Journal of biotechnology最新文献_第6页

CRISPR/Cas9-based gene deletion and targeted metabolomics reveal ectoine flux reprogramming in Halomonas campaniensis 基于CRISPR/ cas9的基因缺失和靶向代谢组学揭示campaniensis外托素通量重编程

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-10-21 DOI: 10.1016/j.jbiotec.2025.10.006

Minhaoxue Zou , Yujie Tao , Bohan Shi , Rong Xu , Derui Zhu , Yongzhen Li , Rui Han , Rong Wang

Ectoine and betaine are widely used compatible solutes. In Halomonas campaniensis XH26, the hom gene is involved in betaine biosynthesis, and the doeA gene participates in ectoine degradation. Deletion of hom and doeA may lead to poorly understood changes in metabolic flux within the ectoine biosynthesis pathway. The metabolically deficient XH26/Δhom and XH26/Δhom/ΔdoeA strains were constructed using a CRISPR/Cas9 approach. Comparative analyses of colony morphology, growth characteristics, and intracellular ectoine yield were conducted to evaluate the regulatory roles of the hom and doeA genes. RT-qPCR and targeted metabolomics were used to assess changes in gene expression related to ectoine biosynthesis and shifts in central carbon metabolic flux. The metabolically deficient strains XH26/Δhom and XH26/Δhom/ΔdoeA were constructed. Compared to the strain XH26, both mutant strains exhibited smaller colony diameters and shorter, broader cells. Intracellular ectoine yield increased by 13.3 % and 33.3 %, respectively, while betaine yield significantly decreased by 73.08 % and 76.92 %. RT-qPCR analysis revealed the significant upregulation of asd, lysC, ectA, ectB, and ectC, suggesting an enhanced metabolic flux toward ectoine biosynthesis. Targeted metabolomics indicated that the differentially abundant metabolites were mainly involved in four key energy metabolism pathways. These results indicate that knocking out the key genes hom and doeA in the ectoine biosynthesis pathway led to the restructuring of carbon metabolic flux in H. campaniensis. More carbon entered the ectoine biosynthesis pathway, resulting in the enhanced production of ectoine and a concomitant reduction in its degradation. These findings offer theoretical support for engineering high-yield ectoine-producing strains.

异托碱和甜菜碱是广泛使用的相容溶质。在campaniensis XH26中，homa基因参与甜菜碱的生物合成，doeA基因参与甜菜碱的降解。hom和doeA的缺失可能导致外托因生物合成途径中代谢通量的变化。利用CRISPR/Cas9方法构建代谢缺陷菌株XH26/Δhom和XH26/Δhom/ΔdoeA。通过对菌落形态、生长特性和胞内外泌素产量的比较分析来评价homa和doeA基因的调控作用。使用RT-qPCR和靶向代谢组学来评估与外托因生物合成相关的基因表达变化和中心碳代谢通量的变化。构建代谢缺陷菌株XH26/Δhom和XH26/Δhom/ΔdoeA。与菌株XH26相比，两种突变株的菌落直径更小，细胞更短、更宽。胞内异位碱产量分别提高13.3%和33.3%，甜菜碱产量显著降低73.08%和76.92%。RT-qPCR分析显示asd、lysC、ectA、ectB和ectC显著上调，表明体外托因生物合成的代谢通量增强。靶向代谢组学表明，差异丰富的代谢物主要涉及四个关键的能量代谢途径。这些结果表明敲除外托碱生物合成途径中的关键基因homa和doeA导致了康帕尼疟原虫碳代谢通量的重组。更多的碳进入了异托因的生物合成途径，导致异托因的产量增加，同时也减少了其降解。这些发现为高产异托氨酸菌株的工程化提供了理论支持。

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引用次数: 0

Impact of humanization scaffold design on the functional activity of FGFR2-targeting chicken scFvs 人源支架设计对靶向fgfr2的鸡scfv功能活性的影响。

