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

• Published in: Bioresources and bioprocessing Vol. 11; no. 1; pp. 103 - 13
• Main Authors: Promubon, Kasimaporn, Tathiya, Kritsada, Panya, Aussara, Pathom-Aree, Wasu, Sattayawat, Pachara
• Format: Journal Article
• Language: English
• Published: Singapore Springer Nature Singapore 26.10.2024; Springer Nature B.V; SpringerOpen
• Subjects: 1-Dodecanol; Acetic acid; Biochemical Engineering; Catalysis; Chemical synthesis; Chemistry; Chemistry and Materials Science; Computational method; Computer applications; Dodecanol; E coli; Engineered E. coli; Environmental Engineering/Biotechnology; Enzymes; Fermentation; Fungi; Glucosides; Glycosyltransferase; Industrial and Production Engineering; Industrial engineering; Lauryl glucoside; Microorganisms; Molecular docking; Molecular structure; Natural products; Novel biosynthetic pathway; Peptides; Predictions; Signal peptides; UDP-glycosyltransferase; Workflow; Engineered; Novel biosynthetic pathway; Lauryl glucoside; UDP-glycosyltransferase; Computational method; 1-Dodecanol; Genome mining; Engineered E. coli
• ISSN: 2197-4365; 2197-4365
• DOI: 10.1186/s40643-024-00820-1

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