Synthetic and Systems Biotechnology最新文献

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Enhancing substrate specificity of microbial transglutaminase for precise nanobody labeling 增强微生物转谷氨酰胺酶的底物特异性，实现精确的纳米抗体标记

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-10-16 DOI: 10.1016/j.synbio.2024.10.003

Xinglong Wang , Kangjie Xu , Haoran Fu , Qiming Chen , Beichen Zhao , Xinyi Zhao , Jingwen Zhou

Streptomyces mobaraenesis transglutaminase (smTG) can be used for site-specific labeling of proteins with chemical groups. Here, we explored the use of modified smTG for the biosynthesis of nanobody-fluorophore conjugates (NFC). smTG catalyzes the conjugation of acyl donors containing glutamine with lysine-containing acceptors, which can lead to non-specific cross-linking. To achieve precise site-specific labeling, we employed molecular docking and virtual mutagenesis to redesign the enzyme's substrate specificity towards the peptide GGGGQR, a non-preferred acyl donor for smTG. Starting with a thermostable and highly active smTG variant (TGm2), we identified that single mutations G250H and Y278E significantly enhanced activity against GGGGQR, increasing it by 41 % and 1.13-fold, respectively. Notably, the Y278E mutation dramatically shifted the enzyme's substrate preference, with the activity ratio against GGGGQR versus the standard substrate CBZ-Gln-Gly rising from 0.05 to 0.93. In case studies, we used nanobodies 1C12 and 7D12 as labeling targets, catalyzing their conjugation with a synthetic fluorophore via smTG variants. Nanobodies fused with GGGGQR were successfully site-specifically labeled by TGm2-Y278E, in contrast to non-specific labeling observed with other variants. These results suggest that engineering smTG for site-specific labeling is a promising approach for the biosynthesis of antibody-drug conjugates.

链霉菌转谷氨酰胺酶（smTG）可用于用化学基团对蛋白质进行位点特异性标记。smTG 催化含谷氨酰胺的酰基供体与含赖氨酸的受体的连接，这可能导致非特异性交联。为了实现精确的位点特异性标记，我们采用了分子对接和虚拟诱变的方法，重新设计了酶对肽 GGGGQR（smTG 的非首选酰基供体）的底物特异性。从一个恒温高活性的 smTG 变体（TGm2）开始，我们发现单个突变 G250H 和 Y278E 显著增强了对 GGGQR 的活性，分别提高了 41% 和 1.13 倍。值得注意的是，Y278E 突变极大地改变了酶的底物偏好，对 GGGGQR 与标准底物 CBZ-Gln-Gly 的活性比从 0.05 上升到 0.93。在案例研究中，我们使用纳米抗体 1C12 和 7D12 作为标记目标，通过 smTG 变体催化它们与合成荧光团的连接。与其他变体的非特异性标记不同，与 GGGGQR 融合的纳米抗体成功地被 TGm2-Y278E 进行了位点特异性标记。这些结果表明，进行位点特异性标记的 smTG 工程是生物合成抗体-药物共轭物的一种可行方法。

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引用次数: 0

Mechanistic insights into the orthogonal functionality of an AHL-mediated quorum-sensing circuit in Yersinia pseudotuberculosis 对耶尔森氏菌假结核中 AHL 介导的法定量感应回路的正交功能的机理认识

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-10-14 DOI: 10.1016/j.synbio.2024.10.002

Boyu Luo , Shanshan Wu , Wei Liu , Dongdong Zhang , Ruicun Liu , Tuoyu Liu , Zhi Sun , Ziqun Wei , Mingyu Liu , Zhiyuan Shi , Niu Huang , Yue Teng

YpsR, a pivotal regulatory protein in the quorum-sensing (QS) of Yersinia pseudotuberculosis(Y. pstb), is essential for molecular signaling, yet its molecular mechanisms remain poorly understood. Herein, this study systematically investigates the interactions between YpsR and acyl-homoserine lactones (AHLs), shedding light on the selective mechanism of YpsR to various AHL molecules. Using molecular docking and surface plasmon resonance (SPR) analysis, we confirmed YpsR's binding affinities, with the strongest observed for 3OC6-HSL, which notably inhibited Y. pstb growth. Additionally, we engineered a whole-cell biosensor based on YpsR-AHL interaction, which exhibited sensitivity to the signal molecule 3OC6-HSL produced by Y. pstb. Furthermore, key YpsR residues (S32, Y50, W54, D67) involved in AHL binding were identified and validated. Overall, this research elucidates the mechanisms of QS signal recognition in Y. pstb, providing valuable insights that support the development of diagnostic tools for detecting Y. pstb infections.