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-10-17 DOI: 10.1016/j.jbiotec.2025.10.005

Shyi-Chun Yii , Tsai-Yu Lin , Fu-Ling Chang , Fu-Shan Jaw , Keng-Chang Tsai , Yun-Shih Lin , Yu-Ching Lee

Fibroblast growth factor receptor 2 (FGFR2) is frequently overexpressed in gastric cancer and represents a promising therapeutic target. We developed FGFR2-specific single-chain variable fragments (scFvs) using a chicken-derived immune library and a refined cell-based panning strategy incorporating FGFR2-knockdown cells for negative selection. The lead clone, scFv R21, exhibited high binding affinity and strong tumor-inhibitory effects in vitro. To enable therapeutic application, R21 was humanized using three distinct framework strategies: CDR grafting, light chain replacement, and structure-guided back-mutation. Structural modeling by AlphaFold3 indicated that the hR21-Bf variant preserved CDR conformation and overall stability. Despite lower expression, hR21-Bf retained moderate FGFR2 binding affinity (K_D) of 38 nM, while other variants showed markedly reduced reactivity. The hR21-Bf construct was further reformatted as a full-length human IgG1 and evaluated in a gastric cancer xenograft model. Mice treated with IgG hR21-Bf showed significant tumor growth inhibition without observable toxicity. Immunohistochemical and biochemical analyses of resected tumors confirmed reduced Ki-67 expression and downregulation of FGFR2-mediated signaling. Our study highlights the impact of scaffold selection on antibody structure and function, supporting a rational approach to antibody humanization using avian-derived libraries for cancer therapy.

成纤维细胞生长因子受体2 （FGFR2）在胃癌中经常过表达，是一个有希望的治疗靶点。我们利用鸡源性免疫文库和一种基于细胞的筛选策略，结合fgfr2敲低细胞进行负选择，开发了fgfr2特异性单链可变片段（scFvs）。该先导克隆scFv R21在体外表现出高结合亲和力和较强的肿瘤抑制作用。为了实现治疗应用，R21采用三种不同的框架策略进行了人源化：CDR嫁接、轻链替换和结构引导的反向突变。AlphaFold3的结构建模显示，hR21-Bf变体保留了CDR构象和整体稳定性。尽管表达较低，hR21-Bf仍保持中等的38nM的FGFR2结合亲和力（KD），而其他变体的反应性明显降低。hR21-Bf构建体进一步重组为全长人IgG1，并在胃癌异种移植模型中进行评估。IgG hR21-Bf对小鼠肿瘤生长有明显抑制作用，无明显毒性。切除肿瘤的免疫组织化学和生化分析证实Ki-67表达降低，fgfr2介导的信号通路下调。我们的研究强调了支架选择对抗体结构和功能的影响，支持了利用禽类衍生文库进行癌症治疗的抗体人源化的合理方法。

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引用次数: 0

Bioconversion for ursodeoxycholic acid using β-cyclodextrin included-chenodeoxycholic acid via immobilized cells of Xanthomonas maltophilia 含β-环糊精-鹅去氧胆酸在嗜麦芽黄单胞菌固定化细胞中转化熊去氧胆酸。

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-10-16 DOI: 10.1016/j.jbiotec.2025.10.004

Zengyan Yang , Caixia Liu , Yan Jin , Jing Wang , Yingying Ma , Juan Zou , Yinlei Han , Yixin Guan , Wenting Zhao , Qingxi Wu

