Synthetic and Systems Biotechnology最新文献_第4页

Leveraging ANXA1 to enhance recombinant protein yields in CHO cells: A UPR-Mediated bioprocessing approach 利用ANXA1提高CHO细胞中的重组蛋白产量：一种upr介导的生物处理方法

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-12-18 DOI: 10.1016/j.synbio.2025.12.001

Qi Zhao , Hui-Jie Zhang , Ming-Ming Han , Jumai Abiti , Yan-Ju Dong , Jia-Ning Wang , Jiang-Tao Lu , Wen Wang , Xi Zhang , Shao-Lei Geng , Le-Le Qiu , Xiao-Yin Wang , Zi-Chun Hua , Tian-Yun Wang , Yan-Long Jia

Chinese hamster ovary (CHO) cells undergo endoplasmic reticulum stress (ERS) during intensive recombinant protein production, triggering the unfolded protein response (UPR) to balance cell survival and protein output. Nevertheless, key regulatory components of this process remain incompletely characterized. In this study, we demonstrate that Annexin A1 (ANXA1) functions as a UPR suppressor in CHO cells. Employing the PiggyBac transposon system, we generated a stable ANXA1-knockdown cell line exhibiting a 4.5-fold increase in recombinant antibody expression and a 4.2-fold increase in specific productivity. Pharmacological inhibition using AC2-26 similarly enhanced recombinant protein expression in low-productivity cell populations. Mechanistically, ANXA1 depletion remodeled the UPR by activating the PERK-eIF2α-ATF4 and IRE1-XBP1 branches. This activation upregulaed ATF4, Bip, and XBP1s; suppressed CHOP; reduced apoptosis; and enhanced autophagic flux. Metabolic profiling revealed increased glucose and lactate utilization, while glutamine consumption and ammonia flux remained unchanged. Collectively, these findings establish that ANXA1 depletion enhances recombinant protein biosynthesis through coordinated pro-survival mechanisms. Targeting ANXA1 thus represents an innovative cell engineering strategy for optimizing CHO cell platforms in industrial biopharmaceutical manufacturing.

中国仓鼠卵巢（CHO）细胞在密集重组蛋白生产过程中经历内质网应激（ERS），触发未折叠蛋白反应（UPR）来平衡细胞存活和蛋白输出。然而，这一过程的关键调控成分仍未完全确定。在这项研究中，我们证明了膜联蛋白A1 （ANXA1）在CHO细胞中作为UPR抑制因子发挥作用。利用PiggyBac转座子系统，我们产生了一个稳定的anxa1敲低细胞系，其重组抗体表达增加了4.5倍，特异性生产力增加了4.2倍。AC2-26在低生产力细胞群中的药理抑制作用同样增强了重组蛋白的表达。机制上，ANXA1缺失通过激活PERK-eIF2α-ATF4和IRE1-XBP1分支来重塑UPR。这种激活上调了ATF4、Bip和xbp1；抑制砍;减少细胞凋亡;增强自噬通量。代谢分析显示葡萄糖和乳酸利用增加，而谷氨酰胺消耗和氨通量保持不变。总之，这些发现表明，ANXA1缺失通过协调的促生存机制增强了重组蛋白的生物合成。因此，针对ANXA1代表了一种创新的细胞工程策略，用于优化工业生物制药制造中的CHO细胞平台。

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引用次数: 0

Corrigendum to “Morphology-engineered alleviation of mycelial aggregation in Streptomyces chassis for potentiated production of secondary metabolites” [Synth Syst Biotechnol 10 (3) (2025) 1059–1069] “形态工程减轻链霉菌底盘中菌丝聚集以增强次级代谢物的生产”的勘误[Synth系统生物技术10 (3)(2025)1059-1069]

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-12-18 DOI: 10.1016/j.synbio.2025.11.006

Shuo Liu , Fei Xiao , Lanxin Lv , Meiyan Wang , Wenli Li , Guoqing Niu

引用次数: 0

Formatotrophic Komagataella phaffii expressing recombinant xylanase via metabolic engineering 通过代谢工程表达重组木聚糖酶

