Protein engineering of carotenoid cleavage dioxygenases to optimize β-ionone biosynthesis in yeast cell factories.

Autor: Werner N; Department of Chemical and Bioprocess Engineering, School of Engineering, Pontificia Universidad Católica de Chile, Avenida Vicuña Mackenna 4860, Santiago, Chile. Electronic address: niwerner@uc.cl., Ramirez-Sarmiento CA; Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Católica de Chile, Avenida Vicuña Mackenna 4860, Santiago, Chile. Electronic address: cesar.ramirez@uc.cl., Agosin E; Department of Chemical and Bioprocess Engineering, School of Engineering, Pontificia Universidad Católica de Chile, Avenida Vicuña Mackenna 4860, Santiago, Chile. Electronic address: agosin@ing.puc.cl.
Jazyk: angličtina
Zdroj: Food chemistry [Food Chem] 2019 Nov 30; Vol. 299, pp. 125089. Date of Electronic Publication: 2019 Jul 08.
DOI: 10.1016/j.foodchem.2019.125089
Abstrakt: Synthesis of β-ionone in recombinant Saccharomyces cerevisiae is limited by the efficiency of Carotenoid Cleavage Dioxygenases (CCD), membrane-tethered enzymes catalyzing the last step in the pathway. We performed in silico design and membrane affinity analysis, focused on single-point mutations of PhCCD1 to improve membrane anchoring. The resulting constructs were tested in a β-carotene hyper-producing strain by comparing colony pigmentation against colonies transformed with native PhCCD1 and further analyzed by β-ionone quantification via RP-HPLC. Two single-point mutants increased β-ionone yields almost 3-fold when compared to native PhCCD1. We also aimed to improve substrate accessibility of PhCCD1 through the amino-terminal addition of membrane destination peptides directed towards the endoplasmic reticulum or plasma membrane. Yeast strains expressing peptide-PhCCD1 constructs showed β-ionone yields up to 4-fold higher than the strain carrying the native enzyme. Our results demonstrate that protein engineering of CCDs significantly increases the yield of β-ionone synthesized by metabolically engineered yeast.
(Copyright © 2019 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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