Ursodeoxycholic acid is a prevalent clinical drug widely used for hepatobiliary diseases. It offers the advantages of good efficacy, high sensitivity, low toxic side effects and multifunctionality. However, its poor water solubility and arduous biopreparation have always been obstacles. This work presents, for the first time, a continuous aerobic-biotransformation system for synthesizing ursodeoxycholic acid. The process utilizes immobilized Xanthomonas maltophilia cells with substrate chenodeoxycholic acid/β-cyclodextrin inclusion complexes. The designed inclusion complexes possessed favourable stability with solubility improved by 122 times comparing to the pure drug. Repeated batches transformation was carried out and reaction parameters like flow speed, fluid direction and filling volume were optimized. The results showed that X. maltophilia cells were successfully immobilized in the microspheres with typical shell-core structure and good abrasion resistance. The optimal reaction conditions were as follows: flow speed of 1.0 mL/min, fluid direction from bottom to top in a reverse orientation, and filling volume set to 1:1. The entire continuous reaction could be recycled in three consecutive batches with no decrease in the conversion of the products. This study provides a new strategy for the preparation of ursodeoxycholic acid via bioconversion, which is valuable to explore the aerobic-based strain for biotransformation.

熊去氧胆酸是临床上广泛应用于肝胆疾病的常用药物。具有疗效好、灵敏度高、毒副作用小、多功能性强等优点。但其水溶性差，生物修复难度大，一直是其发展的障碍。本工作首次提出了一个连续的好氧生物转化系统来合成熊去氧胆酸。该工艺利用固定化嗜麦芽黄单胞菌细胞，底物为鹅脱氧胆酸/β-环糊精包合物。所设计的包合物具有良好的稳定性，其溶解度比纯药物提高了122倍。进行了重复批次转化，优化了流速、流体方向、灌装量等反应参数。结果表明，嗜麦芽葡萄球菌细胞成功地固定在具有典型壳核结构和良好耐磨性的微球中。最佳反应条件为流速1.0mL/min，流体方向由下向上为反向，灌装量为1:1。整个连续反应可连续三批回收，产物转化率不降低。本研究为生物转化法制备熊去氧胆酸提供了一种新的策略，对探索好氧菌株进行生物转化具有一定的价值。

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引用次数: 0

Enhanced interfacial microbial degradation of n-hexane-contaminated waste gas using a novel magnetic silicone oil 新型磁性硅油增强界面微生物对正己烷污染废气的降解

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-10-10 DOI: 10.1016/j.jbiotec.2025.10.002

Guangquan Ou , Fangzhen Ran , Zhengwei Shang , Chenhang Meng , Jiahao Liu , Zhuqiu Sun , Dongzhi Chen , Rongsheng Lin , Lichao Lu

Two-phase partitioning bioreactors have addressed the challenges of treating waste gases contaminated with hydrophobic volatile organic compounds (VOCs), primarily by improving mass transfer. However, the efficient recovery of the non-aqueous phase remains a significant issue. In this study, a novel magnetic silicone oil, designated KH602, was developed as a non-aqueous phase medium in a two-phase partitioning air-lift bioreactor for the treatment of gaseous n-hexane. KH602 improved the oil/water partition coefficient by 1.402-fold compared to conventional silicone oil. When used in the bioreactor, KH602 increased the elimination capacity by 33.3 % at an inlet concentration of 500 mg·m⁻³. KH602 also promoted microbial extracellular polymer secretion, with protein content increasing by 25 %. It stimulated a higher proportion of activated cells and enhanced their capacity to utilize n-hexane metabolic intermediates, particularly esters, which showed a 117.9 % increase. Biomass was enriched at the oil–water interface, facilitating the mass transfer of n-hexane via a “gas–oil–biological” pathway. Additionally, microbial genera with known or potential n-hexane degradation capabilities, including Mycobacterium and Gordonia, were enriched. These findings offer theoretical insights and technical support for the efficient treatment of hydrophobic VOCs.