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-12-18 DOI: 10.1016/j.synbio.2025.11.015

Ziwei Zhou , Bing Liu , Wenjie Cong , Hualan Zhou , Yu Zheng , Jianguo Zhang

Formate utilization as a sustainable carbon source for microbial production of high-value chemicals and heterologous proteins presents considerable safety and environmental benefits over conventional feedstocks. As a low-cost, CO₂-derived compound, formate serves as a non-flammable and non-toxic alternative to methanol for induction of recombinant expression in Komagataella phaffii. However, since native K. phaffii only utilizes formate as an energy source rather than a carbon substrate for biomass synthesis, we engineered a synthetic peroxisomal formate assimilation pathway by introducing heterologous acetyl-CoA synthetase (ACS) and acetaldehyde dehydrogenase (ACDH), thereby enabling formatotrophic growth with formate as the sole carbon source. This chassis was further optimized through (i) co-expression of the transcriptional activator Mit1 to enhance the coupling efficiency of the ACS-ACDH module, and (ii) reinforcement of the Xu5P pathway by overexpressing dihydroxyacetone synthase (DAS1) and ribulose-5-phosphate-3-epimerase (RPE) to redirect metabolic flux. The resulting formatotrophic K. phaffi strain achieved a specific growth rate of 0.012 h⁻¹ in basal salt medium with formate as the sole carbon source, and produced 30.9 U/(mL·OD₆₀₀) of xylanase from Aspergillus niger ATCC 1015 as a model heterologous protein. Furthermore, ¹³C isotopic tracing confirmed the incorporation of formate-derived carbon into central metabolism for the biosynthesis of amino acids, nucleotides, and structural carbohydrates, validating active formate assimilation. This study establishes a microbial platform for formate-based production of heterologous proteins and underscores the potential of metabolic engineering to advance sustainable biomanufacturing from one-carbon feedstocks.

甲酸酯作为微生物生产高价值化学品和异源蛋白质的可持续碳源，与传统原料相比具有相当的安全性和环境效益。甲酸酯是一种低成本的co2衍生化合物，是一种不易燃、无毒的甲醇替代品，可用于诱导法菲Komagataella重组表达。然而，由于原生K. phaffii只利用甲酸作为能量来源，而不是作为生物质合成的碳底物，我们通过引入异源乙酰辅酶a合成酶（ACS）和乙醛脱氢酶（ACDH）设计了合成甲酸过氧化物酶体同化途径，从而实现甲酸作为唯一碳源的形成营养生长。通过(i)共表达转录激活因子Mit1来提高ACS-ACDH模块的偶联效率，以及（ii）通过过表达二羟丙酮合成酶（DAS1）和核酮糖-5-磷酸-3- epimase （RPE）来强化Xu5P途径来重定向代谢通量，进一步优化了该骨架。在以甲酸盐为唯一碳源的基础盐培养基中，得到的富营养化K. phaffi菌株的比生长率为0.012 h−1，以黑曲霉ATCC 1015为模型异源蛋白产生30.9 U/（mL·OD600）的木聚糖酶。此外，13C同位素示踪证实了甲酸衍生碳参与了氨基酸、核苷酸和结构性碳水化合物的生物合成的中心代谢，证实了甲酸的活性同化。本研究建立了一个以甲酸为基础的异源蛋白生产的微生物平台，并强调了代谢工程在促进单碳原料可持续生物制造方面的潜力。

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引用次数: 0

Enhancing delta-tocotrienol production in Saccharomyces cerevisiae via metabolic engineering strategies in conjunction with the mutagenesis of tocopherol cyclase 结合生育酚环化酶诱变的代谢工程策略提高酿酒酵母的三角生育三烯醇产量

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-12-11 DOI: 10.1016/j.synbio.2025.11.013

Ziming Liu , Min Tang , Wanze Zhang , Yanjie Tian , Jianjun Qiao , Mingzhang Wen , Weiguo Li , Qinggele Caiyin

