Journal of biotechnology最新文献_第3页

Engineered Lactococcus lactis expressing antimicrobial peptide HI: Enhanced survival and protection against ETEC in mice 表达抗菌肽HI的工程乳酸乳球菌：提高小鼠的存活率和对ETEC的保护作用。

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2026-01-02 DOI: 10.1016/j.jbiotec.2025.12.019

Mingyang Hu , Chongpeng Bi , Yuwen Li, Yutong Xue, Sina Cha, Lu Zhao, Chenyu Xue, Na Dong

The rising prevalence of antibiotic resistance underscores the urgent need for alternative strategies to manage pathogenic bacteria. Engineered probiotics offer a promising platform for delivering antimicrobial peptides (AMPs); however, their practical application remains constrained by challenges related to maintaining viability and in vivo functionality. This study focused on two main aspects: (1) optimizing a freeze-drying strategy for Lactococcus lactis/pNZC-Usp45-H-6 ×His (L. L/HI), which expresses the AMP HI targeting Escherichia coli, and (2) evaluating its protective efficacy against enterotoxigenic Escherichia coli (ETEC) infection in a murine model. Sorbitol at a concentration of 6 % (w/v) was identified as the most effective cryoprotectant for preserving bacterial viability after freeze-drying. In the ETEC infection model, oral administration of L. L/HI significantly alleviated intestinal injury by reducing bacterial colonization and lipopolysaccharide levels, alleviating inflammation, and restoring the expression of tight junction genes. Moreover, L. L/HI downregulated the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and upregulated the anti-inflammatory cytokine IL-10 in ileal tissues. These findings demonstrate that oral administration of L. L/HI reduced the bacterial burden in the ileum of ETEC–infected mice and indirectly alleviated inflammation and intestinal barrier damage caused by ETEC infection. This study provides a novel approach for the translational application of engineered probiotics.

抗生素耐药性的日益流行突出表明迫切需要采取其他战略来管理致病菌。工程益生菌为提供抗菌肽（amp）提供了一个有前途的平台；然而，它们的实际应用仍然受到与维持活力和体内功能相关的挑战的限制。本研究主要集中在两个方面：(1)优化以大肠杆菌为靶点表达AMP HI的乳酸乳球菌/pNZC-Usp45-H-6×His （L. L/HI）的冷冻干燥策略；(2)在小鼠模型上评价其对产肠毒素大肠杆菌（ETEC）感染的保护作用。山梨醇在6% （w/v）的浓度下被确定为冷冻干燥后保持细菌活力最有效的冷冻保护剂。在ETEC感染模型中，口服L. L. /HI通过减少细菌定植和脂多糖水平、减轻炎症、恢复紧密连接基因的表达，显著减轻肠道损伤。L. L/HI下调回肠组织中促炎因子（TNF-α、IL-1β、IL-6）的表达，上调抗炎因子IL-10的表达。上述结果表明，口服L. L/HI可减轻ETEC感染小鼠回肠细菌负荷，间接减轻ETEC感染引起的炎症和肠屏障损伤。本研究为工程益生菌的转化应用提供了新的途径。

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引用次数: 0

Construction of an E. coli cell factory for ergothioneine through SAM-cycle enhancement and pathway reconstruction 通过sam循环增强和途径重建构建麦角硫因大肠杆菌细胞工厂。

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-12-29 DOI: 10.1016/j.jbiotec.2025.12.018

Xiaoyu Zhang , Jiayue Guan , Yanqi Yang , Shangci Zuo , Chang Liu , Pengchao Wang