两相分配生物反应器解决了处理被疏水性挥发性有机化合物（VOCs）污染的废气的挑战，主要是通过改善传质。然而，非水相的有效回收仍然是一个重要的问题。在本研究中，开发了一种新型磁性硅油，命名为KH602，作为两相分配气升生物反应器中的非水相介质，用于处理气态正己烷。与常规硅油相比，KH602的油水分配系数提高了1.402倍。在生物反应器中，当进口浓度为500 mg·m−3时，KH602的去除率提高了33.3% %。KH602还能促进微生物胞外聚合物分泌，蛋白质含量提高25 %。它刺激了更高比例的活化细胞，增强了它们利用正己烷代谢中间体的能力，特别是酯类，增加了117.9 %。生物质在油水界面富集，促进正己烷通过“气-油-生物”途径传质。此外，具有已知或潜在的正己烷降解能力的微生物属，包括分枝杆菌和戈登菌，也得到了富集。这些发现为疏水挥发性有机化合物的有效治理提供了理论见解和技术支持。

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引用次数: 0

Efficient CRISPR/Cas-based gene editing in cotton induced by cotton leaf crumple virus 基于CRISPR/ cas的棉花皱叶病毒高效基因编辑

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-10-10 DOI: 10.1016/j.jbiotec.2025.10.001

Jiajun Zhang , Peihong Dai , Zheng Weng , Runze Xu , Yue Li , Xiaodong Liu , Jianfeng Lei

Plant viral vectors can replicate autonomously and spread within host cells, making them an ideal tool for the delivery of CRISPR/Cas gene-editing elements. Here, we constructed a cotton CRISPR/Cas system mediated by cotton leaf crumple virus (CLCrV) as a delivery vector. We first inoculated Pro35s::Cas9 and ProUbi::Cas9 cotton with sgRNAs designed to knock out GhAGL16, GhPDS, and GhCLA1 target genes via the CLCrV vector and then compared the effects of these two transformation receptors on the editing efficiency of the same target genes. We next explored the feasibility of simultaneous multi-target editing in cotton via pooled virus inoculation. Finally, we used a cotton line overexpressing nCas9-TadA7.10 as the transformation receptor to explore the feasibility of CLCrV-mediated adenine base editing and verify the specificity of gene editing in this system. Mutation detection and deep sequencing revealed that the Pro35s::Cas9 and ProUbi::Cas9 cotton lines did not differ significantly in editing efficiency, and both could be used as successful receptors for the CLCrV-mediated Cas9 system. Pooled inoculation with CLCrV-sgRNAs enabled the simultaneous editing of multiple target genes in Pro35s::Cas9 and ProUbi::Cas9 cotton, although this approach had somewhat lower editing efficiency than inoculation with single sgRNAs. The CLCrV-mediated adenine base-editing system enabled A-to-G conversion at target sites in cotton GhPEBP and showed high gene-editing specificity. In summary, this study establishes an efficient CLCrV-mediated CRISPR system in cotton, providing a powerful technical tool for editing of multiple target genes and base editing.

植物病毒载体可以在宿主细胞内自主复制和传播，是传递CRISPR/Cas基因编辑元件的理想工具。本研究构建了以棉花叶片皱缩病毒（CLCrV）为传递载体的棉花CRISPR/Cas系统。我们首先通过CLCrV载体接种Pro35s::Cas9和ProUbi::Cas9棉花，将设计的敲除GhAGL16、GhPDS和GhCLA1靶基因的sgrna接种，然后比较这两种转化受体对相同靶基因编辑效率的影响。接下来，我们探索了通过集合病毒接种在棉花中同时进行多靶点编辑的可行性。最后，我们利用过表达nCas9-TadA7.10的棉系作为转化受体，探索clcrv介导腺嘌呤碱基编辑的可行性，验证该系统中基因编辑的特异性。突变检测和深度测序显示，Pro35s::Cas9和ProUbi::Cas9棉系的编辑效率没有显著差异，两者都可以作为clcrv介导的Cas9系统的成功受体。CLCrV-sgRNAs混合接种能够同时编辑Pro35s::Cas9和ProUbi::Cas9棉花中的多个靶基因，尽管这种方法的编辑效率略低于单一sgRNAs接种。clcrv介导的腺嘌呤碱基编辑系统能够在棉花GhPEBP的靶位点实现A-to-G转化，并显示出较高的基因编辑特异性。综上所述，本研究在棉花中建立了高效的clcrv介导的CRISPR系统，为多靶基因编辑和碱基编辑提供了强有力的技术工具。