Delta (δ)-tocotrienol is a member of the vitamin E family and exhibits bioactivities such as antioxidant, anti-inflammatory, and neuroprotective activities. As a nutrient with protective effects on human health, δ-tocotrienol has broad application prospects in food, cosmetic, and pharmaceutical industries. The construction of efficient microbial cell factories capable of δ-tocotrienol production using synthetic biology approaches is an effective strategy for supplementing or even replacing the vitamin E supply chain in the future. The current study successfully enhanced the biosynthesis of δ-tocotrienol in Saccharomyces cerevisiae by combining metabolic engineering and enzyme engineering strategies. Specifically, the substrate channel constructed by the sequential fusion of the enzymes PaCrtE and SyHPT successfully increased the supply of the key precursor MGGBQ, resulting in a significant increase in the production of δ-tocotrienol. In situ extraction and optimization of the expression of transporter protein PDR1 increased the efflux of δ-tocotrienol, directing the metabolic flux toward the product δ-tocotrienol. To enhance the catalytic activity of the key rate-limiting enzyme tocopherol cyclase from Arabidopsis thaliana (AtTC), semirational protein design was conducted herein. The mutant AtTC^T87S was found to increase the production of δ-tocotrienol by 2.3 times compared to that obtained with the wild-type enzyme. AtTC^T87S can thus be universally used for synthetic biology strategies in future studies to enhance the microbial heterologous production of δ-tocotrienol. The strain T08 was finally obtained herein; the numerous metabolic engineering strategies discussed in this study were integrated into this strain, allowing the production of 4337.3 μg/L of δ-tocotrienol in a shake-flask fermentation, which is 8.9 times that of the yield obtained with the initial strain T03. Scaling up to a 5-L fermentation tank resulted in a δ-tocotrienol yield of 16.9 mg/L.

δ (δ)-生育三烯醇是维生素E家族的一员，具有抗氧化、抗炎和神经保护等生物活性。δ-生育三烯醇作为一种对人体健康具有保护作用的营养物质，在食品、化妆品、医药等领域有着广阔的应用前景。利用合成生物学方法构建能够生产δ-生育三烯醇的高效微生物细胞工厂是未来补充甚至取代维生素E供应链的有效策略。本研究采用代谢工程和酶工程相结合的方法，成功地促进了酿酒酵母中δ-生育三烯醇的生物合成。具体来说，通过PaCrtE和SyHPT酶的顺序融合构建的底物通道成功地增加了关键前体MGGBQ的供应，导致δ-生育三烯醇的产量显著增加。原位提取和优化转运蛋白PDR1的表达增加了δ-生育三烯醇的外排，将代谢通量导向产物δ-生育三烯醇。为了提高拟南芥（Arabidopsis thaliana, AtTC）关键限速酶生育酚环化酶（tocop酚环化酶）的催化活性，进行了半分子蛋白设计。与野生型酶相比，突变体AtTCT87S使δ-生育三烯醇的产量增加了2.3倍。因此，在未来的研究中，AtTCT87S可以普遍用于合成生物学策略，以提高微生物异源生产δ-生育三烯醇。最终得到菌株T08；本研究中讨论的多种代谢工程策略被整合到该菌株中，在摇瓶发酵中产生4337.3 μg/L的δ-生育三烯醇，是初始菌株T03产量的8.9倍。扩大到5升的发酵罐，δ-生育三烯醇的产量为16.9 mg/L。

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引用次数: 0

Evaluation of synthetic post-transcription regulatory sequences reveals design principle to enhance mRNA stability and translation efficiency 对合成转录后调控序列的评价揭示了提高mRNA稳定性和翻译效率的设计原则

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-12-08 DOI: 10.1016/j.synbio.2025.11.011

Jin-Peng Zhang , Zi-Lun Mei , Jia-Wei Ren , Xiao-Mei Zhang , Jin-Song Gong , Guo-Qiang Xu , Hui Li , Xiao-Juan Zhang , Zheng-Hong Xu