Ergothioneine (EGT) is a rare sulfur-containing derivative of methionine with potent antioxidant, anti-inflammatory, and neuroprotective properties. Its unique bioactivities make it a promising ingredient for applications in functional foods, nutraceuticals, and cosmetics. Microbial fermentation offers a sustainable alternative to extraction from natural sources, yet challenges such as precursor limitations, cofactor imbalances, and pathway complexity continue to restrict industrial-scale production. In this study, we engineered Escherichia coli (E. coli) as a microbial chassis for efficient de novo synthesis of EGT. By co-expressing key enzymes derived from bacteria and fungi, enhancing cysteine biosynthesis, and improving methionine utilization, we addressed key bottlenecks in precursor supply. Furthermore, the introduction of a methylation cycle significantly improved the regeneration of S-adenosylmethionine (SAM), relieving cofactor limitations. These combined metabolic engineering strategies led to a substantial increase in EGT production. The final engineered strain achieved a titer of 141.3 mg/L in shake flasks, representing a sixfold improvement over the base strain. In a 5-liter fed-batch fermentation, the titer reached 1.95 g/L without precursor supplementation and further increased to 2.52 g/L upon low-dose amino acid feeding. This work establishes a cost-effective and scalable biosynthetic platform for EGT production in E. coli, offering a viable route for its application in food and health-related industries.

麦角硫因（EGT）是一种罕见的含硫蛋氨酸衍生物，具有有效的抗氧化、抗炎和神经保护特性。其独特的生物活性使其在功能性食品、营养保健品和化妆品中应用前景广阔。微生物发酵为天然来源的提取提供了一种可持续的替代方案，但诸如前体限制、辅因子不平衡和途径复杂性等挑战继续限制工业规模的生产。在这项研究中，我们设计了大肠杆菌（E. coli）作为高效从头合成EGT的微生物底盘。通过共表达源自细菌和真菌的关键酶，增强半胱氨酸的生物合成，提高蛋氨酸的利用率，我们解决了前体供应的关键瓶颈。此外，甲基化循环的引入显著改善了s -腺苷蛋氨酸（SAM）的再生，缓解了辅助因子的限制。这些综合代谢工程策略导致EGT产量大幅增加。最终的工程菌株在摇瓶中滴度达到141.3mg/L，比基础菌株提高了六倍。在5升分批补料发酵中，未添加前体时滴度达到1.95g/L，低剂量氨基酸补料后滴度进一步提高到2.52g/L。本研究为大肠杆菌生产EGT建立了一个具有成本效益和可扩展的生物合成平台，为其在食品和健康相关行业的应用提供了一条可行的途径。

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引用次数: 0

In vitro synthesis of β-aspartyl-basic amino acid dipeptides via a multi-enzyme cascade system with ATP regeneration 通过ATP再生的多酶级联系统体外合成β-天冬氨酸-碱性氨基酸二肽。

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-12-24 DOI: 10.1016/j.jbiotec.2025.12.017

Fei-yan Cheng , Long-wei Lou , Zong-lin Li , Zhi-min Li

Cyanophycin, also known as cyanophycin granule polypeptide (CGP), was a natural polyamide synthesized via a nonribosomal pathway from L-aspartic acid and L-arginine. Its derived dipeptide, β-Asp-Arg, holds potential applications in various fields requiring arginine supplementation in feed or food industries. However, conventional CGP production methods were constrained by strict substrate specificity and high costs. To address these challenges, two-step enzymatic cascade system was constructed integrating CGP biosynthesis with dipeptide production. Central to this system is a novel cyanophycin synthetase (CphA1) from Acidobacteria bacterium, which exhibits remarkable substrate promiscuity. This enzyme enables both primer-independent polymerization of poly-L-Arg/L-Lys-poly(L-Asp) backbones and primer-dependent incorporation of non-canonical substrates (L-ornithine and L-citrulline), thereby expanding the repertoire of cyanophycin derivatives. Furthermore, polyphosphate kinase synergizes with CphA1 to regenerate ATP and promote CGP synthesis, reducing ATP consumption by 90 %. Subsequently, the polymer was efficiently hydrolyzed by CphB from Geminocystis herdmanii to yield the target dipeptides. This method achieved complete conversion of CGP into classical dipeptides β-Asp-Arg and β-Asp-Lys, with the highest titer reaching 80 mM, while also generating non-classical products β-Asp-Orn and β-Asp-Cit. These results highlight the robustness and versatility of this strategy, offering a promising route for the scalable synthesis of β-Asp-basic amino acid dipeptides.