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引用次数: 0

Beyond tyrosine feeding: A novel fed-batch cultivation strategy based on tyrosine metabolic engineering in recombinant CHO cells 超越酪氨酸饲养：一种基于酪氨酸代谢工程的重组CHO细胞补料分批培养新策略。

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-10-10 DOI: 10.1016/j.jbiotec.2025.10.003

Lei Cao , Liang Zhao , Qian Ye , Wen-Song Tan

CHO cells dominate monoclonal antibody (mAb) production in fed-batch biomanufacturing, where tyrosine supply is limited by low solubility in neutral media and the complexities of alkaline tyrosine feeds. Existing studies confirm overexpressing pterin-4α-carbinolamine dehydratase 1 (PCBD1) and phenylalanine hydroxylase (PAH) restores tyrosine prototrophy and matches non-engineered cells’ production in tyrosine-supplemented cultures. However, these studies focus on enzyme regulation without resolving feeding-phase supply challenges in high-density scenarios. To address this, this study verified that recombinant CHO (rCHO) cells rely strongly on exogenous tyrosine for growth and production. Multi-level expression analysis further confirmed low PCBD1/PAH levels restrict endogenous tyrosine synthesis, and identified quinoid dihydropteridine reductase (QDPR)—a key tetrahydrobiopterin (BH4) regeneration enzyme—as a previously unrecognized bottleneck, particularly in tyrosine-limited conditions. By co-overexpressing QDPR in high PCBD1/PAHexpressing cells to remodel the tyrosine biosynthesis pathway, a novel fed-batch strategy was established: basal medium with 3.0 mM tyrosine and tyrosine-free singlefeeding medium. Results showed this strategy, effective in high-density fed-batch settings, enabled rCHO cells to reach a final mAb titer of 4.24 g/L, representing a 32.50% increase compared to cells overexpressing only PCBD1 and PAH, and a 10.70 % increase compared to the conventional strategy. In summary, the strategy offers a simplified nutrient alternative by eliminating alkaline tyrosine feeds, highlighting holistic metabolic pathway optimization’s importance in biomanufacturing and targeted value for high-density, tyrosine-limited CHO cell-based mAb production.

在分批投喂生物生产中，CHO细胞主导单克隆抗体（mAb）的生产，酪氨酸在中性培养基中的低溶解度和碱性酪氨酸饲料的复杂性限制了酪氨酸的供应。现有研究证实，在补充酪氨酸的培养中，过表达pterin-4α-碳醇胺脱水酶1 （PCBD1）和苯丙氨酸羟化酶（PAH）可以恢复酪氨酸原生营养，并与非工程细胞的生产相匹配。然而，这些研究集中在酶的调节上，而没有解决高密度情况下的饲喂期供应挑战。为了解决这个问题，本研究证实重组CHO （rCHO）细胞强烈依赖外源酪氨酸来生长和生产。多层次表达分析进一步证实低PCBD1/PAH水平限制内源性酪氨酸合成，并确定醌类二氢蝶呤还原酶（QDPR）是一种关键的四氢生物蝶呤（BH4）再生酶，是以前未被发现的瓶颈，特别是在酪氨酸受限的条件下。通过在PCBD1/ pah高表达细胞中共过表达QDPR，重塑酪氨酸生物合成途径，建立了一种新的补料策略：基础培养基中添加3.0mM酪氨酸和不含酪氨酸的单次补料培养基。结果表明，该策略在高密度补料批设置下有效，使rCHO细胞最终的单抗滴度达到4.24g/L，与仅过表达PCBD1和PAH的细胞相比，提高了33.25%，与传统策略相比，提高了10.70%。总之，该策略通过消除碱性酪氨酸饲料提供了一种简化的营养替代方案，突出了整体代谢途径优化在生物制造中的重要性，以及高密度、酪氨酸受限的CHO细胞单克隆抗体生产的靶向价值。