YpsR是假结核耶尔森菌（Y. pseudotuberculosis，Y. pstb）法定量感应（QS）过程中的关键调控蛋白，对分子信号转导至关重要，但其分子机制仍鲜为人知。本研究系统研究了 YpsR 与酰基高丝氨酸内酯（AHLs）之间的相互作用，揭示了 YpsR 对各种 AHL 分子的选择性机制。通过分子对接和表面等离子体共振（SPR）分析，我们证实了 YpsR 的结合亲和力，其中 3OC6-HSL 的结合亲和力最强，能显著抑制 Y. pstb 的生长。此外，我们还设计了一种基于 YpsR-AHL 相互作用的全细胞生物传感器，它对 Y. pstb 产生的信号分子 3OC6-HSL 具有敏感性。此外，我们还鉴定并验证了参与 AHL 结合的关键 YpsR 残基（S32、Y50、W54、D67）。总之，这项研究阐明了 Y. pstb 的 QS 信号识别机制，为开发检测 Y. pstb 感染的诊断工具提供了有价值的见解。

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引用次数: 0

Revolutionizing biotechnology advances natural products discovery and industrial processing 生物技术的革命性进步推动了天然产品的发现和工业加工

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-10-04 DOI: 10.1016/j.synbio.2024.10.001

Yaojun Tong, Tilmann Weber

引用次数: 0

CRISPR/Cas12a-based genome editing for cyanophage of Anabeana sp. 基于 CRISPR/Cas12a 的蓝藻噬菌体基因组编辑技术

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-10-03 DOI: 10.1016/j.synbio.2024.09.011

Shengjian Yuan , Yanchen Li , Chunhua Kou , YiChen Sun , Yingfei Ma

Efforts have been conducted on cyanobacterial genome editing, yet achieving genome editing in cyanophages remains challenging. Editing cyanophage genomes is crucial for understanding and manipulating their interactions with cyanobacterial hosts, offering potential solutions for controlling cyanobacterial blooms. In this study, we developed a streamlined CRISPR-Cas12a-based method for efficient cyanophage genome editing and then applied this method to the cyanophages A-1(L) and A-4(L) of Anabeana sp. PCC.7120. Multiple hypothetical genes were edited and knocked out from these two cyanophage genomes, generating viable mutants with varying capabilities to inhibit cyanobacterial growth. All these mutants displayed significant inhibitory effects on the host, indicating that these genes were non-essential for phage life cycle and the deletion led to little impairment of the cyanophages in infectious efficiency to their host. By iterative and simultaneous gene knockouts in cyanophage A-4(L), we achieved the minimal genome mutant with a 2400 bp reduction in genome size, representing a 5.75 % decrease compared to the wild type (WT). In conclusion, these cyanophage mutants can facilitate the identification of nonessential genes for cyanophages biology and the insertion of foreign genes for synthetic biology research. This advancement holds promise in addressing the widespread issue of water blooms and the associated environmental hazards.

人们已经在蓝藻基因组编辑方面做出了努力，但在噬菌体中实现基因组编辑仍然具有挑战性。编辑蓝藻噬菌体基因组对于理解和操纵它们与蓝藻宿主的相互作用至关重要，从而为控制蓝藻藻华提供潜在的解决方案。在本研究中，我们开发了一种基于 CRISPR-Cas12a 的简化方法，用于高效编辑蓝藻噬菌体基因组，并将该方法应用于 Anabeana sp. PCC.7120 的蓝藻噬菌体 A-1(L) 和 A-4(L)。这两种蓝藻噬菌体基因组中的多个假定基因被编辑和敲除，产生了具有不同抑制蓝藻生长能力的可行突变体。所有这些突变体都对宿主有明显的抑制作用，表明这些基因对噬菌体的生命周期并不重要，而且基因缺失对蓝藻噬菌体感染宿主的效率几乎没有影响。通过在蓝藻噬菌体 A-4(L)中反复同时进行基因敲除，我们获得了基因组最小的突变体，其基因组大小减少了 2400 bp，与野生型（WT）相比减少了 5.75%。总之，这些蓝藻噬菌体突变体有助于鉴定蓝藻噬菌体生物学中的非必要基因，以及为合成生物学研究插入外来基因。这一进展有望解决普遍存在的水华问题及相关的环境危害。

