Enzyme and Microbial Technology最新文献_第4页

Ovomucoid hydrolysates prepared using alcalase suppress adipogenesis at an early stage of 3T3-L1 cell differentiation 用alcalase制备的卵泡样水解物抑制3T3-L1细胞分化早期的脂肪生成。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-11-14 DOI: 10.1016/j.enzmictec.2025.110781

Seung-Woo Yu , Hyeong-Jin Kim , Su-Yeon Song , Kee-Tae Kim , Dong Uk Ahn , Na-Kyoung Lee , Hyun-Dong Paik

The effects of ovomucoid (OVM), a by-product of egg white, and its hydrolysates on adipocyte differentiation and lipid accumulation were investigated. The OVM hydrolyzed using Alcalase® and pepsin was named AH and PH, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis revealed significant changes in molecular weight of both hydrolysates, with AH showing a higher degree of hydrolysis. AH exhibited a more pronounced inhibitory effect on fat accumulation than PH. In in vitro experiments, AH and PH suppressed lipid accumulation during 3T3-L1 adipocyte differentiation, with AH inhibiting lipid accumulation most effectively. Oil red O staining and triglyceride measurements revealed lipid reduction in AH-treated cells, indicating that AH plays a major role in preventing lipid accumulation in adipocytes. In addition, AH inhibited the expression of lipid transcription factors (CCAAT/enhancer-binding protein alpha (C/EBP-α), peroxisome proliferator-activated receptor gamma (PPAR-γ), and sterol regulatory element-binding proteins (SREBP-1c)), adipogenesis-related factors (fatty acid synthase (FAS) and ACC1), insulin-related factors (insulin receptor substrate (IRS2) and protein kinase B (AKT2)), and lipolysis-related factors (glycerol-3-phosphate acyltransferase (GPAT), CD36, and lipoprotein lipase (LPL)) in a concentration-dependent manner. Specifically, the effect of AH was most pronounced in the early stages of adipocyte differentiation, where it activated AMPK early to associate energy homeostasis and downregulate genes important for cell cycle and lipid formation. This study suggests that OVM hydrolysates prepared using Alcalase® may contribute to the development of new strategies for the obesity treatment market.

本文研究了蛋清的副产物卵黏液（OVM）及其水解产物对脂肪细胞分化和脂质积累的影响。用Alcalase®和pepsin水解的OVM分别命名为AH和PH。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析显示，两种水解产物的分子量都发生了显著变化，AH的水解程度更高。AH对脂肪堆积的抑制作用比PH更明显。在体外实验中，AH和PH对3T3-L1脂肪细胞分化过程中的脂质堆积均有抑制作用，其中AH对脂质堆积的抑制效果最好。油红O染色和甘油三酯测量显示，AH处理的细胞脂质减少，表明AH在防止脂肪细胞脂质积累中起主要作用。此外，AH抑制脂质转录因子(CCAAT/增强子结合蛋白α (C/EBP-α)、过氧化物酶体增殖物激活受体γ （PPAR-γ)和甾醇调节元件结合蛋白（SREBP-1c））、脂肪生成相关因子（脂肪酸合成酶（FAS）和ACC1）、胰岛素相关因子（胰岛素受体底物（IRS2）和蛋白激酶B (AKT2)）和脂溶相关因子(甘油-3-磷酸酰基转移酶（GPAT）、CD36、和脂蛋白脂肪酶（LPL）)以浓度依赖的方式。具体来说，AH的作用在脂肪细胞分化的早期阶段最为明显，它在早期激活AMPK以关联能量稳态并下调对细胞周期和脂质形成重要的基因。该研究表明，使用Alcalase®制备的OVM水解物可能有助于肥胖症治疗市场新策略的发展。

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引用次数: 0

Mutational study-based identification of high activity O-methyltransferase for the regioselective methylation of epigallocatechin gallate 基于突变研究的表没食子儿茶素没食子酸酯区域选择性甲基化高活性o -甲基转移酶鉴定。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-11-13 DOI: 10.1016/j.enzmictec.2025.110779