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引用次数: 0

Modular engineering of the doxorubicin biosynthetic gene cluster based on component functional clustering in Streptomyces 基于链霉菌组分功能聚类的多柔比星生物合成基因簇模块化工程

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-10-03 DOI: 10.1016/j.jbiotec.2025.09.019

Tianqi Cui, Chanjuan Jiang, Jiale Li, Shuo Wang, Lingdi Li, Ji Luan, Hailong Wang

The anticancer drug doxorubicin is synthesized through the synergistic action of the type II polyketide synthases, along with an array of modification enzymes in Streptomyces. The 33 doxorubicin biosynthetic genes are scattered across at least 15 distinct transcriptional units, posing challenges for their regulatory control. In this study, we firstly achieved heterologous production of doxorubicin in a range of Streptomyces hosts by supplementing functional genes essential for biosynthesis of the sugar moiety. Then, employed a modular engineering approach to reconstruct the doxorubicin gene cluster, grouping the genes responsible for each biosynthetic module into 6 well-defined subclusters. Based on these 6 subclusters, we identified that the glycosylation and post-modification subcluster is the module with the greatest capacity to boost doxorubicin production. When introduced into the heterologous host S. albus J1074, the combination of the reconstructed modular gene cluster and the glycosylation and post-modification module resulted in a doxorubicin production rate that was 15 times greater than that of the natural gene cluster. Modular engineering of the doxorubicin gene cluster, which involves clustering based on component functionality, will streamline the regulatory control of the biosynthetic pathway and facilitate its transplantation into desired hosts for production optimization.

抗癌药物阿霉素是通过链霉菌中II型聚酮合成酶与一系列修饰酶的协同作用合成的。33个阿霉素生物合成基因分散在至少15个不同的转录单位中，对其调控控制提出了挑战。在这项研究中，我们首次通过补充糖部分生物合成所必需的功能基因，在一系列链霉菌宿主中实现了阿霉素的异源生产。然后，采用模块化工程方法重建阿霉素基因簇，将负责每个生物合成模块的基因分组为6个定义良好的亚簇。基于这6个亚簇，我们发现糖基化和修饰后亚簇是促进阿霉素生产能力最大的模块。将重组的模块化基因簇与糖基化和修饰后的模块结合，导入到异源寄主S. albus J1074中，阿霉素的产率是天然基因簇的15倍。多柔比星基因簇的模块化工程，包括基于组件功能的聚类，将简化生物合成途径的调控控制，并促进其移植到所需宿主中以优化生产。

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引用次数: 0

Enhancement of natamycin production in Streptomyces gilvosporeus through heavy ion beam mutagenesis and global transcription machinery engineering 通过重离子束诱变和全局转录机械工程提高gilvosporeus链霉菌纳他霉素的产量。

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-10-03 DOI: 10.1016/j.jbiotec.2025.09.017

Yuxiu Xu , Liang Wang , Wei Hu , Jian Xue , Wen Xiao , Hongjian Zhang , Jianhua Zhang , Xusheng Chen

