Synthetic and Systems Biotechnology最新文献_第7页

Putative membrane protein Cgl2168 and its variant enhance thermotolerance in Corynebacterium glutamicum 推测膜蛋白Cgl2168及其变体增强谷氨酸棒状杆菌的耐热性

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-25 DOI: 10.1016/j.synbio.2025.09.018

Ning Gao , Xiaomeng Ni , Jiao Liu , Jiuzhou Chen , Yu Wang , Ping Zheng , Jibin Sun

Corynebacterium glutamicum ATCC 13032 is widely used as the chassis strain for industrial production of various amino acids, but its thermotolerance limits productivity under high temperatures. Comparative genomic analysis in this study identified cgl2168 as a key determinant of enhanced thermotolerance in a laboratory strain (13032-JN) compared to another stock (13032-TJ). A guanine at Residue 270 (G270) deletion in cgl2168 (cgl2168^Δ270G) in 13032-JN significantly improved growth at 40 °C, while reverting this mutation or deleting cgl2168 impaired thermotolerance. Structural analysis revealed the deletion extends the C-terminus with an additional α-helix. Transcriptomic profiling showed cgl2168^Δ270G increased gene expressions involved in oxidative phosphorylation (e.g., atpABCDEFGH), nitrate metabolism (narIJHK), some heat shock protein genes (e.g., dnaK and groES) and deletion of cgl2168 resulted in upregulating sulfur metabolism (sulfonate transporter protein genes ssuBCD, sulfur-containing amino acid cys and met operons) and central carbon metabolism (gltA, pyc), while downregulating gluconeogenesis (pck) and fermentation (ldh) genes. These changes enhanced ATP synthesis, carbon utilization and oxidative tolerance. Predicted interactors of Cgl2168 linked it to respiratory chain function, stress responses, and cell envelope biogenesis. Thus, Cgl2168 and its variant Cgl2168^Δ270G enhance thermotolerance by coordinating energy production, carbon metabolism, and stress adaptation, aiding industrial applications.

谷氨棒状杆菌ATCC 13032被广泛用作各种氨基酸工业生产的基础菌株，但其耐热性限制了高温下的生产能力。本研究的比较基因组分析发现，cgl2168是实验室菌株（13032-JN）与另一个品系（13032-TJ）相比耐热性增强的关键决定因素。13032-JN中cgl2168 （cgl2168Δ270G）中残基270处的鸟嘌呤（G270）缺失显著改善了40°C下的生长，同时恢复该突变或删除cgl2168损伤了耐热性。结构分析显示，该缺失扩展了c端，增加了一个α-螺旋。转录组学分析显示cgl2168Δ270G参与氧化磷酸化（如atpABCDEFGH）、硝酸盐代谢（narIJHK）、一些热休克蛋白基因（如dnaK和groES）的基因表达增加，cgl2168的缺失导致硫代谢（磺酸转运蛋白基因ssuBCD、含硫氨基酸cys和met操纵子）和中心碳代谢（gltA、pyc）基因表达上调，糖异生（pck）和发酵（ldh）基因表达下调。这些变化增强了ATP合成、碳利用和氧化耐受性。预测的Cgl2168相互作用物将其与呼吸链功能、应激反应和细胞包膜生物发生联系起来。因此，Cgl2168及其变体Cgl2168Δ270G通过协调能量产生、碳代谢和逆境适应来增强热耐受性，有助于工业应用。

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引用次数: 0

Structural and functional insights into the iminium ion intermediate in AsKslB-mediated Pictet-Spengler reaction askslb介导的Pictet-Spengler反应中微量离子中间体的结构和功能研究

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-25 DOI: 10.1016/j.synbio.2025.09.017

Yan-Bin Teng , Zhi Qiao , Chunya Xie , Xiaona Yang , Xinyu Liu , Zhengrong Zou , Yunchang Xie , Xuan Zhang , Qi Chen