Gyeongguk Park , Byung-Gee Kim , Joo-Hyun Seo

The site-specific methylation of (-)-epigallocatechin-3-O-gallate (EGCG), especially 3`` hydroxyl group in D-ring, significantly enhances its stability and bioavailability in human body. Therefore, the methylation is the way to make EGCG more orally active and potent nutraceutical agents. In this study, for the effective synthesis of the methylated EGCG, S-adenosyl-L-methionine dependent O-methyltransferase (OMT) was studied to synthesize the methylated EGCG. OMT from Bacillus licheniformis and B. megaterium were known to have methylation activity for EGCG. Because OMT from B. licheniformis (Bl_OMT) showed higher activity and regioselectivity between two OMTs, rational design was tried using Bl_OMT. F163W mutant of Bl_OMT showed 2-fold increased initial velocity for the methylation of EGCG than wild type Bl_OMT. This engineering result was further utilized as a basis for the identification of highly active OMT from sequence database. 100 homolog sequences of OMT of B. licheniformis and 100 homolog sequences of OMT of B. megaterium, were collected using BLAST. Multiple alignment of 202 sequences was used to generate subgroups. Four representative sequences from each subgroup were further studied. As a result, OMTs from Thermolongibacillus altinseunsis and B. subtilis, which were from homolog group of Bl_OMT, showed higher activity than Bl_OMT while showing the same high regioselectivity. OMTs from T. altinseunsis and B. subtilis showed k_cat/K_M of 17.4 M⁻¹s⁻¹ and 11.3 M⁻¹s⁻¹, respectively, while Bl_OMT showed 8.7 M⁻¹s⁻¹. Therefore, we could find that phenylalanine residue of the active site of OMT is very important to make strong binding of hydrophobic moiety of substrate and mutation to tryptophan is able to give higher binding strength.

EGCG(-)-表没食子儿茶素-3- o -没食子酸酯（epigallocatechin-3- o -gallate， EGCG）的位点特异性甲基化，尤其是d环上的3′′羟基甲基化，显著提高了EGCG在人体内的稳定性和生物利用度。因此，甲基化是使EGCG更具口服活性和有效的营养制剂的途径。在本研究中，为了有效合成甲基化EGCG，研究了s -腺苷- l-蛋氨酸依赖的o -甲基转移酶（OMT）来合成甲基化EGCG。已知地衣芽孢杆菌和巨芽孢杆菌的OMT对EGCG具有甲基化活性。由于地衣芽孢杆菌OMT （Bl_OMT）在两种OMT之间具有较高的活性和区域选择性，因此对Bl_OMT进行了合理设计。Bl_OMT突变体F163W的EGCG甲基化初始速度是野生型Bl_OMT的2倍。该工程结果进一步作为从序列数据库中识别高活性OMT的基础。利用BLAST采集了地衣芽孢杆菌OMT的100个同源序列和巨型芽孢杆菌OMT的100个同源序列。对202个序列进行多次比对，生成子群。对每个亚群的4个代表性序列进行进一步研究。结果表明，来自Bl_OMT同源群的高寒热隆杆菌和枯草芽孢杆菌的omt活性高于Bl_OMT，且具有相同的高区域选择性。T. altinseunsis和B. subtilis的omt的kcat/KM分别为17.4 M-1s-1和11.3 M-1s-1， Bl_OMT的kcat/KM分别为8.7 M-1s-1。因此，我们可以发现OMT活性位点的苯丙氨酸残基对底物疏水片段的强结合和突变与色氨酸的强结合非常重要，能够提供更高的结合强度。

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引用次数: 0

Modulation of ADH activity in Zymomonas mobilis provides significant flux redirection away from ethanol 运动单胞菌ADH活性的调节提供了远离乙醇的重要通量重定向。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-11-08 DOI: 10.1016/j.enzmictec.2025.110777

Arun Thapa, Ngangom Pravina Devi, Ashish Misra

Zymomonas mobilis has an extremely high specific glucose uptake rate and produces ethanol at high yields and productivities. For synthesizing products beyond ethanol in Z. mobilis, reducing ethanol yields is a major challenge. Previous efforts have sought to decrease PDC activity to minimize ethanol formation. Here, we sought to modulate ADH (encoded by adhA and adhB genes) activity to redirect flux away from ethanol. We found that deletion of adhB combined with co-expression of a heterologous NAD(P) regenerating enzyme (L-LDH) diverted more than 50 % of carbon flux away from ethanol formation. The sequential deletion of adhA in the adhB-deleted strain led to ∼90 % reduction in ADH activity. During batch growth, the strain showed ∼90 % reduction in ethanol titres (27 mM) compared to WT (209 mM) and significant flux redirection toward L-LA formation (169 mM). The results demonstrate that modulation of ADH activity by deletion of adhA and adhB genes is an effective strategy for rediverting flux toward products beyond ethanol in Z. mobilis.