The Shine-Dalgarno (SD) sequence and its adjacent flanking regions, including the translation standby site (TSS) and the N-terminal coding sequence (NCS), play critical roles in regulating ribosome recruitment, translation efficiency, and mRNA stability against RNase degradation. However, structure-activity relationships governing these regions remain poorly characterized, and their functional interplay introduces substantial complexity. In this study, we employed one-pot technology to build a post-transcriptional regulatory component (PTRC) library of 576 variants to clarify the relationship between sequence variants and both protein expression and mRNA levels via high-throughput sequencing. Our results show that although unstructured TSSs do not enhance mRNA stability, they markedly increase translation efficiency, causing a 16 %–100 % rise in protein expression. In contrast, structured TSSs increase mRNA levels by 43 %–90 %. Additionally, highly conserved SD sequences boost translation efficiency by up to 10 % and mRNA abundance by up to 12 %. Moreover, it was found that optimized linear N-terminal coding sequence (NCS) positively affects protein expression and mRNA levels. The effects of these optimized regulatory components were verified in the expression control of the nrk and sam2 genes, resulting in enhanced production. These findings underscore the crucial role of structural optimization, guiding the rational design of synthetic post-transcriptional regulatory elements.

sine - dalgarno （SD）序列及其相邻的侧翼区域，包括翻译备用位点（TSS）和n端编码序列（NCS），在调节核糖体招募、翻译效率和mRNA抗rna酶降解的稳定性方面发挥着关键作用。然而，控制这些区域的结构-活性关系仍然很不清楚，它们的功能相互作用引入了大量的复杂性。在本研究中，我们采用一锅技术构建了576个变异的转录后调控成分（PTRC）文库，通过高通量测序来阐明序列变异与蛋白表达和mRNA水平的关系。我们的研究结果表明，尽管非结构化的tss不提高mRNA的稳定性，但它们显著提高了翻译效率，导致蛋白质表达增加16% - 100%。相反，结构化的tss使mRNA水平增加43% - 90%。此外，高度保守的SD序列可使翻译效率提高10%，mRNA丰度提高12%。此外，优化后的线性n端编码序列（NCS）对蛋白质表达和mRNA水平有积极影响。这些优化后的调控成分在调控nrk和sam2基因的表达中得到了验证，从而提高了产量。这些发现强调了结构优化的关键作用，指导合理设计合成的转录后调控元件。

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引用次数: 0

Efficient whole-cell biocatalytic synthesis of 2′-deoxy-2′-fluoroadenosine, a key building block for nucleic acid drugs 高效全细胞生物催化合成2 ' -脱氧-2 ' -氟腺苷，核酸药物的关键组成部分

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-12-05 DOI: 10.1016/j.synbio.2025.11.014

Dandan Feng , Tianshu Zhang , Guan Zhou , Yanli Cao , Quan Luo , Huifang Xu , Xuefeng Lu

2′-Deoxy-2′-fluoroadenosine (2′-F-dA) is a nucleoside analogue used as a key building block for oligonucleotide drugs. It can be biosynthesized from a low-cost 2′-deoxy-2′-fluorouridine via one-pot transglycosylation catalyzed by a thymidine phosphorylase (TP) and a purine nucleoside phosphorylase (PNP). However, reliance on purified enzymes and low space-time yields present challenges for industrial application of the process. Here, we develop a whole-cell-based biocatalytic system employing TP and PNP from Escherichia coli, which demonstrates high catalytic efficiency and operational simplicity in scaled-up reaction. In particular, a thermal pretreatment of TP- and PNP-expressing whole cells, determined as 50 °C for 3 h, effectively suppressed endogenous deamination side reaction while enhancing 2′-F-dA yield. Subsequent optimization of enzyme and substrate loadings and their relative ratios achieved an unprecedented space-time yield of 1.22 g/L/h with 88.1 g/L product titer in a 500 mL scaled-up reaction, manifesting a highest total conversion of 68.2 %. An integrated purification process yielded gram-scale solid powder of 2′-F-dA with 98.0 % chemical purity and 85.0 % recovery. This novel whole-cell biocatalytic process demonstrates significant industrial potential for the production of 2′-F-dA.