藻青素又称藻青素颗粒多肽（CGP），是由l -天冬氨酸和l -精氨酸经非核糖体途径合成的天然聚酰胺。其衍生的二肽β-Asp-Arg在饲料或食品工业中需要精氨酸补充的各种领域具有潜在的应用前景。然而，传统的CGP生产方法受到严格的底物特异性和高成本的限制。为了解决这些问题，我们构建了将CGP生物合成与二肽生产相结合的两步酶级联系统。该系统的核心是一种来自酸杆菌的新型蓝藻素合成酶（CphA1），它表现出显著的底物混杂性。该酶既可以独立于引物的聚l -精氨酸/ l -赖氨酸聚（L-Asp）骨架聚合，也可以依赖于引物的非规范底物（l -鸟氨酸和l -瓜氨酸）的结合，从而扩大了蓝藻素衍生物的范围。此外，多磷酸激酶与CphA1协同再生ATP，促进CGP合成，减少90%的ATP消耗。随后，该聚合物被来自herdmanii的CphB高效水解，以产生目标二肽。该方法实现了CGP完全转化为经典二肽β-Asp-Arg和β-Asp-Lys，最高滴度达到80mM，同时还生成了非经典产物β-Asp-Orn和β-Asp-Cit。这些结果突出了该策略的稳健性和多功能性，为大规模合成β- asp -碱性氨基酸二肽提供了一条有前途的途径。

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引用次数: 0

Lyophilized chloroplasts as molecular capsules: A scalable platform for stable and functional human α-lactalbumin production 冻干叶绿体作为分子胶囊：稳定和功能性人α-乳清蛋白生产的可扩展平台。

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-12-23 DOI: 10.1016/j.jbiotec.2025.12.016

Maryam Ehsasatvatan , Bahram Baghban Kohnehrouz

The growing world population and the increasing demand for proteins present significant challenges to food security. Advances in synthetic biology have enabled the development of transplastomic plants engineered to produce milk proteins, offering a promising solution for the large-scale, economical production of alternative protein sources. This study demonstrated the extended stability of human α-lactalbumin (hLA) in freeze-dried transplastomic tobacco chloroplasts. hLA was expressed at high levels, constituting 39.1 % of the total soluble protein in chloroplasts (66.5 mg/g isolated chloroplasts). Western blot analysis of lyophilized tobacco chloroplasts stored at room temperature for up to six months indicated that the hLA protein remained stable, soluble, and properly folded over extended periods. The freeze-drying process of isolated chloroplasts resulted in a 34-fold increase in hLA protein concentrations. Importantly, the biological functionality of purified hLA from lyophilized chloroplasts was confirmed through an in vitro lactose synthesis assay, where recombinant hLA successfully activated galactosyltransferase, leading to efficient lactose production at level comparable to commercial standard. Lyophilized chloroplasts, free from nicotine and bacterial contamination, are suitable candidates for the oral administration of the dietary protein alpha-lactalbumin. These findings suggest that recombinant protein production in transplastomic plants could serve as a viable food source and alternative to conventional sources. Lyophilization further reduces expenses and streamlines downstream processing, purification, and storage. These methods facilitate relevant formulation practices for these compounds to meet the demand-oriented requirements. Future research should prioritize translation of this technology to edible plant hosts to enable direct human consumption and undertake comprehensive in vivo and clinical studies to validate safety, bioavailability, and efficacy.

世界人口的增长和对蛋白质需求的增加对粮食安全构成了重大挑战。合成生物学的进步使转基因植物能够生产牛奶蛋白，为大规模、经济地生产替代蛋白质来源提供了一个有希望的解决方案。本研究证实了人α-乳清蛋白（hLA）在冻干转质体烟草叶绿体中的扩展稳定性。hLA高表达，占叶绿体可溶性蛋白总量的39.1% （66.5mg/g）。在室温下保存6个月的烟草冻干叶绿体的Western blot分析表明，hLA蛋白在较长时间内保持稳定、可溶和适当折叠。冻干分离的叶绿体导致hLA蛋白浓度增加34倍。重要的是，从冻干叶绿体中纯化的hLA的生物学功能通过体外乳糖合成实验得到了证实，重组hLA成功地激活了半乳糖转移酶，导致了与商业标准相当的高效乳糖生产。冻干的叶绿体，没有尼古丁和细菌污染，是膳食蛋白-乳清蛋白口服的合适人选。这些发现表明，在转基因植物中生产重组蛋白可以作为一种可行的食物来源和替代传统来源。冻干法进一步降低了费用，简化了下游加工、净化和储存。这些方法有助于这些化合物的相关配方实践，以满足需求导向的要求。未来的研究应优先将该技术转化为可食用植物宿主，使人类能够直接食用，并进行全面的体内和临床研究，以验证安全性、生物利用度和有效性。