活动单胞菌具有极高的特定葡萄糖摄取率，并以高产量和生产率生产乙醇。在草中合成乙醇以外的产物，降低乙醇产量是一个主要的挑战。之前的研究一直致力于降低PDC活度，以减少乙醇的生成。在这里，我们试图调节ADH（由adhA和adhB基因编码）活性，使其从乙醇中转移出去。我们发现，adhB的缺失与异种NAD(P)再生酶（L-LDH）的共表达将超过50% %的碳通量从乙醇形成中转移开。在adhb -缺失菌株中，adhA的顺序缺失导致ADH活性降低~ 90 %。在批量生长过程中，与WT（209 mM）相比，菌株的乙醇滴度降低了约90 %(27 mM)，并显著地向L-LA形成方向转移（169 mM）。结果表明，通过删除adhA和adhB基因来调节ADH活性是一种有效的策略，可以将流量重新转向乙醇以外的产物。

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引用次数: 0

Non-native genetic configuration of Gluconobacter oxydans dehydrogenases drives 2-keto-L-gulonic acid production in recombinant Escherichia coli 脱氧葡萄糖杆菌脱氢酶的非天然遗传结构驱动重组大肠杆菌生产2-酮- l -谷醛酸。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-11-07 DOI: 10.1016/j.enzmictec.2025.110776

Saloni Samant , Prashant S. Kharkar , Shreerang V. Joshi , Shamlan M.S. Reshamwala

Commercial production of 2-keto-L-gulonic acid (2-KGA), a crucial precursor in the synthesis of vitamin C, is carried out using the two-step fermentation process. This process uses a dual-strain system in the second step (conversion of L-sorbose to 2-KGA), necessitating complex process control. In this study, we report development of a recombinant Escherichia coli strain capable of producing 2-KGA from L-sorbose. L-Sorbose dehydrogenase (sdh) and L-sorbosone dehydrogenase (sndh) genes from Gluconobacter oxydans were expressed in E. coli as a synthetic operon in three different configurations under the control of the strong inducible T7 promoter. High-density whole-cell biotransformation was carried out to produce 2-KGA from L-sorbose, resulting in a yield of 0.69 g/g when the two genes were arranged in the non-native sdh-sndh configuration. Our design eliminates the need for multi-organism co-culture, intricate redox balancing, or nutrient-enriched media, representing a robust and scalable alternative to conventional production. This work demonstrates the feasibility of employing E. coli as a chassis strain for vitamin C precursor biosynthesis and offers a modular operon framework adaptable to other dehydrogenase-driven bioconversions. This approach enables efficient, scalable production of the vitamin C precursor in a genetically tractable host.

2-酮- l -谷醛酸（2-KGA）的商业化生产是合成维生素C的关键前体，采用两步发酵工艺进行。该工艺在第二步（L-sorbose到2-KGA的转换）中使用双应变系统，需要复杂的过程控制。在这项研究中，我们报道了一种重组大肠杆菌菌株的发展，该菌株能够从l -山梨糖中产生2-KGA。在强诱导型T7启动子的控制下，从氧葡萄糖杆菌中提取的l -山梨糖脱氢酶（sdh）和l -山梨糖脱氢酶（sndh）基因在大肠杆菌中以三种不同构型的合成操纵子表达。利用l -山梨糖进行高密度全细胞生物转化生产2-KGA，当这两个基因以非天然的sdh-sndh构型排列时，产量为0.69 g/g。我们的设计消除了对多生物共培养，复杂的氧化还原平衡或营养丰富的培养基的需求，代表了传统生产的强大和可扩展的替代方案。这项工作证明了利用大肠杆菌作为维生素C前体生物合成的基础菌株的可行性，并提供了一个适用于其他脱氢酶驱动的生物转化的模块化操纵子框架。这种方法使维生素C前体在遗传上易于处理的宿主中高效、可扩展地生产成为可能。

