Construct Phenylethanoid Glycosides Harnessing Biosynthetic Networks, Protein Engineering and One‐Pot Multienzyme Cascades

Angewandte Chemie, Год журнала: 2024, Номер 136(25)

Опубликована: Апрель 15, 2024

Abstract Phenylethanoid glycosides (PhGs) exhibit a multitude of structural variations linked to diverse pharmacological activities. Assembling various PhGs via multienzyme cascades represents concise strategy over traditional synthetic methods. However, the challenge lies in identifying comprehensive set catalytic enzymes. This study explores biosynthetic PhG reconstruction from natural precursors, aiming replicate and amplify their diversity. We discovered 12 enzymes, including four novel 6′‐OH glycosyltransferases three new polyphenol oxidases, revealing intricate network biosynthesis. Subsequently, crystal structure CmGT3 (2.62 Å) was obtained, guiding identification conserved residue 144# as critical determinant for sugar donor specificity. Engineering this (FsGT61, CmGT3, FsGT6) altered recognition. Finally, one‐pot cascade established, where combined action acyltransferases boosted conversion rates by up 12.6‐fold. facilitated 26 with ranging 5–100 %, 20 additional detectable mass spectrometry. extra glycosyl hydroxyl modules demonstrated notable liver cell protection. work not only provides tools biosynthesis, but also serves proof‐of‐concept cell‐free enzymatic construction products.

Язык: Английский

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A Flavone-5-O-glycosyltransferase Obtained through Domain Shuffling and Its Application in the Bioproduction of Luteolin-5-O-glucoside

ACS Sustainable Chemistry & Engineering, Год журнала: 2024, Номер 12(42), С. 15418 - 15431

Опубликована: Окт. 7, 2024

Luteolin-5-O-glucoside is a rare luteolin glycoside compound with enormous potential for application. In order to promote sustainability and circular economy, environmentally friendly catalysts catalytic processes are in demand produce luteolin-5-O-glucoside. Using glycosyltransferase transformation an efficient method synthesizing glycosylated flavonoids. However, most reported glycosyltransferases have not been able catalyze glycosylation on the 5-OH site, sustainable production of luteolin-5-O-glucoside has established. this paper, DoUGT71A15 was cloned from Dichanthelium oligosanthes. The N-terminal fragment ZmUGT707A8 (Met1–Met93) inserted into N-terminus by domain shuffling, biological catalyst ZmDo obtained. could convert Km kcat/Km values 0.13 mM 1355.8 s–1·M–1, respectively. Then, mechanism region selectivity studied using bioinformatics methods, key site E89 identified through alanine scanning. Finally, strain BSZ containing UDPG circulatory system, which use cellobiose as substrate recycle UDP synthesize UDPG, used luteolin-5-O-glucoside, yield 2253 mg/L molar conversion rate 95.8%. This research obtained novel flavone-5-OH produced bioconversion, provided possibility application

Язык: Английский

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Computational-guided discovery of UDP-glycosyltransferases for lauryl glucoside production using engineered E. coli

Bioresources and Bioprocessing, Год журнала: 2024, Номер 11(1)

Опубликована: Окт. 26, 2024

Abstract Defining suitable enzymes for reaction steps in novel synthetic pathways is crucial developing microbial cell factories non-natural products. Here, we developed a computational workflow to identify C12 alcohol-active UDP-glycosyltransferases. The involved three steps: (1) assembling initial candidates of putative UDP-glycosyltransferases, (2) refining selection by examining conserved regions, and (3) 3D structure prediction molecular docking. Genomic sequences from Candida , Pichia Rhizopus Thermotoga known lauryl glucoside synthesis via whole-cell biocatalysis, were screened. Out 240 predicted glycosyltransferases, 8 annotated as glycosyltransferases selected after filtering out those with signal peptides identifying UDP-glycosyltransferase regions. These proteins underwent docking 1-dodecanol. RO3G, candidate delemar RA 99–880 relatively high ChemPLP fitness score, was expressed Escherichia coli BL21 (DE3). It further characterized using feeding experiment Results confirmed that the RO3G-expressing strain could convert 1-dodecanol glucoside, quantified HPLC identified targeted LC-MS. Monitoring growth fermentation profiles engineered revealed RO3G expression did not affect growth. Interestingly, acetate, major product, reduced compared GFP-expressing strain, suggesting redirection flux acetate other pathways. Overall, this work presents successful discovering activity toward production. Graphical abstract

Язык: Английский

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Genome-wide study of UDP-glycosyltransferases gene family in Cannabis sativa

3 Biotech, Год журнала: 2024, Номер 14(8)

Опубликована: Июль 22, 2024

Язык: Английский

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Efficient and green production of flavone-5-O-glycosides by glycosyltransferases in Escherichia coli

International Journal of Biological Macromolecules, Год журнала: 2024, Номер 277, С. 134477 - 134477

Опубликована: Авг. 6, 2024

Язык: Английский

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