Synthetic and Systems Biotechnology最新文献_第5页

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 : 2026-06-01 Epub 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

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 : 2026-06-01 Epub 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

Multi-omics elucidation of resource allocation for enhanced ethanol production via precise glucose control in anaerobic Saccharomyces cerevisiae fermentation 厌氧发酵过程中葡萄糖精确控制提高乙醇产量的资源分配多组学研究

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2026-06-01 Epub Date: 2025-11-11 DOI: 10.1016/j.synbio.2025.10.015

Zhihao Liu , Linghong Zheng , Xiaofei Yu , Yingping Zhuang , Guan Wang

Ethanol, a high-demand clean energy source, is primarily produced via fed-batch fermentation in industrial settings. Although our previous study identified an optimal glucose concentration of 30 g/L for maximal ethanol yield, the mechanisms underlying glucose-dependent cellular adaptation remain unclear. Here, we performed an integrated multi-omics analysis, including transcriptomics, proteomics, metabolomics, and fluxomics, to compare yeast cells under glucose-controlled and uncontrolled conditions. Our results indicate that high glucose stress triggers the regulation of transporters with different affinities and the upregulation of heat shock proteins (HSPs), trehalose, and amino acids. In contrast, protein turnover was reduced under glucose-controlled conditions, suggesting more efficient resource allocation. This metabolic reallocation enhances carbon flux through glycolysis, potentially providing additional energy and NADH to support biomass growth and ethanol production. These findings advance our understanding of yeast regulatory mechanisms under glucose stress and provide insights for metabolic engineering and process optimization.

乙醇是一种高需求的清洁能源，在工业环境中主要通过补料分批发酵生产。虽然我们之前的研究确定了最大乙醇产量的最佳葡萄糖浓度为30 g/L，但葡萄糖依赖性细胞适应的机制尚不清楚。在这里，我们进行了综合多组学分析，包括转录组学、蛋白质组学、代谢组学和通量组学，以比较葡萄糖控制和非葡萄糖控制条件下的酵母细胞。我们的研究结果表明，高葡萄糖应激触发了不同亲和力的转运蛋白的调节，以及热休克蛋白（HSPs）、海藻糖和氨基酸的上调。相比之下，在葡萄糖控制的条件下，蛋白质周转减少，表明更有效的资源分配。这种代谢再分配通过糖酵解增强碳通量，可能提供额外的能量和NADH来支持生物质生长和乙醇生产。这些发现促进了我们对酵母在葡萄糖胁迫下的调节机制的理解，并为代谢工程和工艺优化提供了见解。

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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 : 2026-06-01 Epub 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

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 : 2026-06-01 Epub 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

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 : 2026-06-01 Epub 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

Antibiotic-free high-yield (-)-α-bisabolol production in Serratia marcescens via metabolic engineering and genomic integration of mevalonate pathway genes 通过代谢工程和甲羟戊酸途径基因的基因组整合，粘质沙雷菌无抗生素高产(-)-α-双abolol

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2026-06-01 Epub Date: 2025-12-31 DOI: 10.1016/j.synbio.2025.12.011

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

(-)-α-Bisabolol, a valuable monocyclic sesquiterpene alcohol, has garnered significant attention in the pharmaceutical and cosmetic industries due to its remarkable anti-inflammatory, antibacterial, and skin-care properties. In this study, Serratia marcescens HBQA7 (S. marcescens HBQA7), a non-model strain resistant to terpenoid toxicity, was used as the production host, and the expression intensities of different integration sites were screened. The complete (-)-α-bisabolol synthesis pathway was integrated into these sites, achieving a production titer of 3.5 g L⁻¹. On this basis, by knocking out competitive pathway genes (such as slaB and adhE) and global regulatory factors (arcA and iclR), and introducing efficient glucose transport and activation (glf and glk), the shake flask fermentation titer was increased to 7.21 g L⁻¹. Through optimization of fermentation culture by orthogonal experiments and others, the titer was further increased to 9.90 g L⁻¹. Finally, through the fed-batch fermentation process conducted in a 50 L bioreactor, a titer of 102.3 g L⁻¹ was achieved after 110 h of cultivation. The productivity reached 0.93 g L⁻¹ h⁻¹. This study not only establishes the most efficient microbial production system for (-)-α-bisabolol reported to date, but also demonstrates the outstanding potential of S. marcescens as a chassis for terpenoid biosynthesis. It provides a novel strategy for the industrial production of high-value terpenoids.