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引用次数: 0

Developing polycistronic expression tool in Yarrowia lipolytica 在脂溶性亚罗酵母中开发多聚组蛋白表达工具

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-09-28 DOI: 10.1016/j.synbio.2024.09.010

Donghan Li , Jianhui Liu , Lingxuan Sun , Jin Zhang , Jin Hou

Unconventional oleaginous yeast Yarrowia lipolytica has gained widespread applications as a microbial cell factory for synthesizing various chemicals and natural products. The construction of efficient cell factories requires intricate metabolic engineering. However, multi-gene expression in Y. lipolytica is labor-intensive. To facilitate multi-gene expression, we developed the polycistronic expression tool using 2A peptides. We first compared different 2A peptides in Y. lipolytica and identified two 2A peptides with high cleavage efficiency: P2A and ERBV-1. The effect of 2A peptides on the expression level of upstream and downstream genes was then determined. Ultimately, we applied the identified 2A peptides to express four genes in canthaxanthin biosynthetic pathway within one expression cassette for canthaxanthin production. This study enriches the multi-gene expression tools of Y. lipolytica, which will facilitate the cell factory construction of Y. lipolytica.

非常规油脂酵母亚罗酵母（Yarrowia lipolytica）作为合成各种化学品和天然产品的微生物细胞工厂，已获得广泛应用。构建高效细胞工厂需要复杂的代谢工程。然而，在脂溶性酵母中进行多基因表达是一项劳动密集型工作。为了促进多基因表达，我们利用 2A 肽开发了多聚组表达工具。我们首先比较了脂溶性酵母中不同的 2A 肽，确定了两种裂解效率较高的 2A 肽：P2A和ERBV-1。然后确定了 2A 肽对上游和下游基因表达水平的影响。最终，我们利用所鉴定的 2A 肽在一个表达盒中表达了角黄素生物合成途径中的四个基因，以生产角黄素。这项研究丰富了溶脂酵母的多基因表达工具，有助于溶脂酵母细胞工厂的构建。

引用次数: 0

Synthetic biotechnology for C1 bio-refinery 用于 C1 生物精炼厂的合成生物技术

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-09-23 DOI: 10.1016/j.synbio.2024.09.009

Yongjin Zhou (Chair Professor), Benjamin Woolston (Assistant Professor)

引用次数: 0

Transcriptome analysis reveals methanol metabolism variations for the growth damage caused by overexpression of chimeric transactivators in Pichia pastoris 转录组分析揭示了甲醇代谢变化对过量表达嵌合转录因子的 Pichia pastoris 生长造成的损害

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-09-23 DOI: 10.1016/j.synbio.2024.09.008

Qi Liu , Ziyu He , Menghao Cai

Methanol is a promising substrate for sustainable biomanufacturing, and Pichia pastoris has become a commonly used yeast for methanol utilization due to its powerful methanol metabolic pathways and methanol inducible promoter. Previous reconstruction of gene circuits highly improved transcriptional activity, but excessive expression of chimeric transactivator damaged cell growth on methanol. Here we employed transcriptome analysis to investigate the effects of chimeric transactivator overexpression on cellular metabolism and regulatory networks. The results showed that strong expression of chimeric transactivator unexpectedly downregulated methanol metabolism, especially the alcohol oxidase 1 (AOX1), but without remarkable changes in expression of transcriptional factors. Meanwhile, the synthesis of peroxisomes also varied with chimeric transactivator expression. In addition, the enrichment analysis of differentially expressed genes revealed their impact on cellular metabolism. The gene expression patterns caused by different expression levels of chimeric transactivators have also been clarified. This work provides useful information to understand the transcriptional regulation of the AOX1 promoter and methanol signaling. It revealed the importance of balancing transcription factor expression for the host improvement.