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引用次数: 0

Revolutionizing recombinant protein production in prokaryotic platforms – Methodologies and advances 原核平台重组蛋白生产的革命性变革-方法和进展

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-11-05 DOI: 10.1016/j.enzmictec.2025.110778

Shrinidhi Bhat, Senthamizh R, Mayur Mahindra Kedare, Sanjukta Patra

Recombinant protein production in prokaryotic systems remains a major topic in biotechnology because of their rapid growth, cost-effectiveness, and ease of genetic manipulation. However, the production of functionally active proteins still faces significant challenges due to folding failures, insolubility, and the lack of the capability of most prokaryotes for complex post-translational processing. This review dwells into both traditional and emerging strategies for optimizing recombinant protein expression in various prokaryotic systems. It also highlights recent advances in genetic engineering and synthetic biology for expanding the toolkit available for protein production, which include refined expression vectors, engineered hosts with improved folding capabilities, and high-throughput screening platforms. Additionally, it provides a thorough discussion of how to optimize heterologous expression using fusion tag approaches, codon bias elimination, promoter and ribosome binding site (RBS) engineering, and chaperone-assisted folding. This review explores diverse prokaryotic expression systems that offer unique advantages for heterologous expression that extend far beyond the limitations of traditional hosts. Additionally, this review also emphasizes the need for the selection of the right expression system and optimizing conditions to fulfill the increasing demands for recombinant protein production in various fields.

原核系统中重组蛋白的生产由于其快速生长、成本效益和易于遗传操作而成为生物技术的一个主要课题。然而，由于折叠失败、不溶性以及大多数原核生物缺乏复杂的翻译后加工能力，功能活性蛋白的生产仍然面临着重大挑战。本文综述了在各种原核系统中优化重组蛋白表达的传统策略和新兴策略。它还强调了基因工程和合成生物学的最新进展，以扩大可用于蛋白质生产的工具包，其中包括改进的表达载体，具有改进折叠能力的工程宿主和高通量筛选平台。此外，它还深入讨论了如何使用融合标签方法、密码子偏差消除、启动子和核糖体结合位点（RBS）工程以及伴侣辅助折叠来优化异源表达。这篇综述探讨了多种原核表达系统，这些系统为异源表达提供了独特的优势，远远超出了传统宿主的限制。此外，本文还强调了选择合适的表达体系和优化表达条件的必要性，以满足各领域对重组蛋白生产日益增长的需求。

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引用次数: 0

Identification of a 3-deoxy-D-manno-octulosonic acid kinase of lipid A in Vibrio parahaemolyticus 副溶血性弧菌脂质a 3-脱氧-d -甘露糖醛酸激酶的鉴定

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-11-04 DOI: 10.1016/j.enzmictec.2025.110775

Lingyan Chen , Danyang Huang , Xinrui Zhang , Hongchen Yin , Xiaoyuan Wang

Vibrio parahaemolyticus is a major seafood-associated foodborne pathogen whose lipopolysaccharide (LPS) plays an important role in virulence and antimicrobial resistance. The LPS of V. parahaemolyticus contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) sugar with phosphorylation. Previously, we have characterized the gene VP_RS01035 is responsible for the addition of Kdo; in this study, we characterized another gene VP_RS00960 which is responsible for the Kdo phosphorylation of V. parahaemolyticus LPS. To investigate its function, we first constructed an LPS-deficient Escherichia coli WH600 strain using CRISPR/Cas9. Heterologous expression of VP_RS01035 alone or in combination with VP_RS00960 yielded recombinant strains WH600/pB1–1 and WH600/pB2–12, respectively. Analysis of total lipids from the recombinant strains by thin-layer chromatography and high-performance liquid chromatography-tandem mass spectrometry demonstrated that the VP_RS00960 gene encodes a Kdo kinase responsible for phosphorylating the 4-OH site of the Kdo sugar in V. parahaemolyticus. To further validate its role, we deleted VP_RS00960 gene in V. parahaemolyticus, resulting in mutant ΔRS00960. Notably, ΔRS00960 failed to produce polysaccharide-linked lipid A, although free lipid A synthesis remained unaffected. Furthermore, defective long-chain LPS assembly compromised outer membrane integrity, increasing permeability and hydrophobicity while reducing biofilm formation. Consequently, ΔRS00960 exhibited heightened susceptibility to membrane-targeting antibiotics, such as erythromycin and novobiocin. Macrophage infection assays using RAW264.7 cells revealed that VP_RS00960 deletion attenuated bacterial pathogenicity. These findings enhance the understanding of the pathogenicity and drug resistance of V. parahaemolyticus, and provide novel insights and strategies for addressing antibiotic resistance and food safety challenges posed by V. parahaemolyticus.