2 ' -脱氧-2 ' -氟腺苷（2 ' -F-dA）是一种核苷类似物，用作寡核苷酸药物的关键构建块。它可以通过胸苷磷酸化酶（TP）和嘌呤核苷磷酸化酶（PNP）催化的一锅转糖基化反应，以低成本的2 ' -脱氧-2 ' -氟吡啶为原料进行生物合成。然而，对纯化酶的依赖和低时空产率为该工艺的工业应用带来了挑战。在这里，我们开发了一种基于全细胞的生物催化系统，该系统采用来自大肠杆菌的TP和PNP，在放大反应中表现出高催化效率和操作简单。特别是，对表达TP-和pnp的全细胞进行50℃、3 h的热预处理，可以有效抑制内源性脱胺副反应，同时提高2′- f - da的产率。随后对酶和底物的负载及其相对比例进行优化，在500 mL放大反应中获得了前所未有的时空产率1.22 g/L/h，产物滴度为88.1 g/L，最高总转化率为68.2%。综合纯化工艺可制得2′-F-dA克级固体粉末，化学纯度为98.0%，回收率为85.0%。这种新型的全细胞生物催化工艺显示了生产2′-F-dA的巨大工业潜力。

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引用次数: 0

Functions and optimization of soft law in the international governance of synthetic biology: The predicament of hard law vs. the rise of soft law 软法在合成生物学国际治理中的作用与优化：硬法的困境与软法的兴起

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-12-04 DOI: 10.1016/j.synbio.2025.11.005

Yu Qin , Jiaxiang Hu , Kezhen Su

Synthetic biology, as an emerging field that integrates life sciences and engineering technology, is driving profound transformations in global science, ethics, and legal systems. In international legal framework, the Biological Weapons Convention (BWC) and the Convention on Biological Diversity (CBD) have established initial hard law governance systems. However, these frameworks still face structural limitations in terms of technical adaptability, the scope of provisions, and institutional coordination. Soft law, with its flexibility, non-binding nature, and ability to build consensus, is increasingly becoming an essential supplement to the international response to the ethical risks of synthetic biology. International organizations, industry alliances, and non-governmental actors are constructing a multi-layered soft law governance network through ethical guidelines, policy recommendations, and codes of conduct, providing institutional support for risk identification, technology classification, and behavioral guidance. Soft law is well-suited to perform the roles of guiding and providing feedback in governance, while hard law should focus on the construction of systems of rights and responsibilities and the establishment of obligations. There is a collaborative governance model that integrates both soft and hard law. This model, characterized by “soft law guidance, hard law consolidation, and soft law feedback,” aims to create a flexible and enforceable governance framework. This approach ensures that soft law provides a timely and adaptive starting point, hard law offers a uniform and accountable foundation, and a feedback loop allows for continuous adjustment based on practical experience.

合成生物学作为一个融合生命科学和工程技术的新兴领域，正在推动全球科学、伦理和法律体系的深刻变革。在国际法律框架内，《禁止生物武器公约》和《生物多样性公约》已初步建立起硬法治理体系。然而，这些框架在技术适应性、规定范围和机构协调方面仍然面临结构性限制。软法律具有灵活性、非约束性和建立共识的能力，正日益成为国际社会应对合成生物学伦理风险的重要补充。国际组织、行业联盟和非政府行动者正在通过道德准则、政策建议和行为准则构建多层次的软法律治理网络，为风险识别、技术分类和行为指导提供制度支持。软法适合在治理中发挥引导和反馈的作用，而硬法应侧重于权利和责任制度的构建和义务的确立。有一种集成了软法和硬法的协作治理模型。该模式以“软法律指导、硬法律巩固和软法律反馈”为特征，旨在创建一个灵活且可执行的治理框架。这种方法确保软法提供了一个及时和适应性的起点，硬法提供了一个统一和负责任的基础，反馈回路允许根据实际经验进行持续调整。