甲醇是一种很有前景的可持续生物制造底物，而 Pichia pastoris 因其强大的甲醇代谢途径和甲醇诱导启动子而成为甲醇利用的常用酵母。以前的基因回路重建大大提高了转录活性，但嵌合转录因子的过度表达会破坏细胞在甲醇中的生长。在此，我们利用转录组分析研究了嵌合型转录激活因子过度表达对细胞代谢和调控网络的影响。结果表明，嵌合型转录因子的强表达意外地下调了甲醇代谢，尤其是醇氧化酶1（AOX1），但转录因子的表达没有明显变化。同时，过氧化物酶体的合成也随嵌合转座子的表达而变化。此外，差异表达基因的富集分析显示了它们对细胞代谢的影响。此外，还阐明了嵌合转座因子不同表达水平所导致的基因表达模式。这项工作为了解 AOX1 启动子的转录调控和甲醇信号转导提供了有用的信息。它揭示了平衡转录因子表达对宿主改良的重要性。

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引用次数: 0

Efficient biosynthesis of β-caryophyllene in Saccharomyces cerevisiae by β-caryophyllene synthase from Artemisia argyi 艾蒿中的β-石竹烯合成酶在酿酒酵母中高效生物合成β-石竹烯

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-09-22 DOI: 10.1016/j.synbio.2024.09.005

Zhengping Li , Yuhong Gan , Changyu Gou , Qiongyu Ye , Yang Wu , Yuhong Wu , Tingxing Yang , Baolian Fan , Aijia Ji , Qi Shen , Lixin Duan

Artemisia argyi H. Lév. & Vaniot is an important traditional Chinese medicinal plant known for its volatile oils, which are the main active components of A. argyi, including monoterpenes, sesquiterpenes and their derivatives. Despite its medicinal significance, the biosynthesis of sesquiterpenoids in A. argyi remains underexplored. In this study, we identified four β-caryophyllene synthases from A. argyi. A high-yield β-caryophyllene engineered Saccharomyces cerevisiae cell factory has been built in this study. By fusing ERG20 and AarTPS88 with a flexible linker (GGGS)₂ and enhancing metabolic flux in the MVA pathway (HIF-1, tHMGR, and UPC2-1), we achieved a titer of β-caryophyllene reached 15.6 g/L by fed-batch fermentation in a 5 L bioreactor. To our knowledge, this represents the highest reported titer of β-caryophyllene in yeast to date. This study provides a valuable tool for the industrial-scale production of β-caryophyllene.

阿尔基蒿 H. Lév.青蒿是一种重要的传统中药植物，以其挥发油而闻名，挥发油是青蒿的主要活性成分，包括单萜、倍半萜及其衍生物。尽管阿胶具有重要的药用价值，但其倍半萜类化合物的生物合成仍未得到充分探索。在这项研究中，我们从 A. argyi 中发现了四种 β-加里叶烯合成酶。本研究建立了一个高产的β-石竹烯工程酿酒酵母细胞工厂。通过将 ERG20 和 AarTPS88 与柔性连接子（GGGS）2 融合，并增强 MVA 通路（HIF-1、tHMGR 和 UPC2-1）的代谢通量，我们在 5 L 生物反应器中通过喂料批量发酵使 β-石竹烯的滴度达到 15.6 g/L。据我们所知，这是迄今为止报道的酵母中β-石竹烯的最高滴度。这项研究为β-石竹烯的工业化生产提供了宝贵的工具。

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引用次数: 0

Development of a green Komagataella phaffii cell factory for sustainable production of plant-derived sesquiterpene (–)-α-bisabolol 开发绿色 Komagataella phaffii 细胞工厂，以可持续方式生产植物源倍半萜 (-)-α-bisabolol

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-09-17 DOI: 10.1016/j.synbio.2024.09.006

Jintao Cheng , Zhongji Pu , Jiali Chen , Dingfeng Chen , Baoxian Li , Zhengshun Wen , Yuanxiang Jin , Yanlai Yao , Kan Shao , Xiaosong Gu , Guiling Yang