Pictet-Spengler (PS) reactions are pivotal in the biosynthesis of bioactive alkaloids and pharmaceuticals, yet key structural details underlying their enzymatic catalysis remain insufficiently understood. We identified AsKslB from Actinosynnema sp. ALI-1.44 as a Pictet-Spenglerase with broad substrate scope that catalyzes the stereoselective condensation of l-tryptophan (l-Trp) and α-ketoglutarate (α-KG) to form kitasetalic acid (KA), a tetrahydro-β-carboline (THβC). High-resolution crystal structures of apo, substrate-, intermediate-, and product-bound forms elucidate the full catalytic trajectory and key residues. Crucially, the elusive iminium ion intermediate (IM-1) and a synchronously released water molecule are captured, providing direct structural evidence for the initiating cyclization step of Pictet-Spengler reaction. Glu276 undergoes conformational changes essential for catalysis. These findings offer detailed mechanistic insights into Pictet-Spenglerase function and establish AsKslB as a promising biocatalyst for stereoselective N-heterocycle synthesis.

Pictet-Spengler （PS）反应在生物活性生物碱和药物的生物合成中是至关重要的，但其酶催化的关键结构细节仍未得到充分的了解。我们从Actinosynnema sp. i -1.44中鉴定出AsKslB是一种具有广泛底物范围的picet - spenglerase，它催化l-色氨酸（l-Trp）和α-酮戊二酸（α-KG）的立体选择性缩合，形成四氢β-碳碱（th -β c） kitasetalic acid （KA）。载脂蛋白、底物结合、中间结合和产物结合形式的高分辨率晶体结构阐明了完整的催化轨迹和关键残基。关键是，捕获了难以捉摸的im1和同步释放的水分子，为Pictet-Spengler反应的起始环化步骤提供了直接的结构证据。Glu276经历了催化所必需的构象变化。这些发现为Pictet-Spenglerase功能提供了详细的机制见解，并确立了AsKslB作为立体选择性n -杂环合成生物催化剂的前景。

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引用次数: 0

A smart gene circuit for precise regulation of tumor cell behavior 精确调控肿瘤细胞行为的智能基因回路

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-22 DOI: 10.1016/j.synbio.2025.09.016

Hao Fu , Zhiwei Zhou , Lan Fang , Qing Wang , Xin Tang , Wei Su , Xuancai Chen , Yachun Tang , Qun Zhou

Personalized gene circuit is a robust mode of cellular regulation that can manipulate intracellular gene expression to achieve desired functional regulation. However, the construction of logic circuits that automatically sense the characteristics of a particular environment within a cell is often difficult and lacking in sensitivity. Here, we synthesize from scratch specific promoters capable of sensing in cells, and use the combination of different types of promoters to construct smart gene circuits that can regulate gene expression in specific cell types sensitively. In detail, the tumour-specific promoter and the prostate tissue-specific promoter were constructed to be combined together into generating an artificial AND-gate gene circuit using the CRISPR-Cas9 system which could identify prostate cancer selectively. We then utilized this artificial gene circuit to drive targeted genes expression, such as P21, E-cadherin and Bax, to inhibit multifunctional prostate cancer cells but not other cells. Moreover, we applied gene circuits to redirect endogenous genes within cells and significantly and specifically suppressed the tumor growth of prostate cancer in vivo. Overall, these results highlight the clinical potential of these gene circuits as specific tools for prostate cancer detection and treatment, which is a new method for specifically reprogramming prostate cancer cells in vivo and may serve as a promising treatment strategy.

个性化基因回路是一种强大的细胞调控模式，可以操纵细胞内基因表达以实现所需的功能调控。然而，在细胞内自动感知特定环境特征的逻辑电路的构建通常是困难的，而且缺乏灵敏度。在这里，我们从无到有地合成了能够在细胞中感知的特异性启动子，并利用不同类型启动子的组合构建了智能基因回路，可以敏感地调节特定细胞类型中的基因表达。具体而言，构建肿瘤特异性启动子和前列腺组织特异性启动子，利用CRISPR-Cas9系统将它们组合在一起，形成一个人工and -gate基因回路，可以选择性地识别前列腺癌。然后，我们利用这种人工基因回路驱动P21、E-cadherin和Bax等靶向基因的表达，抑制多功能前列腺癌细胞而非其他细胞。此外，我们利用基因回路在细胞内重定向内源性基因，在体内显著特异性抑制前列腺癌的肿瘤生长。总之，这些结果突出了这些基因回路作为前列腺癌检测和治疗的特异性工具的临床潜力，这是一种在体内特异性重编程前列腺癌细胞的新方法，可能是一种有前景的治疗策略。