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引用次数: 0

Multi-parameter process optimization for high yield AAV2 vector production using scale-down multi-parallel bioreactor systems and high-throughput analytical tools 利用多平行生物反应器系统和高通量分析工具对AAV2载体生产进行多参数优化。

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-12-19 DOI: 10.1016/j.jbiotec.2025.12.007

Nazgul Wagner, Jonas Austerjost, Julia Niemann

Adeno-associated virus (AAV) vectors are recognized as highly efficient carriers for genetic material. Among the various manufacturing platforms, transient production is the most established one for AAV production. Despite this, the transient production is influenced by numerous variables that can significantly impact the outcome. In this study, we employed 15 ml multi-parallel scale-down bioreactor system to evaluate critical process parameters like cell densities and plasmid DNA concentration. Additionally, a 250 ml bioreactor system was used to explore factors, such as bioprocess mode, comparing batch versus perfusion processes under optimized conditions. Results showed that in low cell density AAV production, where cell specific productivity is highest, the perfusion process yielded over three-fold lower functional titers compared to the batch process at 37°C. Furthermore, the impact of temperature-shifts, a yield improvement strategy that is rarely explored in the context of AAV production, was examined, resulting in increased functional AAV yields - almost two-fold in batch process and over three-fold increase in perfusion process. The temperature-shift not only increased total number of functional AAV particles but also improved the ratio of functional to capsid titers, suggesting enhanced encapsidation or reduced genomic loss from filled particles. After identifying the optimal setup, the process was successfully scaled up to a benchtop bioreactor, demonstrating the scalability and reproducibility of the optimized process. Production results were rapidly assessed using high-throughput analytical techniques, evaluating capsid titers via the biolayer interferometry (BLI)-based Octet® platform and functional titers through an in vitro assay using the high-throughput imaging-based Incucyte® system.

腺相关病毒（AAV）载体被认为是遗传物质的高效载体。在各种制造平台中，暂态生产是AAV生产中最成熟的一种。尽管如此，瞬态生产受到许多变量的影响，这些变量可能会对结果产生重大影响。在这项研究中，我们采用15ml的多平行缩小生物反应器系统来评估细胞密度和质粒DNA浓度等关键工艺参数。此外，使用250ml生物反应器系统探索生物工艺模式等因素，比较优化条件下的批处理与灌注处理。结果表明，在低细胞密度的AAV生产中，细胞特异性生产力最高，与37°C的批量生产相比，灌注过程的功能滴度降低了三倍以上。此外，研究人员还研究了温度变化的影响，这是一种在AAV生产中很少被探索的产率提高策略，结果表明，AAV的功能性产率增加了——在批量生产过程中几乎增加了两倍，在灌注过程中增加了三倍以上。温度的变化不仅增加了功能AAV颗粒的总数，而且提高了功能与衣壳滴度的比率，表明填充颗粒增强了衣壳化或减少了基因组损失。在确定了最佳设置后，该工艺成功地扩展到台式生物反应器，展示了优化工艺的可扩展性和可重复性。使用高通量分析技术快速评估生产结果，通过基于生物层干涉法（BLI）的Octet®平台评估衣壳滴度，并使用基于高通量成像的Incucyte®系统通过体外检测评估功能滴度。

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引用次数: 0

Heavy ion beam irradiation-induced mutational profiles in Saccharomyces cerevisiae and their dependencies on dose and intracellular state inform an enhanced microbial breeding strategy 重离子束辐照诱导的酿酒酵母突变谱及其对剂量和胞内状态的依赖关系为强化微生物育种策略提供了依据。

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-12-19 DOI: 10.1016/j.jbiotec.2025.12.015