副溶血性弧菌是一种主要的海产食源性病原菌，其脂多糖（LPS）在毒力和耐药性中起着重要作用。副溶血性弧菌的LPS含有一个磷酸化的3-脱氧-d -甘露糖醛酸（Kdo）糖。之前，我们已经鉴定了VP_RS01035基因负责Kdo的添加；在这项研究中，我们鉴定了另一个基因VP_RS00960，该基因负责副溶血性弧菌LPS的Kdo磷酸化。为了研究其功能，我们首先利用CRISPR/Cas9构建了一株缺乏lps的大肠杆菌WH600菌株。VP_RS01035单独或与VP_RS00960联合异种表达分别产生重组菌株WH600/ pB1-1和WH600/ pB2-12。利用薄层色谱和高效液相色谱-串联质谱法对重组菌株的总脂质进行了分析，结果表明VP_RS00960基因编码Kdo激酶，负责磷酸化副溶血性弧菌Kdo糖的4-OH位点。为了进一步验证其作用，我们在副溶血性弧菌中删除VP_RS00960基因，导致突变ΔRS00960。值得注意的是，ΔRS00960不能产生多糖连接的脂质A，尽管游离脂质A的合成不受影响。此外，有缺陷的长链LPS组装破坏了外膜的完整性，增加了渗透性和疏水性，同时减少了生物膜的形成。因此，ΔRS00960对膜靶向抗生素（如红霉素和新生物素）表现出更高的敏感性。RAW264.7细胞的巨噬细胞感染实验显示，VP_RS00960缺失降低了细菌的致病性。这些发现增强了对副溶血性弧菌致病性和耐药性的认识，并为解决副溶血性弧菌的抗生素耐药性和食品安全挑战提供了新的见解和策略。

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引用次数: 0

Overexpression, biochemical characterization, and structural modeling of polyhydroxybutyrate depolymerase from Nocardiopsis dassonvillei 多羟基丁酸解聚合酶的过表达、生化表征和结构建模。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-10-25 DOI: 10.1016/j.enzmictec.2025.110767

H. Anjulal , Aritri Saha , Vitthal T. Barvkar , Kshitija Pawar , Manali Joshi , Smita S. Zinjarde

The ability of Nocardiopsis dassonvillei NCIM 5124 to synthesize polyhydroxybutyrate depolymerase (PHBD) was recently reported. In this investigation, in vitro codon optimized gene synthesis, overexpression, and biochemical characterization of this enzyme along with molecular docking studies are presented. The sequence of the PHBD was inserted in pET-28a(+) along with the PelB_Signal and His₆ tag to generate the recombinant vector pET-Nd-pelB_PHBD. The transformed Escherichia coli BL21(DE3) could produce active PHBD. This enzyme was purified using Ni-NTA affinity chromatography, producing a product with a molecular weight of roughly 50 kDa. The optimum temperature and pH of the recombinant enzyme were 35°C and 8.0, respectively. Triton X100 and Tween 20 inhibited the enzyme activity by 90 %, indicating the role of hydrophobic residues in the active site of the enzyme, as also noted during docking studies. On the basis of the Michaelis-Menten equation, apparent K_m and V_max of recombinant PHBD were found to be 1.782 mg/mL and 4.79 U/mL/min, respectively. Molecular docking studies indicated that the hydrophobic amino acids Cys 39, Ala 40, Cys 77, Phe 158, Met 161, Val 201, Ala 272, and Tyr 273 present in the catalytic site were providing the necessary hydrophobic environment for binding of the ligand. The purified enzyme could also degrade films of PHB and poly (3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate (PHBVH). As far as we are aware, this is the first report on the overexpression of PHBD from Nocardiopsis sp.