(–)-α-Bisabolol is a plant-derived sesquiterpene derived from Eremanthus erythropappus, which can be used as a raw material in cosmetics and has anti-inflammatory function. In this study, we designed six mutation sites of the (–)-α-bisabolol synthase BOS using the plmDCA algorithm. Among these, the F324Y mutation demonstrated exceptional performance, increasing the product yield by 73 %. We constructed a de novo (–)-α-bisabolol biosynthesis pathways through systematic synthetic biology strategies, including the enzyme design of BOS, selection of different linkers in fusion expression, and optimization of the mevalonate pathway, weakening the branching metabolic flow and multi-copy strategies, the yield of (–)-α-bisabolol was significantly increased, which was nearly 35-fold higher than that of the original strain (2.03 mg/L). The engineered strain was capable of producing 69.7 mg/L in shake flasks. To the best of our knowledge, this is the first report on the biosynthesis of (–)-α-bisabolol in Komagataella phaffii, implying this is a robust cell factory for sustainable production of other terpenoids.

(-)-α-双羟基苯甲酸酯是一种植物源倍半萜，来自红花楹，可用作化妆品原料，具有抗炎作用。在这项研究中，我们利用 plmDCA 算法设计了 (-)-α-bisabol 合成酶 BOS 的六个突变位点。其中，F324Y 突变位点表现出优异的性能，使产物产量提高了 73%。我们通过系统的合成生物学策略，包括BOS的酶设计、融合表达中不同连接子的选择、甲羟戊酸途径的优化、弱化分支代谢流和多拷贝策略，构建了一条全新的(-)-α-二羟基苯并酚生物合成途径，(-)-α-二羟基苯并酚的产率显著提高，比原始菌株（2.03 mg/L）提高了近35倍。工程菌株在摇瓶中的产量为 69.7 毫克/升。据我们所知，这是首次报道 Komagataella phaffii 中 (-)-α-bisabol 的生物合成，这意味着这是一个可持续生产其他萜类化合物的强大细胞工厂。

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引用次数: 0

Functional investigation of the SAM-dependent methyltransferase RdmB in anthracycline biosynthesis 蒽环类生物合成中依赖于 SAM 的甲基转移酶 RdmB 的功能研究

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2024-09-12 DOI: 10.1016/j.synbio.2024.09.002

Moli Sang , Qingyu Yang , Jiawei Guo , Peiyuan Feng , Wencheng Ma , Wei Zhang

A novel sub-class of S-adenosyl-l-methionine (SAM)-dependent methyltransferases catalyze atypical chemical transformations in the biosynthesis of anthracyclines. Exemplified by RdmB from Streptomyces purpurascens, it was found with 10-decarboxylative hydroxylation activity on anthracyclines. We herein investigated the catalytic activities of RdmB and discovered a previously unknown 4-O-methylation activity. The site-directed mutagenesis studies proved that the residue at position R307 and N260 are vital for the decarboxylative hydroxylation and 4-O-methylation, respectively, which define two distinct catalytic centers in RdmB. Furthermore, the multifunctionality of RdmB activity was found as cofactor-dependent and stepwise. Our findings expand the versatility and importance of methyltransferases and should aid studies to enrich the structural diversity and bioactivities of anthracyclines.

一种新型的 S-腺苷-l-蛋氨酸（SAM）依赖性甲基转移酶亚类在蒽环类化合物的生物合成过程中催化非典型化学转化。以紫链霉中的 RdmB 为例，它对蒽环类化合物具有 10-脱羧羟化活性。我们在此研究了 RdmB 的催化活性，并发现了一种之前未知的 4-O 甲基化活性。定点突变研究证明，R307 和 N260 位残基分别对脱羧羟基化和 4-O 甲基化起着至关重要的作用，从而确定了 RdmB 中两个不同的催化中心。此外，研究还发现 RdmB 活性的多功能性取决于辅助因子，并且是逐步进行的。我们的发现拓展了甲基转移酶的多功能性和重要性，有助于研究丰富蒽环类化合物的结构多样性和生物活性。

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