Xiaopeng Guo , Xiuyue Xiao , Xuexia Dong , Runsheng Yin , Shengli Zhang , Miaomiao Zhang , Guanghong Luo , Yonggang Wang , Dong Lu

Efficient microbial mutagenesis using heavy-ion beam (HIB) radiation is crucial for breeding. Here, Saccharomyces cerevisiae was irradiated with HIB across medium and high doses. Based on 65 randomly selected isolates, we systematically characterized the mutagenic features and preliminarily explored the influence of gene transcriptional activity on mutation susceptibility, while also modulating the intracellular state to optimize strain breeding. High-dose irradiation (120–210 Gy) resulted in a mutation frequency more than double that of the medium-dose (90 Gy), with minimal overlapping mutations between doses. Although mutation site numbers correlated with chromosome length broadly, they were not uniformly distributed at a finer scale. The overall expression of genes associated with mutation sites moderately exceeded the genome-wide background level (p < 0.05). By coupling radiation with osmotic stress, osmoregulatory-related genes were induced to express highly during irradiation. The proportion of osmotolerant mutants obtained from each coupled treatment group (averaging 27.62 %) was higher than that from the radiation-only group (11.43 %). Inference and validation indicated that early selection pressure alone could not fully account for this improvement, highlighting the importance of the intracellular state. Compared to radiation alone, coupled radiation-osmotic stress increased the distribution of mutations in osmotically inducible osmoregulatory-related genes. We propose that the enhanced transcriptional activity may alter local chromatin conformation, together with pre-activation of shared osmotic-radiation response genes, reshape the damage-repair-mutagenesis balance. The coupled treatment produced genetically stable, highly osmotolerant mutants with mutations synergistically regulating carbon metabolism, ion homeostasis, cell adhesion, and DNA replication. This work supports developing high-efficiency microbial breeding strategies.

利用重离子束（HIB）辐射进行高效的微生物诱变对育种至关重要。在这里，用中剂量和高剂量的HIB照射酿酒酵母。基于随机选取的65株菌株，系统表征了其致突变性特征，并初步探讨了基因转录活性对突变易感性的影响，同时调节胞内状态，优化菌株选育。高剂量照射（120-210Gy）导致的突变频率是中剂量照射（90Gy）的两倍多，剂量之间的重叠突变最小。虽然突变位点数与染色体长度有广泛的相关性，但它们在更细的尺度上分布并不均匀。突变位点相关基因的总体表达量略高于全基因组背景水平（p < 0.05）。通过辐射与渗透胁迫的耦合作用，诱导渗透调控相关基因在辐照过程中大量表达。各偶联处理组获得的渗透耐受突变体比例（平均27.62%）高于单辐射处理组（11.43%）。推断和验证表明，早期的选择压力本身并不能完全解释这种改善，强调了细胞内状态的重要性。与单独辐射相比，辐射-渗透耦合胁迫增加了渗透诱导的渗透调节相关基因的突变分布。我们提出，增强的转录活性可能会改变局部染色质构象，连同共享渗透辐射应答基因的预激活，重塑损伤-修复-突变平衡。偶联处理产生了遗传稳定、高度耐渗透的突变体，突变体协同调节碳代谢、离子稳态、细胞粘附和DNA复制。这项工作为开发高效的微生物育种策略提供了支持。

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引用次数: 0

Rational design of a bifunctional glycosyltransferase for enhanced substrate promiscuity and thermostability 合理设计双功能糖基转移酶，增强底物混杂性和热稳定性。

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-12-19 DOI: 10.1016/j.jbiotec.2025.12.009

Dekai Liu , Ruifang Zhang , Peiqin Chen , Chunying Jin , Lingzhi Zhang , Xiao Gu , Li Wen , Lijuan Zhang , Guangya Zhang , Zhongkun Wu , Wei Jiang