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引用次数: 0

Engineering an ATP-saving mevalonate pathway for high-efficiency S-(+)-linalool production in Serratia marcescens 在粘质沙雷氏菌中设计高效S-(+)-芳樟醇生产的节省atp的甲羟戊酸途径

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-12-04 DOI: 10.1016/j.synbio.2025.11.010

Yue Chen , Linbo Gou , Di Liu , Shengfang Wu , Xiuwen Zhou , Tai-Ping Fan , Long Wang , Yujie Cai

(S)-(+)-linalool, a valuable acyclic monoterpenol secondary metabolite of plants, finds extensive applications in the food, flavor and fragrance, pharmaceutical, and daily chemical industries. Microbial synthesis offers two pathways for its production, among which the mevalonate (MVA) pathway derived from halophilic archaea is more widely employed. However, the traditional Haloarchaea-type MVA pathway relies on bifunctional enzyme catalysis, consuming 3 molecules of ATP. Moreover, the catalytic activity of natural linalool synthase (LIS) is generally low, failing to meet industrial requirements. Additionally, linalool exhibits significant toxicity to microbial hosts, thereby limiting the production capacity of conventional chassis microorganisms. To address these bottlenecks, this study implemented systematic optimizations: Firstly, the Archaeal mevalonate pathway was reconstructed by replacing the original bifunctional enzyme with two monofunctional enzymes, successfully reducing ATP consumption to 2 molecules. Secondly, through directed screening and rational design, a high-activity linalool synthase mutant, CsMLIS^I331V/I444L, derived from Coriandrum sativum, was obtained. Furthermore, an enzyme fusion strategy was adopted, involving the introduction of a long flexible linker between key genes, which significantly enhanced catalytic efficiency. Finally, S. marcescens HBQA7ΔsIaAB-pyc, a strain previously screened in our laboratory with broad-spectrum tolerance to terpenoids, was selected as the novel chassis cell. Collectively, these efforts resulted in the construction of a microbial cell factory for the efficient synthesis of (S)-(+)-linalool, laying a solid foundation for industrial-scale production.

(S)-(+)-芳樟醇是一种有价值的植物无环单萜醇次生代谢物，在食品、香精香料、制药和日化工业中有着广泛的应用。微生物合成有两种途径生产甲羟戊酸，其中来自嗜盐古菌的甲羟戊酸（MVA）途径应用较为广泛。然而，传统的盐古菌型MVA途径依赖于双功能酶催化，消耗3分子ATP。此外，天然芳樟醇合成酶（LIS）的催化活性普遍较低，达不到工业要求。此外，芳樟醇对微生物宿主表现出显著的毒性，从而限制了传统底盘微生物的生产能力。为了解决这些瓶颈，本研究进行了系统优化：首先，用两个单功能酶取代原来的双功能酶，重构了古菌甲羟戊酸途径，成功地将ATP消耗减少到2分子。其次，通过定向筛选和合理设计，从芫荽中获得了一个高活性的芳樟醇合成酶突变体CsMLISI331V/I444L。此外，采用酶融合策略，在关键基因之间引入一个长而灵活的连接体，显著提高了催化效率。最后，本实验室筛选的对萜类化合物具有广谱耐受性的粘质S. marcescens HBQA7ΔsIaAB-pyc菌株被选为新型底盘细胞。总的来说，这些努力的结果是建立了一个微生物细胞工厂，用于高效合成(S)-(+)-芳樟醇，为工业规模生产奠定了坚实的基础。

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引用次数: 0

Transfer learning with pre-trained language models for protein expression level prediction in Escherichia coli 迁移学习与预训练语言模型在大肠杆菌蛋白表达水平预测中的应用

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-12-04 DOI: 10.1016/j.synbio.2025.11.012