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引用次数: 0

Enhanced microbial production of pyridoxine (Vitamin B6) in Bacillus subtilis via pathway and process optimization 优化枯草芽孢杆菌产吡哆醇（维生素B6）的途径和工艺

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-16 DOI: 10.1016/j.synbio.2025.09.014

Ai-Tong Jiang , Guang-Qing Du , Xu-Yang Huang , Zheng-Zi Ji , Si-Riguleng Qian , Lin-Xia Liu , Da-Wei Zhang

Vitamin B₆ refers to a family of water-soluble B vitamin, which is essential for various physiological functions, including amino acid metabolism, neurotransmitter synthesis, and hemoglobin synthesis. The biosynthesis of pyridoxine (PN), a commercial form of vitamin B₆, through microbial fermentation has garnered widespread attention owing to its environmentally friendly and safe production methods, as well as its mild reaction conditions. However, the low yield of natural strains limits their application. This study focused on constructing a high-yielding strain of PN through pathway engineering and process optimization. Firstly, five key deoxyxylulose-5-phosphate-dependent pathway genes (epd, pdxB, serC, pdxA, and pdxJ) were overexpressed in Bacillus subtilis ARTP, which improved the PN titer by 3.2-fold to 2.9 mg/L. Subsequently, pdxST genes involved in the DXP-independent pathway were screened from various strains. Ribosome binding site (RBS) sequences were optimized to regulate their expression, which further increased the PN titer to 24.6 mg/L. Finally, systematic medium optimization was identified as a critical strategy for enhancing PN biosynthesis, leading to a remarkable 1.8-fold improvement in PN production. Under optimized fermentation conditions, the engineered strain achieved a PN titer of 174.6 mg/L in fed-batch fermentation, which represents the highest level reported to date in B. subtilis. Overall, this study presents an effective strategy combining pathway engineering and medium optimization for significantly improving PN production, offering valuable insights for the industrial development of PN biosynthesis.

维生素B6是指水溶性B族维生素，对氨基酸代谢、神经递质合成、血红蛋白合成等多种生理功能至关重要。吡哆醇（pyridoxine， PN）是维生素B6的一种商业形式，由于其生产方法环保、安全、反应条件温和而受到广泛关注。然而，天然菌株的低产量限制了它们的应用。本研究主要通过途径工程和工艺优化来构建高产的PN菌株。首先，枯草芽孢杆菌ARTP中5个关键的脱氧葡糖-5-磷酸依赖通路基因（epd、pdxB、serC、pdxA和pdxJ）过表达，使PN效价提高3.2倍，达到2.9 mg/L。随后，从不同菌株中筛选了参与dxp独立通路的pdxST基因。通过优化核糖体结合位点（RBS）序列调控其表达，使PN效价进一步提高至24.6 mg/L。最后，系统培养基优化被确定为促进PN生物合成的关键策略，导致PN产量显著提高1.8倍。在优化的发酵条件下，工程菌株在补料分批发酵中获得了174.6 mg/L的PN滴度，这是迄今为止报道的枯草芽孢杆菌的最高水平。总的来说，本研究提出了一种有效的途径工程和培养基优化相结合的策略，可以显著提高PN的产量，为PN生物合成的工业发展提供有价值的见解。

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引用次数: 0

Development of a CRISPR/Cas12a genome editing toolbox in Kluyveromyces marxianus and its application in succinic acid biosynthesis 马氏克卢维菌CRISPR/Cas12a基因组编辑工具箱的开发及其在琥珀酸生物合成中的应用

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-16 DOI: 10.1016/j.synbio.2025.09.015

Hao Zha , Yanjie Li , Zhongmei Hu, Jiacheng Li, Yujie Xie, Mingtao Zhao, Lili Ren, Biao Zhang