Glycosylation of natural products significantly enhances their physicochemical properties, with glycosyltransferases (GTs) serving as the catalytic core of this biotransformation. Rational design of tailored GTs aligned with green chemistry principles is pivotal for the targeted synthesis of high-value glycosides, yet precise functional engineering remains challenging. This study employed a multi-scale computational strategy (molecular docking, multiple sequence alignment, molecular dynamics simulations) to systematically re-engineer the substrate recognition and stability modules of the bacterial GT BsGT-1. By reverse-engineering the active pocket of a plant-derived hyperpromiscuous GT (FiGT-2), we orthogonally mapped and identified six functional hotspots in BsGT-1. Site-directed mutagenesis and screening yielded the double variant S128T/T229S, achieving dual optimization: improved substrate scope (121–140 % increase in conversion with UDP-Gal/UDP-Rha compared to wild-type) and enhanced thermostability (>70 % residual activity after 4 h at 50℃). Structural dynamics analyses revealed that mutation-induced global conformational rigidity and localized hydrogen-bond network optimization primarily drove thermostability improvement and substrate affinity enhancement, respectively. This work establishes a closed-loop engineering paradigm of "computational prediction → rational mutagenesis → mechanistic decoding", providing a scalable framework for precision engineering of GTs and glycoside biomanufacturing.

天然产物的糖基化显著提高了其物理化学性质，而糖基转移酶（GTs）是这种生物转化的催化核心。合理设计符合绿色化学原则的定制gt对于有针对性地合成高价值糖苷至关重要，但精确的功能工程仍然具有挑战性。本研究采用多尺度计算策略（分子对接、多序列比对、分子动力学模拟）对细菌GT BsGT-1的底物识别和稳定性模块进行了系统重构。通过对植物衍生的超混杂GT的活性口袋进行逆向工程（图2），我们正交绘制并鉴定了BsGT-1中的6个功能热点。定点诱变和筛选产生了双变体S128T/T229S，实现了双重优化：提高了底物范围（与野生型相比，与UDP-Gal/UDP-Rha的转化率提高了121-140%），增强了热稳定性（在50℃下4小时后的残余活性达到70%）。结构动力学分析表明，突变诱导的整体构象刚性和局部氢键网络优化分别是热稳定性改善和底物亲和力增强的主要驱动因素。本研究建立了“计算预测→理性突变→机制解码”的闭环工程范式，为gt和糖苷生物制造的精密工程提供了可扩展的框架。

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引用次数: 0

Aluminum phthalocyanine chloride loaded poly (lactic-co-glycolic acid) nanoparticles based photodynamic therapy for antibacterial and anticancer applications 负载聚乳酸-羟基乙酸-酞菁铝纳米粒子的光动力疗法在抗菌和抗癌中的应用。

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-12-18 DOI: 10.1016/j.jbiotec.2025.12.014

Anum Jamil , Aleena Zahid , Rafaqat Ali Khan , Muhammad Saleem , Syed Mujtaba Ul Hassan , Shahzad Anwar , Qura Tul Ain

Photodynamic therapy (PDT) and antibacterial Photodynamic therapy (aPDT) have become a viable non-invasive treatment against cancer and bacterial infections. By using light-activated photosensitizers (PS), PDT effectively destroys cancer cells and pathogenic bacteria. However, many PS agents, such as Aluminum Phthalocyanine chloride (AlPc), face limitations like poor solubility and low bioavailability. To overcome these challenges, AlPc-loaded PLGA nanoparticles (AlPc NPs) have been synthesized, where PS is encapsulated in Poly (lactic-co-glycolic acid) (PLGA) to enhance drug delivery and therapeutic efficacy. PLGA was selected for its well-established biomedical applications, including targeted drug delivery and antibacterial activity. This study focuses on synthesizing 200 nm AlPc NPs by using the single emulsion solvent evaporation method and characterizing them via spectroscopic and microscopic techniques. This study demonstrates that AlPc NPs' encapsulation efficiency is 82 % and drug loading efficiency is 10 %. The drug release profile shows 52 % release of AlPc NPs within 24 h. Cellular uptake of free AlPc and AlPc NPs was examined using confocal fluorescence imaging. The in vitro investigation used Hep-2C human laryngeal cancer cells to assess the dark and phototoxic effects of free AlPc and AlPc NPs for PDT. MTT results show that the cells treated with AlPc NPs had lower cell viability than cells treated with free AlPc at the same drug doses, which confirms improved efficacy of AlPc NPs. Moreover, AlPc NPs improved the antibacterial potential against the Escherichia coli (E. coli) bacterial strain and increased their bioavailability.