最近报道了达森维尔诺卡opsis NCIM 5124合成多羟基丁酸解聚合酶（PHBD）的能力。在这项研究中，体外密码子优化基因的合成，过表达，生化表征以及分子对接研究。将PHBD序列与PelB_Signal和His6标签一起插入pET-28a（+）中，生成重组载体pET-Nd-pelB_PHBD。转化后的大肠杆菌BL21（DE3）可产生活性PHBD。利用Ni-NTA亲和层析纯化该酶，得到分子量约为50 kDa的产物。重组酶最适温度为35℃，最适pH为8.0。Triton X100和Tween 20抑制了90 %的酶活性，表明疏水残基在酶活性位点的作用，对接研究中也注意到这一点。根据Michaelis-Menten方程，重组PHBD的表观Km和Vmax分别为1.782 mg/mL和4.79 U/mL/min。分子对接研究表明，存在于催化位点的疏水氨基酸Cys 39、Ala 40、Cys 77、Phe 158、Met 161、Val 201、Ala 272和Tyr 273为配体的结合提供了必要的疏水环境。纯化后的酶还能降解PHB和聚3-羟基丁酸-co-3-羟基戊酸-co-3-羟基己酸酯（PHBVH）膜。据我们所知，这是第一次报道Nocardiopsis sp.的PHBD过表达。

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引用次数: 0

Highly efficient spermidine production system in Escherichia coli BL21 (DE3) based on precursor metabolic modules optimization and carboxyaminopropylagmatine pathway construction 基于前体代谢模块优化和羧胺丙基lagmatine途径构建的大肠杆菌BL21 （DE3）高效亚精胺生产体系

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-10-24 DOI: 10.1016/j.enzmictec.2025.110763

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

Spermidine, a naturally occurring polyamine compound, has garnered significant attention due to its versatile physiological functions, including induction of cellular autophagy, antioxidant activity, and maintenance of mitochondrial homeostasis. In this study, we established a novel spermidine biosynthesis system in E. coli BL21 (DE3) by heterologously introducing the carboxyaminopropylagmatine (CAPA) pathway derived from cyanobacterium. To enhance precursor supply, we overexpressed key enzymes in the L-aspartate β-semialdehyde and agmatine branch metabolic pathways within the precursor metabolic module, while simultaneously knocking out competing metabolic pathways to redirect metabolic flux toward spermidine biosynthesis. To address the challenge of intracellular spermidine accumulation and its associated cytotoxicity, the high-efficiency spermidine efflux pump protein AmvA from Acinetobacter baumannii was heterologously expressed in E. coli BL21 (DE3). This engineering strategy enabled efficient extrusion of spermidine from the cells, alleviating the toxic effects of high intracellular spermidine concentrations on the host strain. Through these metabolic and efflux pump engineering modifications, the engineered strain SPD06-P5-P6 was constructed. Following 48 h of shake flask fermentation, SPD06-P5-P6 achieved a spermidine titer of 163.11 mg/L, which further increased to 1164.22 mg/L after 96 h of scale-up cultivation in a 5 L bioreactor.

亚精胺是一种天然存在的多胺化合物，由于其多种生理功能，包括诱导细胞自噬、抗氧化活性和维持线粒体稳态，已经引起了广泛的关注。在本研究中，我们通过异源引入源自蓝藻的羧胺丙基lagmatine （CAPA）途径，在大肠杆菌BL21 （DE3）中建立了一个新的亚精胺生物合成系统。为了增强前体供应，我们在前体代谢模块中过度表达l -天冬氨酸β-半醛和胍丁氨酸分支代谢途径中的关键酶，同时敲除竞争性代谢途径，将代谢通量转向亚精胺生物合成。为了解决细胞内亚精胺积累及其相关的细胞毒性问题，我们在大肠杆菌BL21 （DE3）中异种表达了鲍曼不动杆菌高效亚精胺外排泵蛋白AmvA。这种工程策略可以有效地从细胞中挤出亚精胺，减轻细胞内高浓度亚精胺对宿主菌株的毒性作用。通过这些代谢泵和外排泵的工程改造，构建了工程菌株SPD06-P5-P6。摇瓶发酵48 h后，SPD06-P5-P6的亚精胺滴度达到163.11 mg/L，在5 L的生物反应器中放大培养96 h后，亚精胺滴度进一步提高到1164.22 mg/L。