Natamycin is an antifungal agent produced by Streptomyces and is widely used in food, pharmaceuticals, and other fields. However, the low production of natamycin limits its application in various fields, making it crucial to enhance the biosynthetic capacity of natamycin-producing strains. In this study, Streptomyces gilvosporeus ATCC 13326 was used to construct a chassis for the efficient production of natamycin through strain engineering. A mutant strain S. gilvosporeus Z1403 with a titer of 1.7 g·L⁻¹ was obtained by heavy ion beam radiation and LiCl resistance screening, 70.0 % higher than that of the wild-type strain S. gilvosporeus ATCC 13326. Subsequently, transcription factor screening identified whiG as a key regulator of natamycin biosynthesis. Targeting whiG via global transcription machinery engineering further enhanced natamycin production of S. gilvosporeus Z1403. Finally, the high-production mutant strain EP-whiG was obtained with a titer of 2.2 g·L⁻¹, 29.4 % and 120.0 % higher than that of the parent strain S. gilvosporeus Z1403 and the wild-type strain, respectively. S. gilvosporeus EP-whiG achieved a natamycin production of 13.1 g·L⁻¹ in a 5-L bioreactor within 120 h by fed-batch fermentation, which was 61.7 % higher than that of the wild-type strain. These results suggest that combining heavy ion beam mutagenesis with global transcription machinery engineering is an effective strategy for strain improvement, laying a theoretical foundation for enhancing the production of secondary metabolites in Streptomyces.

纳他霉素是链霉菌产生的抗真菌剂，广泛应用于食品、医药等领域。然而，纳他霉素的低产量限制了其在各个领域的应用，因此提高产纳他霉素菌株的生物合成能力至关重要。本研究以gilvosporeus链霉菌ATCC 13326为原料，通过菌株工程构建了高效生产纳他霉素的基质。通过重离子束辐射和LiCl抗性筛选，获得了突变株S. gilvosporeus Z1403，其效价为1.7g·L-1，比野生型菌株S. gilvosporeus ATCC 13326提高了70.0%。随后，转录因子筛选发现whg是纳他霉素生物合成的关键调节因子。通过全球转录机械工程靶向whg进一步提高了S. gilvosporeus Z1403纳他霉素的产量。最终获得高产突变株ep - whg，其滴度分别比亲本菌株S. gilvosporeus Z1403和野生型菌株高29.4%和120.0%。S. gilvosporeus ep -在5-L的生物反应器中分批补料发酵120h，产纳他霉素13.1g·L-1，比野生型提高61.7%。这些结果表明，将重离子束诱变与全局转录机械工程相结合是一种有效的菌株改良策略，为提高链霉菌次生代谢产物的产量奠定了理论基础。

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引用次数: 0

Optimizing Chlorella vulgaris production and exploring its impact on germination through microalga-N2-fixing bacteria consortia 优化小球藻产量及探讨微藻-固氮菌群对其萌发的影响。

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-10-01 DOI: 10.1016/j.jbiotec.2025.09.018

Ana Sanchez-Zurano , Silvia Vilaró-Cos , Daniel Figueiredo , Lusine Melkonyan , Alice Ferreira , Francisco Gabriel Acién , Tomas Lafarga , Luisa Gouveia

Microalgal biomass is increasingly valued in industrial and agricultural sectors due to its bioactive compounds. However, large-scale production remains costly, mainly due to nitrogen fertilizer expenses. A promising sustainable alternative is co-cultivation with N₂-fixing bacteria, capable of supplying biologically available nitrogen. In this study, Chlorella vulgaris was grown in synthetic medium with and without nitrogen, as well as in co-culture with three different N₂-fixing bacteria in nitrogen-free medium. Microalgal growth was assessed by dry weight, Fv/Fm ratio, and flow cytometry, which also allowed evaluation of population dynamics and cell viability. Biomass composition (proteins, carbohydrates, lipids, chlorophyll, and carotenoids) was analyzed under all conditions. Co-cultures in nitrogen-free medium showed comparable biomass productivity to nitrogen-supplemented controls, although Fv/Fm values indicated physiological stress in some cases. Moreover, the agricultural potential of the resulting biomass and supernatants was evaluated through germination bioassays using lettuce seeds. All cultures tested at 0.2 g·L⁻¹ significantly improved the germination index. Also, applying the culture supernatant (biomass removed) also yielded positive effects, with GI increases exceeding 40 %. These results suggest that co-cultivation with N₂-fixing bacteria can support efficient microalgal production while generating biomass and supernatants with biostimulant potential, contributing to sustainable agriculture and circular bioeconomy strategies.