光动力疗法（PDT）和抗菌光动力疗法（aPDT）已成为一种可行的非侵入性治疗癌症和细菌感染的方法。PDT通过使用光活化光敏剂（PS），有效地破坏癌细胞和致病菌。然而，许多PS制剂，如酞菁氯化铝（AlPc），面临溶解度差和生物利用度低的局限性。为了克服这些挑战，人们合成了装载AlPc的PLGA纳米颗粒（AlPc NPs），其中PS被包裹在聚乳酸-羟基乙酸（PLGA）中，以增强药物传递和治疗效果。PLGA因其完善的生物医学应用而被选中，包括靶向药物输送和抗菌活性。本研究主要采用单乳液溶剂蒸发法制备了200nm的AlPc NPs，并对其进行了光谱和显微表征。本研究表明，AlPc NPs的包封效率为82%，载药效率为10%。药物释放谱显示AlPc NPs在24小时内释放52%。用共聚焦荧光成像检测游离AlPc和AlPc NPs的细胞摄取。体外研究采用Hep-2C人喉癌细胞，评估游离AlPc和AlPc NPs对PDT的暗毒性和光毒性作用。MTT结果显示，在相同药物剂量下，AlPc NPs处理的细胞活力低于游离AlPc处理的细胞，证实了AlPc NPs的疗效提高。此外，AlPc NPs提高了对大肠杆菌（E. coli）菌株的抗菌潜力，提高了其生物利用度。

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引用次数: 0

Systematic engineering of cell wall for improving single cell protein (SCP) production 提高单细胞蛋白（SCP）产量的细胞壁系统工程。

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-12-16 DOI: 10.1016/j.jbiotec.2025.12.011

Linhai Xie , Shuo Tian , Zhehao Jin , Tiantian Zou , Mingxin Chang , Hongting Tang , Tao Yu , Zhoukang Zhuang

As an emerging alternative protein, Single cell protein (SCP) has gained considerable traction in livestock feed applications. Furthermore, it presents substantial potential for replacing traditional dietary proteins for human consumption. Yet current research remains focused on strain screening with limited advances in cellular metabolic engineering. Due to the complexity of protein synthesis and processing, enhancing the SCP production efficiency in microbial chassis remains a major challenge. Here, we present a metabolic engineering approach to systematic engineer the cell wall of Saccharomyces cerevisiae for improving SCP production. In this study, we constructed a strain by gene modifications of genes related to cell wall biogenesis and associated signaling pathways, achieving a 32.6 % increase in cellular protein content. Additionally, comparative transcriptomics analysis uncovered the regulatory mechanism whereby remodeling of carbon and nitrogen metabolism governs cellular protein biosynthesis, providing new insight for the rational design of microbial cell factories with enhanced protein content.

单细胞蛋白（Single cell protein， SCP）作为一种新兴的替代蛋白，在家畜饲料中得到了广泛的应用。此外，它为人类消费提供了替代传统膳食蛋白质的巨大潜力。然而，目前的研究仍然集中在菌株筛选上，细胞代谢工程的进展有限。由于蛋白质合成和加工的复杂性，提高微生物底盘的SCP生产效率仍然是一个主要挑战。在这里，我们提出了一种代谢工程方法来系统地改造酿酒酵母的细胞壁，以提高SCP的产量。在这项研究中，我们通过对细胞壁生物发生相关基因和相关信号通路进行基因修饰构建了一个菌株，使细胞蛋白含量增加了32.6%。此外，比较转录组学分析揭示了碳氮代谢重塑控制细胞蛋白质生物合成的调控机制，为合理设计提高蛋白质含量的微生物细胞工厂提供了新的见解。

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引用次数: 0

Engineered BDH–NOx co-expression in Escherichia coli enables highly efficient in vivo cascade catalysis for the transformation of racemic epoxides to α-hydroxyketones 改造后的BDH-NOx在大肠杆菌中的共表达能够高效地在体内级联催化将外消旋环氧化合物转化为α-羟基酮。