Chunhe Yang , YuLing Zhao , Ruoyu Wang , Haoran Li , Xiaoping Liao , Hongwu Ma

Accurately predicting recombinant protein expression in Escherichia coli remains a long-standing challenge due to the multifactorial nature of gene regulation and translation. Existing computational approaches typically emphasize either codon usage or protein sequence features, limiting predictive accuracy and generalizability. Here we present TLCP-EPE, a transfer learning framework that, for the first time, fuses codon- and protein-level pre-trained language models to jointly capture determinants of expression. By fine-tuning CaLM and ProtT5 with low-rank adaptation (LoRA) and integrating their embeddings through a BiGRU-MLP predictor, TLCP-EPE learns expression-aware representations that outperform state-of-the-art methods. Across two independent test datasets, TLCP-EPE achieved robust performance (AUC 0.835 on codon data; AUC 0.713 on protein data), consistently surpassing conventional codon-based metrics and deep learning baselines. Our results demonstrate that dual-modal modeling of codon and protein sequences enables more accurate and generalizable prediction of expression levels, providing a powerful foundation for rational protein design and biomanufacturing applications.

由于基因调控和翻译的多因子特性，准确预测重组蛋白在大肠杆菌中的表达仍然是一个长期存在的挑战。现有的计算方法通常强调密码子使用或蛋白质序列特征，限制了预测的准确性和通用性。在这里，我们提出了TLCP-EPE，这是一个迁移学习框架，首次融合密码子和蛋白质水平的预训练语言模型来共同捕获表达决定因素。通过对CaLM和ProtT5进行低秩自适应（LoRA）微调，并通过BiGRU-MLP预测器整合它们的嵌入，TLCP-EPE学习了优于最先进方法的表情感知表征。在两个独立的测试数据集上，TLCP-EPE取得了稳健的性能（密码子数据的AUC为0.835，蛋白质数据的AUC为0.713），始终超过传统的基于密码子的指标和深度学习基线。我们的研究结果表明，密码子和蛋白质序列的双模态建模可以更准确、更普遍地预测表达水平，为合理的蛋白质设计和生物制造应用提供了有力的基础。

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引用次数: 0

Programmable large-cargo integration: Overcoming size constraints for next-generation gene therapy 可编程大货物集成：克服下一代基因治疗的尺寸限制

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-12-02 DOI: 10.1016/j.synbio.2025.11.008

Lifang Yu , Mario Andrea Marchisio

The emergence of base and prime editors—genome editing tools that avoid double-strand breaks (DSBs)—has enabled precise point mutations, insertions, inversions, deletions, and substitutions, which accelerates the development of single-intervention therapies and advances individualized genomic medicine. However, their limited efficiency in inserting large DNA fragments has restricted applications for correcting diverse pathogenic mutations within a single gene. In this review, we explore three recently developed strategies for efficient large DNA cargo insertion (>1 kb): CRISPR-associated Tn7-like transposases (CASTs), PE-integrase systems, and R2 retrotransposon fusions (nCas9-R2). We examine the applications of these systems in both bacterial and mammalian contexts and discuss their respective advantages and current limitations. Finally, we address persistent challenges and propose potential directions to guide future research.

碱基和引物编辑器——避免双链断裂（dsb）的基因组编辑工具——的出现，使得精确的点突变、插入、倒位、缺失和替换成为可能，从而加速了单干预疗法的发展，推进了个体化基因组医学的发展。然而，它们插入大DNA片段的效率有限，限制了在单个基因中纠正多种致病突变的应用。在这篇综述中，我们探讨了最近开发的三种有效的大DNA货物插入策略（> 1kb）： crispr相关的tn7样转座酶（cast）， pe整合酶系统和R2反转录转座子融合（nCas9-R2）。我们研究了这些系统在细菌和哺乳动物环境中的应用，并讨论了它们各自的优势和当前的局限性。最后，我们解决了持续存在的挑战，并提出了指导未来研究的潜在方向。

引用次数: 0