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引用次数: 0

Development of high-efficiency hybrid strains for cellulolytic enzyme production via interspecific and intergeneric protoplast fusion of Trichoderma and Penicillium species 木霉与青霉原生质体种间和属间融合生产纤维素水解酶的高效杂交菌株的研究。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-10-24 DOI: 10.1016/j.enzmictec.2025.110764

Mohammad J. Alsarraf , Aisha S.M. Al-Wahaibi , Steven L. Stephenson , Najla A. Alshaikh , Fuad Ameen

Forty-five fungal strains from decomposing wood, representing eight genera, were isolated. Among them, 27 cellulolytic strains were identified. The genera Trichoderma, Penicillium, and Phanerochaete (6, 2, and 4 isolates, respectively) demonstrated the highest cellulase production. The isolates TW-25, TW-28, and TW-33 exhibited superior enzyme activities, CMCase (5.4–6.5 U/mL), FPase (3.2–3.8 U/mL), pNPCase (2.6–3.0 U/mL), and pNPGase (3.5–4.0 U/mL) and were selected for further study. Internal transcribed spacer (ITS) region sequencing, combined with phenotypic characteristics, identified these strains as Trichoderma sp. TW-25, Trichoderma sp. TW-28, and Penicillium sp. TW-33. Maximum protoplast release was observed in Trichoderma sp. TW-25 (3.6 × 10⁶ protoplasts/mL), followed by Trichoderma sp. TW-28 (3.0 × 10⁶ protoplasts/mL) and Penicillium sp. TW-33 (2.8 × 10⁶ protoplasts/mL). Fusion frequencies were 2.8 × 10⁻³ for TW-25 × TW-28, 2.0 × 10⁻³ for TW-25 × TW-33, and 1.8 × 10⁻³ for TW-28 × TW-33. A total of 13 colonies obtained from TW-25 × TW-28, and 18 from intergeneric fusions (10 from TW-25 × TW-33 and 8 from TW-28 × TW-33). The cellulase activity of the fusants TWF1/1, TWF1/6, and TWF2/5 was the same as TW-25 and the fusants TWF1/3 and TWF3/2 the same as TW-28 while none of the fusants had the cellulase activity of TW-33. Fusants differed from their parental strains in their DNA content (3.25–3.65 µg/mg dry weight) and showed high cellulase activities in general. Among them, TWF1/10 demonstrated the highest enzymatic activity, producing CMCase, FPase, pNPCase, and pNPGase (10.5, 6.5, 5.8, and 7.5 U/mL), respectively, followed by TWF1/13, TWF2/8, TWF2/10, and TWF3/8. DNA banding patterns of TWF1/10, TWF1/13, TWF2/8, TWF2/10, and TWF3/8, analyzed using four RAPD and three ISSR primers, differed from their parental strains, except for ISSR-3 with fusants TWF1/10 and TWF1/13. These variations underscore the effectiveness of interspecific and intergeneric protoplast fusion. The supernatant of the hybrid strain TWF1/10 was concentrated and purified via ultrafiltration, and SDS-PAGE and zymogram assays confirmed its cellulase activity using CMC as the substrate.