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of biotechnology

Pub Date : 2025-12-16 DOI: 10.1016/j.jbiotec.2025.12.012

Bochun Hu , Xiaoqi Xi , Qianjin Hou , Jiaxin Ou , Xiaoxuan Shen , Zining Wang , Jiahui Zhao , Cunduo Tang , Jihong Huang

To eliminate the requirement for pyruvate supplementation and to avoid lactate accumulation, a multi-enzyme cascade was systematically restructured by replacing lactate dehydrogenase (LDH) with water-forming NADH oxidase (EfNOx). In this revised system, RpEH^L360V catalyzed the enantioconvergent hydrolysis of rac-4-chlorostyrene oxide (rac-1a) to (R)-1-(4-chlorophenyl)-1,2-ethanediol (1b), followed by stereoselective oxidation of (R)-1b to 4’-chloro-2-hydroxyacetophenone (1c) by BsBDHA^{I49L/V266L/G292A}. Concurrently, EfNOx reduced molecular oxygen to water, regenerating NAD⁺ and sustaining redox balance. To further enhance intracellular cofactor turnover, BsBDHA^{I49L/V266L/G292A} and EfNOx were co-expressed in Escherichia coli, generating the recombinant strain E. coli/Cbn. Through single-factor and L9(3³) orthogonal array optimization, the one-pot in vivo cascade achieved maximal efficiency with lyophilized whole cells of E. coli/rpeh^L360V (5 mg DCW/mL) and E. coli/Cbn (35 mg DCW/mL) in sodium phosphate buffer (10 mM, pH 7.0) containing 5 mM NAD⁺ and 5 % (v/v) Tween-20 at 25 °C for 8 h under controlled agitation and aeration. Under these conditions, 25 mM rac-1a was converted to 1c with a yield of 92.1 % and a space-time yield (STY) of 2.9 mmol/L/h, representing a 3.7-fold increase relative to the original LDH-based system. The applicability of the cascade was further demonstrated using additional substrates (rac-2a to rac-7a), yielding the corresponding α-hydroxyketones at 39.1–94.5 % with STYs of 1.2–3.0 mmol/L/h. These findings establish BDH–NOx co-expression as a robust strategy for cofactor self-sufficiency and provide a scalable framework for the efficient in vivo synthesis of structurally diverse α-hydroxyketones.

为了消除对丙酮酸补充的需求并避免乳酸积累，通过用形成水的NADH氧化酶（EfNOx）取代乳酸脱氢酶（LDH）来系统地重组多酶级联。在改进后的体系中，RpEHL360V催化rac-4-氯苯乙烯氧化物（rac-1a）对映聚合水解为(R)-1-（4-氯苯基）-1,2-乙二醇（1b），随后BsBDHAI49L/V266L/G292A将(R)-1b立体选择性氧化为4'-氯-2-羟基苯乙酮（1c）。同时，EfNOx将分子氧还原为水，再生NAD+并维持氧化还原平衡。为了进一步促进细胞内辅助因子的转换，BsBDHAI49L/V266L/G292A和EfNOx在大肠杆菌中共表达，生成重组菌株E. coli/Cbn。通过单因素和L9（33）正交阵列优化，大肠杆菌/rpehL360V （5mg DCW/mL）和大肠杆菌/Cbn （35mg DCW/mL）冻干全细胞在含有5mM NAD+和5% (v/v) Tween-20的磷酸钠缓冲液（10mM, pH 7.0）中，在25°C、可控搅拌和曝气条件下，在8小时内进行一锅体内级联，效率最高。在此条件下，25mM rac-1a转化为1c，产率为92.1%，空时产率（STY）为2.9mmol/L/h，比原来的ldh体系提高了3.7倍。利用其他底物（rac-2a至rac-7a）进一步验证了该级联的适用性，得到相应的α-羟基酮，产率为39.1%至94.5%，STYs为1.2至3.0mmol/L/h。这些发现证实了BDH-NOx共表达是一种辅助因子自给自足的强大策略，并为结构多样的α-羟基酮的高效体内合成提供了可扩展的框架。

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引用次数: 0