Kluyveromyces marxianus is a promising thermotolerant yeast for industrial biotechnology, but lacks efficient genome engineering tools. A CRISPR/Cas12a genome editing toolbox for K. marxianus was developed for the first time in this study. A plasmid-free transient system achieved single-gene knockout efficiencies of about 50 %–100 % in Δku70 strain. Even with homology arms as short as 35 bp, the knockout efficiency remained 66.67 %. Chromosomal integration of Cas12a enabled single-to-triple fragment knock-ins efficiency of 82.93–85.70 % and 94.50 % for large fragment (>5 kb) integrations. Applying this system, the roles of succinate dehydrogenase (SDH) genes SDH1-SDH5 were elucidated. Combinatorial SDH genes knockouts redirected carbon flux toward succinic acid (SA), but increased glycerol/acetate byproducts. Subsequent GPD1/ACH1/ADH2A co-knockout in a Δsdh1,3,5,4A,2 strain with NDE1 overexpression (YZH43) yielded a chassis producing 32.38 g/L SA from glucose at 37 °C, which is the highest reported titer in K. marxianus, while reducing ethanol, acetate, and glycerol by 60.79 %, 89.24 %, and 67.5 %, respectively. At 46 °C, YZH43 produced 20.51 g/L SA through simultaneous saccharification and fermentation (SSF) using cellulose as substrate. This work provides a high-efficiency CRISPR/Cas12a platform for K. marxianus, enabling rapid metabolic engineering for value-added chemical production, and demonstrates its utility in developing thermotolerant SA-overproducing strains.

马氏克鲁维酵母是一种很有应用前景的耐热酵母，但缺乏有效的基因组工程工具。本研究首次构建了马氏乳杆菌CRISPR/Cas12a基因组编辑工具箱。无质粒瞬时系统在Δku70菌株中实现了约50% - 100%的单基因敲除效率。即使同源臂短至35 bp，敲除效率仍为66.67%。Cas12a染色体整合使单片段到三片段的敲入效率为82.93 - 85.70%，大片段（>5 kb）整合的敲入效率为94.50%。利用该系统，研究了琥珀酸脱氢酶（SDH）基因SDH1-SDH5的作用。组合SDH基因敲除将碳通量转向琥珀酸（SA），但增加了甘油/乙酸副产物。随后，在NDE1过表达（YZH43）的Δsdh1、3、5、4A、2菌株（YZH43）中共敲除GPD1/ACH1/ADH2A，在37℃下从葡萄糖中产生32.38 g/L SA，这是在马氏K. marxianus中报道的最高滴度，同时乙醇、乙酸和甘油分别减少了60.79%、89.24%和67.5%。在46℃下，YZH43以纤维素为底物，通过同时糖化发酵（SSF）产生20.51 g/L SA。这项工作为K. marxianus提供了一个高效的CRISPR/Cas12a平台，实现了增值化学品生产的快速代谢工程，并证明了其在培养耐热sa过量菌株方面的实用性。

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引用次数: 0

Corrigendum to “Overproduction of endusamycin in Streptomyces endus subsp. aureus” [Synth Syst Biotechnol 10 (2) (2025) 523–531] “endusamycin在endus链霉菌亚种中的过量生产”的勘误。[Synth系统生物技术10 (2)(2025)523-531]

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-15 DOI: 10.1016/j.synbio.2025.09.001

Yingying Chang , Zhen Liu , Zixin Deng , Tiangang Liu

引用次数: 0

Efficient synthesis of l-DOPA in Escherichia coli via cofactor and enzyme engineering 利用辅助因子和酶工程在大肠杆菌中高效合成左旋多巴

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-12 DOI: 10.1016/j.synbio.2025.09.011

Lihao Deng , Jurong Ping , Zhuoyuan Liu , Kai Linghu , Heng Zhang , Xiaoyu Shan , Weizhu Zeng , Jianghua Li , Jingwen Zhou

