Indigo is an important biobased colorant used in textile dyeing and as a precursor for blue food colorants; however, its microbial production is often limited by cytotoxicity and intracellular accumulation of insoluble products. Here, we established a systems-level host engineering framework in Escherichia coli to improve indigo biosynthesis. CRISPRi screening of cell envelope-associated genes identified targets including sulA, ftsZ, fabF, and pgi. Mechanistic analyses showed that modulation of cell division and central carbon metabolism alleviates envelope stress and enhances reducing power for indigo formation, while electron microscopy confirmed reduced cellular perturbation caused by intracellular indigo accumulation. A second CRISPRi library targeting transcription factors revealed coordinated regulation of BolA, ArcA, and RpoN to balance metabolism and stress responses. Integration of targets enabled 9.38 g/L indigo production in a 5 L fed-batch bioreactor, a 2.55-fold improvement over the parental strain. This work provides a promising engineering strategy for microbial indigo production.
Heng Hu, Meng-Ying Jiang, Yan Zheng et al.· Journal of Agricultural and...· 0 citations
Zearalenone (ZEN) lactonases are promising biocatalysts for ZEN detoxification, yet the catalytic mechanism underlying ZEN hydrolysis remains poorly understood. Here, we combined structural analysis, quantum-mechanical (QM) calculations, and molecular dynamics (MD) simulations to elucidate the catalytic mechanism and guide enzyme engineering. QM and MD analyses identified a near-attack conformation of the catalytic His245 as essential for proton transfer. Crystal structure analysis revealed that mutations within the active pocket enhanced the local hydrophobic microenvironment, thereby optimizing the reactive conformation through an improved substrate positioning and catalytic residue alignment. Engineering the hydrophobic microenvironment significantly enhanced the activity of ZENM toward multiple substrates. Transfer of the engineered region to another ZEN lactonase, ZHD101, also significantly improved the hydrolytic activity, supporting the potential general applicability of this strategy. Hydrophobic microenvironment engineering might represent a promising strategy for modulating proton transfer and provide a potential framework for improving the activity of ZEN lactonases for food and feed detoxification.
Binbin Ouyang, Zhao Huang, Chenshuo Song et al.· Journal of Agricultural and...· 0 citations
Ginsenoside Rh2, a potential anticancer agent originally isolated from the medicinal plant Panax ginseng, has low natural abundance and is difficult to extract, making biosynthetic production a promising alternative. UDP-glycosyltransferases (UGTs) that specifically catalyze protopanaxadiol (PPD) glycosylation to produce Rh2 have been widely identified in plants and microorganisms. In this study, we report that Barbarea vulgaris UGT73C21 effectively catalyzes this reaction. The enzyme was expressed in Escherichia coli and purified to electrophoretic homogeneity via Ni2⁺-affinity chromatography. It exhibited optimal activity at pH 8.0 (100 mM HEPES) and 45 °C. Mn2⁺, Mg2⁺, and Ca2⁺ significantly enhanced enzyme activity, whereas other tested metal ions reduced it. The kinetic parameters Km, Vmax, kcat, and kcat/Km were determined as 231.92 μM, 5.68 μM min-1, 0.053 s-1, and 228 M-1 s-1, respectively. In a reaction containing 0.2 mg/mL UGT73C21, 1 mM PPD, and 5 mM UDP-Glucose in HEPES buffer (pH 8.0), 0.55 mM Rh2 was produced within 1 h, corresponding to a 55% conversion rate. These results demonstrate that B. vulgaris UGT73C21 is an efficient biocatalyst for ginsenoside Rh2 biosynthesis.
Fan Xu, Yuting Liao, Yulei Zhang et al.· Enzyme and Microbial Technol...· 0 citations
α-Bisabolol, a natural sesquiterpene alcohol with notable physiological activities, exhibits broad application prospects in the pharmaceutical, cosmetic, and flavor industries. Traditional plant extraction suffers from low efficiency and resource scarcity, while chemical synthesis faces stereoisomerism and environmental issues. In contrast, microbial synthesis overcomes these limitations by utilizing renewable agricultural feedstocks to address plant resource scarcity and enabling highly stereoselective biosynthesis to produce the more bioactive (-)-α-bisabolol. This review summarizes the physiological activities of α-bisabolol, describes its microbial biosynthetic pathway, and focuses on the key enzyme (-)-α-bisabolol synthase. Unlike previous reviews focused on pharmacological effects or chemical synthesis, this review uniquely integrates recent metabolic engineering strategies for enhancing (-)-α-bisabolol production, including chassis cell development, metabolic flux regulation, competitive pathway knockout, cofactors and global regulatory factors optimization, and fermentation scale-up. Finally, current challenges and future directions are discussed, with an emphasis on green biomanufacturing through transporter engineering, enzyme evolution, and subcellular compartmentalization.
Junsong Xiao, Zeyu Li, Jiali Zhang et al.· Journal of Agricultural and...· 0 citations
2'-Fucosyllactose (2'-FL), the most abundant human milk oligosaccharide (HMO), has attracted considerable interest for its prebiotic and immunomodulatory functions, with broad applications in infant nutrition. In this study, we report the development of a high-yield, genome-integrated 2'-FL-producing strain based on Escherichia coli MG1655 through systematic modular optimization. Starting from a single-copy BKHT strain (MGC06), we first optimized the copy number of the α-1,2-fucosyltransferase (α-1,2-FT) gene BKHT. Subsequently, the GDP-L-fucose supply was enhanced through coordinated genomic integration of the gene clusters cpsG-cpsB and gmd-fcl, while the multidrug efflux transporter gene mdfA was integrated to improve product export and strain robustness. BKHT copy number was then re-evaluated in the optimized background, with four copies yielding the highest production. The final engineered strain, harboring all genetic modifications stably integrated into the chromosome, produced 17.18 g/L 2'-FL in shake-flask culture. In fed-batch fermentation using a 5-L bioreactor, this strain achieved a titer of 154.12 g/L after 60 h, with a productivity of 2.57 g/L/h. Notably, throughout the entire fermentation process, no antibiotics or inducers were supplemented, underscoring the genetic stability and regulatory compliance of this plasmid-free system. To our knowledge, this represents the highest 2'-FL titer reported to date, positioning our engineered strain as a promising candidate for commercial 2'-FL production.
Rou-Lin Chen, Longhao Yang, Hao Wang et al.· Enzyme and Microbial Technol...· 0 citations
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