微藻生物量由于其生物活性化合物在工业和农业部门越来越受到重视。然而，大规模生产的成本仍然很高，主要是由于氮肥的费用。一个有希望的可持续替代方案是与能够提供生物可利用氮的固氮细菌共同培养。本研究分别在含氮和不含氮的合成培养基中培养普通小球藻，并在无氮培养基中与三种不同的固氮细菌共培养。微藻生长通过干重、Fv/Fm比和流式细胞术进行评估，也可以评估群体动态和细胞活力。在所有条件下分析生物量组成（蛋白质、碳水化合物、脂类、叶绿素和类胡萝卜素）。在无氮培养基中共培养的生物量生产力与补氮对照相当，尽管Fv/Fm值在某些情况下表明生理应激。此外，通过使用生菜种子进行萌发生物测定，评估了所得生物量和上清液的农业潜力。在0.2g·L-1浓度下，所有培养物的萌发指数均显著提高。此外，施用培养上清（去除生物量）也产生了积极的影响，GI增加超过40%。这些结果表明，与固氮细菌共同培养可以支持高效的微藻生产，同时产生具有生物刺激素潜力的生物量和上清，有助于可持续农业和循环生物经济战略。

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引用次数: 0

Komagataella phaffii as a microbial cell factory for antimicrobial peptide production Komagataella phaffii作为抗菌肽生产的微生物细胞工厂。

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-09-30 DOI: 10.1016/j.jbiotec.2025.09.015

Michel Lopes Leite , Kamila Botelho Sampaio de Oliveira , Letícia Ferreira Lima , Nadielle Tamires Moreira Melo , José Brango-Vanegas , Hugo Costa Paes , Octávio Luiz Franco

In recent decades, the methylotrophic yeast Komagataella phaffii has emerged as a powerful host for the heterologous production of antimicrobial peptides (AMPs) as an economical and scalable platform. K. phaffii combines several important advantages for a recombinant expression system, such as rapid growth, high-density cell culture, efficient protein secretion, and the ability to perform essential post-translational modifications. The methanol-inducible AOX1 promoter (P_AOX1), generally employed through the pPICZalpha vector, allows strong and tightly regulated heterologous expression and is a central factor in the expression system of this yeast. However, several strategies have been employed to produce recombinant AMPs in K. phaffii, among them codon optimization, engineered methanol-resistant strains, alternative promoters, and the use of different secretory peptides. This review highlights the latest advances and practical considerations in the use of K. phaffii for AMP production, discussing challenges such as peptide stability, proteolytic degradation, and yield optimization. The insights provided contribute to the expansion of biotechnological applications of K. phaffii, reinforcing its potential as an efficient and safe platform for large-scale production of therapeutic AMPs.

近几十年来，甲基营养酵母Komagataella phaffii作为一种经济且可扩展的平台，已成为异种生产抗菌肽（amp）的强大宿主。法菲氏K. phaffii结合了重组表达系统的几个重要优势，如快速生长，高密度的细胞培养，高效的蛋白质分泌，以及进行必要的翻译后修饰的能力。甲醇诱导的AOX1启动子（PAOX1）通常通过pPICZalpha载体使用，它允许强的和严格调控的异源表达，是该酵母表达系统的中心因子。然而，已经采用了几种策略来在菲氏K. phaffii中产生重组AMPs，其中包括密码子优化、工程甲醇抗性菌株、替代启动子和使用不同的分泌肽。本文重点介绍了利用菲氏酵母生产AMP的最新进展和实际考虑，讨论了肽稳定性、蛋白水解降解和产率优化等挑战。所提供的见解有助于扩大菲氏K. phaffii的生物技术应用，增强其作为大规模生产治疗性amp的有效和安全平台的潜力。

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引用次数: 0