The global incidence of Parkinson's disease continues to rise. Levodopa (l-DOPA) is the core therapeutic drug, and efficient and sustainable production methods are needed. However, the complex metabolic pathways and the low catalytic efficiency of enzymes limit biosynthesis of l-DOPA in microorganisms. To address this issue, this study significantly enhanced the production efficiency of l-DOPA through a multi-dimensional, integrated metabolic and enzyme engineering approach. Firstly, the de novo synthesis pathway for l-DOPA was established through optimization of the promoter, ribosome-binding site (RBS), plasmid copy number, and tighly accurately regulating the expression level of key enzymes. Secondly, combined with metabonomic analysis, carbon metabolic flow was diverted, increasing the l-DOPA titer by 36.7 %. Glucose dehydrogenase (BmgdH) and gluconate kinase (gntK) were introduced to construct a cofactor regeneration system, which synergistically enhanced the supply of NADH and FADH₂, increasing the l-DOPA conversion rate by 18 %. Next, the substrate tunnel of 4-hydroxyphenylacetic acid-3-monooxygenase subunit B (HpaB) was subjected to rational design, and mutant T292A significantly expanded the substrate channel, improved catalytic efficiency, and decreased l-tyrosine by 87 %. Finally, through the process optimization in a 5 L bioreactor (involving phased pH control and induction timing adjustment) achieved an l-DOPA titer of 60.73 g/L, the highest reported to date for de novo microbial synthesis. This research offers a novel approach for industrial biosynthesis of l-DOPA, and broadens engineering concepts for efficient synthesis of aromatic compounds.

帕金森氏症的全球发病率持续上升。左旋多巴（l-DOPA）是核心治疗药物，需要高效、可持续的生产方法。然而，复杂的代谢途径和酶的低催化效率限制了微生物对左旋多巴的生物合成。为了解决这一问题，本研究通过多维、综合的代谢和酶工程方法，显著提高了左旋多巴的生产效率。首先，通过优化启动子、核糖体结合位点（RBS）、质粒拷贝数，严格准确调控关键酶的表达水平，建立l-DOPA从头合成途径。其次，结合代谢组学分析，改变了碳代谢流量，使左旋多巴滴度提高了36.7%。引入葡萄糖脱氢酶（BmgdH）和葡萄糖酸激酶（gntK）构建辅助因子再生体系，协同增强NADH和FADH2的供应，使l-DOPA转化率提高18%。接下来，对4-羟基苯基乙酸-3-单加氧酶亚基B （HpaB）的底物通道进行合理设计，突变体T292A显著扩大了底物通道，提高了催化效率，使l-酪氨酸降低了87%。最后，通过在5l生物反应器中进行工艺优化（包括分阶段pH控制和诱导时间调节），获得了60.73 g/L的L - dopa滴度，这是迄今为止报道的微生物从头合成的最高滴度。本研究为左旋多巴的工业生物合成提供了新的途径，拓宽了芳香族化合物高效合成的工程概念。

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引用次数: 0

Enhanced PrrAB system activation and restricted farnesyl pyrophosphate diversion underlie high coenzyme Q10 accumulation in Rhodobacter sphaeroides HY01 在球形红杆菌HY01中，PrrAB系统激活增强和法尼脂基焦磷酸转移受限是辅酶Q10高积累的基础

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-12 DOI: 10.1016/j.synbio.2025.09.012

Xinwei He , Huangwei Wang , Mindong Liang , Weishan Wang , Biqin Chen , Dan Li , Lixin Zhang , Gao-Yi Tan

The industrial Rhodobacter sphaeroides HY01 accumulates an exceptionally high level of coenzyme Q₁₀ (Q₁₀), but the underlying mechanisms remain incompletely understood. Given the central role of Q₁₀ in respiratory electron transport, previous observation of reduced expression of cbb₃-type cytochrome c oxidase genes in HY01 suggested a potential mechanistic link. In this study, we found that cbb₃ oxidase activity in HY01 was only 21.8–32.8 % of that in the wild-type 2.4.1, and restoring this activity led to a 64.4 % decrease in Q₁₀ accumulation, demonstrating a strong inverse correlation. This correlation was found to be mediated by the activation of the PrrAB two-component regulatory system, which is negatively regulated by cbb₃ oxidase. However, disruption of cbb₃ oxidase in 2.4.1 alone was insufficient to reproduce the high Q₁₀ accumulation phenotype, indicating that additional factors may be required. Previous research also revealed restricted synthesis of geranylgeranyl diphosphate (GGPP) in HY01, which likely reduces the diversion of the Q₁₀ precursor farnesyl diphosphate (FPP). Reconstituting this metabolic constraint in wild-type strain, combined with fine-tuning of PrrAB system activation, resulted in up to a 218.0 % increase in Q₁₀ accumulation, achieving a level nearly identical to HY01. Combining mechanistic investigation and inverse metabolic engineering, this study demonstrates that the high Q₁₀ accumulation in HY01 results from the synergistic effects of enhanced PrrAB activation and restricted FPP diversion, providing new insights into the key factors underlying high-level Q₁₀ accumulation in R. sphaeroides.