从腐解木材中分离到8属45株真菌。其中鉴定出27株纤维素降解菌。木霉属、青霉菌属和平革菌属（分别为6株、2株和4株）纤维素酶产量最高。菌株TW-25、TW-28和TW-33的CMCase（5.4 ~ 6.5 U/mL）、FPase（3.2 ~ 3.8 U/mL）、pNPCase（2.6 ~ 3.0 U/mL）和pNPGase（3.5 ~ 4.0 U/mL）的酶活性均较好，被选为进一步研究的菌株。内部转录间隔区（ITS）测序结合表型特征，鉴定菌株为Trichoderma sp. TW-25、Trichoderma sp. TW-28和Penicillium sp. TW-33。原生质体释放量最大的是木霉sp. TW-25(3.6 × 10 26原生质体/mL)，其次是木霉sp. TW-28（3.0 × 10 26原生质体/mL）和青霉sp. TW-33（2.8 × 10 26原生质体/mL）。融合频率2.8 ×10 ⁻³ 为TW-25 × TW-28, 2.0×10 ⁻³ 为TW-25 × TW-33,和1.8 ×10 ⁻³ 为TW-28 × TW-33。共有13个菌落来自TW-25 × TW-28, 18个菌落来自属间融合（10个来自TW-25 × TW-33, 8个来自TW-28 × TW-33）。融合体TWF1/1、TWF1/6和TWF2/5的纤维素酶活性与TW-25相同，融合体TWF1/3和TWF3/2与TW-28相同，而融合体均不具有TW-33的纤维素酶活性。融合体的DNA含量（3.25 ~ 3.65 µg/mg干重）与亲本菌株不同，且纤维素酶活性普遍较高。其中，TWF1/10酶活性最高，分别产生CMCase、FPase、pNPCase和pNPGase(10.5、6.5、5.8和7.5 U/mL)，其次是TWF1/13、TWF2/8、TWF2/10和TWF2/8。利用4条RAPD引物和3条ISSR引物分析TWF1/10、TWF1/13、TWF2/8、TWF2/10和TWF2/8的DNA带型，除ISSR-3与融合体TWF1/10和TWF1/13存在差异外，其余均与亲本菌株存在差异。这些变化强调了种间和属间原生质体融合的有效性。对杂交菌株TWF1/10的上清液进行浓缩和超滤纯化，以CMC为底物，通过SDS-PAGE和酶谱分析证实其纤维素酶活性。

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引用次数: 0

Study on bio-imprinted Aspergillus niger lipase cross-linked aggregates and catalytic synthesis of Vitamin E succinate 生物印迹黑曲霉脂肪酶交联聚集体及其催化合成维生素E琥珀酸酯的研究。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-10-23 DOI: 10.1016/j.enzmictec.2025.110766

Junqing Qian, Zhengze Yang, Aomei Huang, Qian Li, Hui Guo

To enhance the esterification and stability of lipase in organic solvents, the bio-imprinted Aspergillus niger lipase combined with cross-linked aggregate immobilization was investigated. The bio-imprinted lipase cross-linked aggregates were applied to the catalytic esterification for the synthesis of Vitamin E succinate in N, N-dimethylformamide (DMF) solution. Lauric acid, serving as a succinic acid analogue, was selected as the bio-imprinting molecule, 0.10 g lauric acid was added to 36 mL of 2.10 mg/mL lipase solution, imprinting 40 mins at pH 8.0, the immobilization yield achieved 91.5 % with cross-linked aggregates by glutaraldehyde. The catalytic activity of the bio-imprinted lipase cross-linked aggregates was significantly enhanced, achieving an esterification yield of 87.4 ± 0.43 % for Vitamin E succinate. Moreover, the bio-imprinted lipase cross-linked aggregates maintained their catalytic activity over five consecutive reaction cycles in DMF. Fluorescence spectroscopy analysis revealed that bio-imprinting promoted the exposure of the lipase active sites, which corresponded with the observed increase in esterification activity. In addition, the mechanism of the substrate analogue-imprinted lipase was characterized. This study provides a theoretical foundation for improving the catalytic esterification performance of lipase as well as a process basis for the enzymatic synthesis of Vitamin E succinate.

为了提高脂肪酶在有机溶剂中的酯化和稳定性，研究了生物印迹黑曲霉脂肪酶与交联聚集体固定化的结合。应用生物印迹脂肪酶交联聚集体在N， N-二甲基甲酰胺（DMF）溶液中催化酯化合成维生素E琥珀酸酯。选择琥珀酸类似物月桂酸作为生物印迹分子，将0.10 月桂酸加入到36 mL 2.10 mg/mL脂肪酶溶液中，在pH 8.0条件下印迹40 min，戊二醛交联聚体的固定化率达到91.5 %。生物印迹脂肪酶交联聚集体的催化活性显著增强，维生素E琥珀酸酯的酯化率为87.4 ± 0.43 %。此外，生物印迹脂肪酶交联聚集体在DMF中连续五个反应循环中保持其催化活性。荧光光谱分析表明，生物印迹促进了脂肪酶活性位点的暴露，这与观察到的酯化活性增加相对应。此外，还对底物类似物印迹脂肪酶的作用机理进行了表征。本研究为提高脂肪酶的催化酯化性能提供了理论基础，也为酶促合成维生素E琥珀酸酯提供了工艺基础。

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引用次数: 0