动子替换，三个nadph再生模块同时增强：膜结合的转氢酶（pntAB）、氧化戊糖磷酸途径（zwf编码的葡萄糖-6-磷酸脱氢酶）和脱羧分支（gnd编码的6-磷酸葡萄糖酸脱氢酶）。在500 mL摇瓶中发酵54小时后，工程菌株NXBG-20的胞苷滴度达到7.83 g/L，比初始菌株提高了9.10倍。系统的多组学分析显示，代谢网络发生了实质性的变化。这些改变的特点是糖酵解通量向核苷酸前体物质的重定向和核糖-5-磷酸生物合成的增强。这种工程方法不仅为胞苷生物生产建立了一个新的微生物平台，而且为辅因子驱动的代谢通量控制提供了机制见解。

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引用次数: 0

Development of a suite of activatable plant synthetic promoter systems using a bacterial LysR-type transcriptional regulator FdeR 利用细菌lysr型转录调控因子FdeR开发一套可激活的植物合成启动子系统

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2026-01-02 DOI: 10.1016/j.synbio.2025.12.005

Yinan Wu , Curtis Chen , Sijin Li

Advancing plant synthetic biology requires an abundant supply of orthogonal and tunable genetic parts to express multiple genes simultaneously. Current genetic parts, particularly promoters, used in plants are still limited and often suffer from tissue specificity and endogenous regulation in planta. Synthetic promoter systems that combine engineered plant-compatible transcription factors (TFs) with synthetic promoters provide a promising alternative approach to enrich current plant synthetic biology toolkits. Leveraging the feature that LysR-type TFs usually bind to the operators regardless of the presence or absence of ligands, we present a systematic approach to develop and characterize a large suite of synthetic promoter systems based on a single LysR-type bacterial TF. Using FdeR from the soil bacterium Herbaspirillum seropedicae and its corresponding operator, fdeO, we developed 52 synthetic promoter systems regulated by four FdeR-derived synthetic activators. Transient expression assays in Nicotiana benthamiana showed that the synthetic promoters were constitutively activated by synthetic activators in a ligand-independent manner, and the resulting promoters spanned a wide range of expression activities, with the lowest equivalent to minimal CaMV 35S promoter and the highest with ∼65% of CaMV 35S activity. We also developed modular workflows and pipelines to accelerate the development of the synthetic transcription platform, including a MoClo-based plug-and-play assembly system for plant-compatible promoter construction and a yeast-based prescreening platform for rapid TF evaluation in eukaryotic cells. This work has demonstrated a scalable framework for developing a large set of synthetic promoter systems from minimal genetic components, leveraging bacterial TF diversity to expand the plant synthetic biology toolkit for robust, orthogonal gene expression.

植物合成生物学的发展需要大量的正交可调基因片段来同时表达多个基因。目前在植物中使用的遗传部分，特别是启动子，仍然有限，并且在植物中经常受到组织特异性和内源调控的影响。将工程植物相容转录因子（TFs）与合成启动子相结合的合成启动子系统为丰富当前的植物合成生物学工具包提供了一种有前途的替代方法。利用lysr型TF通常与操作符结合的特征，无论是否存在配体，我们提出了一种系统的方法来开发和表征基于单个lysr型细菌TF的大型合成启动子系统。利用土壤细菌Herbaspirillum seropedicae的FdeR及其相应的操作符fdeO，我们开发了52个由4个FdeR衍生的合成激活剂调节的合成启动子体系。在benthamiana中的瞬时表达实验表明，合成的启动子以不依赖于配体的方式被合成的激活子激活，并且合成的启动子具有广泛的表达活性，最低的启动子相当于最小的CaMV 35S启动子，最高的启动子相当于CaMV 35S活性的约65%。我们还开发了模块化的工作流程和管道来加速合成转录平台的开发，包括一个基于moclos的即插即用组装系统，用于植物兼容启动子的构建，以及一个基于酵母的真核细胞TF快速评估预筛选平台。这项工作已经证明了一个可扩展的框架，可以从最小的遗传成分中开发出大量的合成启动子系统，利用细菌TF多样性来扩展植物合成生物学工具包，以实现稳健的、正交的基因表达。

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引用次数: 0