工业球形红杆菌HY01积累了异常高水平的辅酶Q10 (Q10)，但其潜在的机制尚不完全清楚。鉴于Q10在呼吸电子传递中的核心作用，先前观察到HY01中cbb3型细胞色素c氧化酶基因表达减少，表明可能存在潜在的机制联系。在本研究中，我们发现HY01的cbb3氧化酶活性仅为野生型2.4.1的21.8 - 32.8%，恢复该活性导致Q10积累减少64.4%，表现出很强的负相关关系。这种相关性被发现是通过激活PrrAB双组分调控系统介导的，该系统由cbb3氧化酶负调控。然而，仅在2.4.1中破坏cbb3氧化酶不足以重现高Q10积累表型，这表明可能需要其他因素。先前的研究还发现，在HY01中，geranylgeranyl diphosphate （GGPP）的合成受到限制，这可能减少了Q10前体farnesyl diphosphate （FPP）的转移。在野生型菌株中重建这种代谢约束，并结合PrrAB系统激活的微调，导致Q10积累增加了218.0%，达到与HY01几乎相同的水平。本研究结合机制研究和逆向代谢工程研究，证明了在HY01中Q10的高积累是PrrAB激活增强和FPP转移受限的协同作用的结果，为探究球棘草Q10高积累的关键因素提供了新的见解。

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引用次数: 0

MicroDFBEST: A dCas12b-derived dual-function base editor with programmable editing characteristics for microbial genetic engineering MicroDFBEST：一种dcas12b衍生的双功能碱基编辑器，具有可编程编辑特性，用于微生物基因工程

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-12 DOI: 10.1016/j.synbio.2025.09.013

Wen-Liang Hao , De-Zhi Geng , Yu-Feng Liu , Lai-Chuang Han , Zhe-Min Zhou , Wen-Jing Cui

Base editors (BEs) enable precise genome editing, but their use in microbes remains limited by restricted mutagenesis capabilities and narrow editing windows. Here, we reported MicroDFBEST, a novel dual-function base editor (DFBE) for microbes, by fusing the high-activity deaminases evoCDA1 and TadA9 with nuclease-deficient Cas12b from Bacillus hisashii (dBhCas12b). This engineered system enables simultaneous C-to-T and A-to-G editing within a 26–33 nt window, the broadest range reported for microbial DFBEs. The editing characteristics of MicroDFBEST can be easily adjusted by changing fusion protein expression and editing generations to create diverse mutant libraries. We show that the MicroDFBEST system enables both flexible gene expression modulation via random promoter (P_ylbP) diversification and targeted protein evolution through mutational hotspot scanning in native genomic contexts. This study offers a versatile platform enabling in situ gene regulation (e.g., biosynthetic gene clusters activation) and protein evolution (e.g., chassis optimization), with broad synthetic biology utility.

碱基编辑器（BEs）能够实现精确的基因组编辑，但它们在微生物中的应用仍然受到有限的诱变能力和狭窄的编辑窗口的限制。在这里，我们报道了MicroDFBEST，一种新的微生物双功能碱基编辑器（DFBE），通过将高活性脱氨酶evoCDA1和TadA9与来自hisashii芽孢杆菌的核酸酶缺陷Cas12b （dBhCas12b）融合。该工程系统能够在26 - 33nt的窗口内同时进行C-to-T和a -to- g编辑，这是目前报道的微生物DFBEs中范围最广的。通过改变融合蛋白表达和编辑代数，可以轻松调整MicroDFBEST的编辑特性，从而创建不同的突变文库。我们发现MicroDFBEST系统既可以通过随机启动子（PylbP）多样化灵活地调节基因表达，也可以通过突变热点扫描在原生基因组环境中实现靶向蛋白进化。该研究为原位基因调控（如生物合成基因簇激活）和蛋白质进化（如底盘优化）提供了一个多功能平台，具有广泛的合成生物学实用性。