（-）-α-比abolol是一种有价值的单环倍半萜醇，由于其显著的抗炎、抗菌和护肤特性，在制药和化妆品行业引起了极大的关注。本研究以抗萜类毒性的非模式菌株粘质Serratia marcescens HBQA7 （S. marcescens HBQA7）为生产宿主，筛选不同整合位点的表达强度。完整的(-)-α-双abolol合成途径被整合到这些位点，生产滴度达到3.5 g L−1。在此基础上，通过敲除竞争通路基因（如slaB和adhE）和全局调控因子（arcA和iclR），引入高效葡萄糖运输和激活（glf和glk），摇瓶发酵滴度提高到7.21 g L−1。通过正交试验等优化发酵培养，进一步将滴度提高到9.90 g L−1。最后，在50 L的生物反应器中进行补料分批发酵，培养110 h后滴度达到102.3 g L−1。产率达到0.93 g L−1 h−1。本研究不仅建立了迄今为止报道的最有效的(-)-α-双abolol微生物生产系统，而且还证明了S. marcescens作为萜类生物合成基质的巨大潜力。为高值萜类化合物的工业化生产提供了新的思路。

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引用次数: 0

Metabolic reprogramming of abscisic acid-producing strain Botrytis cinerea TB-31 toward terpenoid biosynthesis using a CRISPR/Cas9 ribonucleoprotein system 利用CRISPR/Cas9核糖核蛋白系统对脱落酸产生菌株葡萄孢TB-31进行萜类生物合成的代谢重编程

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2026-06-01 Epub Date: 2025-12-31 DOI: 10.1016/j.synbio.2025.12.002

Xiao-Nan Hou , Dan Shu , Tian-Fu Li , Qi Yang , Zhe-Min Li , Di Luo , Jie Yang , Zhi-Ying Yan , Hong Tan

Compared with conventional microbial hosts, filamentous fungi have distinct advantages for the industrial-scale biosynthesis of high-value chemical compounds. However, current research on strain engineering and fermentation optimization strategies for synthetic biology applications is limited in filamentous fungi, especially in industrial production strains. In this study, we established a CRISPR/Cas9-based gene editing system in Botrytis cinerea strain TB-31, an important filamentous fungal platform for the study of the biosynthesis and regulation of the sesquiterpenoid abscisic acid (ABA). This system enables efficient single- and multigene knockout, large-fragment deletion, and heterologous protein expression. Among the engineered mutant strains, the △bcaba1234 strain with complete ablation of the ABA biosynthetic gene cluster (BGC) demonstrated significant metabolic flux rewiring, redirecting cellular resources toward terpenoid precursor biosynthesis; this metabolic reprogramming proves pivotal for high-value terpenoid biosynthesis. This study not only establishes an efficient genome editing tool for the ABA-producing strain B. cinerea TB-31 but also provides a foundation for its development as a new potential terpenoid-producing chassis strain.

与传统的微生物宿主相比，丝状真菌在工业规模生物合成高价值化合物方面具有明显的优势。然而，目前用于合成生物学应用的菌株工程和发酵优化策略的研究仅限于丝状真菌，特别是工业生产菌株。本研究基于CRISPR/ cas9基因编辑技术，在研究倍半萜类脱落酸（ABA）生物合成与调控的重要丝状真菌平台灰霉病菌TB-31中建立了基于CRISPR/ cas9基因编辑系统。该系统可实现高效的单基因和多基因敲除、大片段删除和异源蛋白表达。在工程突变菌株中，完全切除ABA生物合成基因簇（BGC）的△bcaba1234菌株表现出显著的代谢通量重连接，将细胞资源重定向到萜类前体的生物合成；这种代谢重编程被证明是高价值萜类生物合成的关键。本研究不仅为产aba菌株B. cinerea TB-31建立了高效的基因组编辑工具，也为其作为一种潜在的新型萜类化合物生产基质菌株的开发奠定了基础。

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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 : 2026-06-01 Epub 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

Metabolic engineering enables Escherichia coli to grow on 1,3-propanediol 代谢工程使大肠杆菌在1,3-丙二醇上生长

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2026-06-01 Epub Date: 2025-12-22 DOI: 10.1016/j.synbio.2025.11.009

Nga Yu Poon , Anthony J. Sinskey , Kang Zhou

1,3-propanediol (1,3-PDO) is used to synthesize plastics used in many consumer products. As the demand and production of such plastics increase, a technology will be needed to utilize 1,3-PDO released from the plastics after their disposal. In our previous study, we developed the strain (BA07Δ) that could use malonate semialdehyde (MSA, an important intermediate in the 1,3-PDO assimilation pathway) as the major carbon source. Here, we present construction of PA16, a strain which could grow to an OD₆₀₀ of 7 by consuming 6.5 g/L of 1,3-PDO within 72 h in M9-based medium supplemented with 1 g/L of complete supplement mixture (CSM). This was achieved by adaptive laboratory evolution (ALE) after extending the pathway in BA07Δ through the introduction of a 1,3-propanediol dehydrogenase from Klebsiella pneumoniae (KpDhaT), an aldehyde dehydrogenase from E. coli (EcPuuC) and a 3-hydroxypropionate dehydrogenase from Halomonas bluephagenesis (HbDddA). Comparing the transcriptome of PA16 and its ancestor in the ALE (PA1) revealed the upregulation of two genes, threonine dehydrogenase (EcTdh) and 2-amino-3-ketobutyrate CoA ligase (EcKbl) responsible for threonine degradation. The overexpression of these genes in PA1 resulted in a 5-fold increase in the 72-h cell density. This finding helped simplify the growth medium of PA16: the supplement mixture containing more than 10 amino acids/nucleobases was reduced to just having 0.1 g/L threonine. PA16's OD₆₀₀ reached 3 when it grew in a defined medium containing 10 g/L 1,3-PDO and 0.1 g/L threonine as carbon sources. E. coli PA16 should be a useful strain to the subsequent research on upcycling 1,3-PDO derived from plastic wastes.

1,3-丙二醇（1,3- pdo）用于合成许多消费品中使用的塑料。随着此类塑料的需求和产量的增加，需要一种技术来利用塑料处理后释放的1,3- pdo。在我们之前的研究中，我们开发了以丙二酸半醛（MSA, 1,3- pdo同化途径中的重要中间体）为主要碳源的菌株（BA07Δ）。在此，我们构建了PA16菌株，该菌株在添加1 g/L的完全补充混合物（CSM）的m9培养基中，在72 h内消耗6.5 g/L的1,3- pdo，可以生长到OD600为7。这是通过引入肺炎克雷伯菌的1,3-丙二醇脱氢酶（KpDhaT）、大肠杆菌的醛脱氢酶（EcPuuC）和蓝色嗜盐单胞菌的3-羟丙酸脱氢酶（HbDddA），在BA07Δ中扩展途径后通过适应性实验室进化（ALE）实现的。比较PA16及其在ALE中的祖先（PA1）的转录组，发现负责苏氨酸降解的苏氨酸脱氢酶（EcTdh）和2-氨基-3-酮丁酸辅酶a连接酶（EcKbl）两个基因上调。这些基因在PA1中的过表达导致72 h细胞密度增加5倍。这一发现有助于简化PA16的生长培养基：含有10个以上氨基酸/核碱基的补充混合物被减少到只有0.1 g/L的苏氨酸。PA16在以10 g/L 1,3- pdo和0.1 g/L苏氨酸为碳源的培养基中生长时，其OD600达到3。大肠杆菌PA16应成为今后塑料废弃物中1,3- pdo升级回收研究的有益菌株。

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