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引用次数: 0

Whole-cell biosensors with regulatory circuits based on a synthetic dual-input promoter enabling the highly sensitive detection of ultra-trace Cd2+ 具有基于合成双输入启动子的调节电路的全细胞生物传感器，能够高度灵敏地检测超痕量Cd2+

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-11 DOI: 10.1016/j.synbio.2025.09.008

Yuke Qin , Shuting Hu , Yibin Yang , Xiaoqiang Jia

The research on whole-cell biosensors tailored for trace and ultra-trace detection remains limited and the biosensors based on natural bacterial heavy metal resistance mechanisms have common issues of low sensitivity. In this study, we designed a promoter P_T7-cadO using the Cadmium ions (Cd²⁺)-binding protein binding site cadO with the T7 promoter at first and constructed a single-input whole-cell biosensor, which was named CP100, whose detection limit for Cd²⁺ met the WHO requirement, yet its response and sensitivity were quite low. We further introduced the lacI and lac operator (lacO) as the signal amplifier to construct a dual-input promoter P_{T7-cadO-lacO-cadO} and developed a biosensor named LC100, whose response and sensitivity were significantly improved, but background leakage became a new problem. Then we redesigned the gene circuit based on the regulatory circuit LCPM-2, which has the structure of “CadR-P_J23100-P_{T7-cadO-lacO-cadO}-mRFP1-LacI”, with the LacI protein as the autoregulatory negative feedback model and finally obtained the biosensor named LC100-2, which achieved the detection of ultra-trace Cd²⁺ (0.00001–0.02 nM), with the sensitivity of 3748.22 times that of CP100. Moreover, LC100-2 demonstrated excellent specificity to Cd²⁺ among four other divalent metal ions and good anti-interference capability in the mixed divalent metal ions system. The results of the real water sample tests demonstrated that precise quantitative detection of Cd²⁺ with a final concentration of 0.001–0.02 nM could be achieved by adding only a small volume of the sample (1 μL). This finding showed promising application potential in the field of trace detection. Additionally, the regulatory circuit LCPM-2 based on the unique dual-input promoter enhanced responses and reduced background leakage simultaneously, thereby providing an innovative strategy for the design and simplification of biosensors’ circuits.

针对痕量和超痕量检测的全细胞生物传感器的研究仍然有限，基于天然细菌重金属抗性机制的生物传感器普遍存在灵敏度低的问题。本研究首先利用镉离子（Cd2+）结合蛋白结合位点cadO与T7启动子设计了启动子PT7-cadO，构建了单输入全细胞生物传感器CP100，该传感器对Cd2+的检出限符合WHO要求，但其响应和灵敏度较低。我们进一步引入lacI和lac算子（lacO）作为信号放大器，构建双输入启动子PT7-cadO-lacO-cadO，并开发了LC100生物传感器，其响应和灵敏度得到了显著提高，但背景泄漏成为新的问题。然后基于结构为“CadR-PJ23100-PT7-cadO-lacO-cadO-mRFP1-LacI”的调控电路LCPM-2重新设计基因电路，以LacI蛋白为自调控负反馈模型，最终获得命名为LC100-2的生物传感器，实现了超微量Cd2+ (0.00001-0.02 nM)的检测，灵敏度为CP100的3748.22倍。LC100-2对其他四种二价金属离子中的Cd2+具有良好的特异性，在混合二价金属离子体系中具有良好的抗干扰能力。实际水样测试结果表明，只需加入少量样品（1 μL），就可以实现对Cd2+的精确定量检测，最终浓度为0.001 ~ 0.02 nM。这一发现在痕量检测领域具有广阔的应用前景。此外，基于独特双输入启动子的调控电路LCPM-2增强了响应，同时减少了背景泄漏，从而为生物传感器电路的设计和简化提供了一种创新策略。

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