Beneficial base substitutions in Escherichia coli fucO gene for enhancement of glycolic acid production.

Autor: Nemoto M; Faculty of Agriculture, Department of Biological Chemistry and Food Science, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan., Muranushi W; Faculty of Agriculture, Department of Biological Chemistry and Food Science, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan., Shuting C; Faculty of Agriculture, Department of Biological Chemistry and Food Science, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan., Saito Y; Faculty of Agriculture, Department of Biological Chemistry and Food Science, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan., Sugimori D; Materials Science Course, Faculty of Symbiotic Systems Science and Technology, Fukushima University, 1 Kanayagawa, Fukushima 960-1296, Japan., Yamada M; Faculty of Agriculture, Department of Biological Chemistry and Food Science, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan; Agri-Innovation Center, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan. Electronic address: myamada@iwate-u.ac.jp.
Jazyk: angličtina
Zdroj: Journal of bioscience and bioengineering [J Biosci Bioeng] 2024 Oct; Vol. 138 (4), pp. 301-307. Date of Electronic Publication: 2024 Jul 29.
DOI: 10.1016/j.jbiosc.2024.06.007
Abstrakt: Microbial production of glycolic acid (GA) from ethylene glycol is extensively used in a variety of industries because ethylene glycol is not only an inexpensive raw material but also the main component of industrial wastes. In this study, we produced GA from ethylene glycol using Escherichia coli overexpressing the endogenous 1,2-propanediol oxidoreductase (fucO) and lactaldehyde dehydrogenase (aldA) genes. To increase GA productivity, we screened a random mutant library generated using an error-prone polymerase chain reaction of fucO and obtained FucO mutants MF2-9 and MF6-9 with enhanced GA production in Lysogeny Broth medium containing 800 mM ethylene glycol. MF2-9 contained three amino acid substitutions (D23E, E222K, and G363S) and two synonymous mutations (coding DNA [c.] 93G > A and c.1131T > C) in fucO. MF6-9 contained one amino acid substitution (L377H) in FucO. An amino acid substitution (L377H) and a single synonymous mutation (c.1131T > C) in fucO contributed to the enhancement in GA production. Notably, cell lysates from E. coli harboring a synonymous mutation (c.1131T > C) or amino acid substitution (L377H) in fucO showed that only AldA activity was 1.3-fold higher than that of the cell lysate from E. coli harboring the wild-type fucO. We confirmed that c.1131T > C and L377H mutations increased aldA expression in E. coli. Analysis of mRNA levels and simulation of mRNA stabilization indicated that base substitutions at positions c.1130T, which corresponds to L377H amino acid substitution, and c.1131T increased aldA expression due to partial destabilization of the mRNA. These findings will be useful for the large-scale microbial production of GA from industrial waste.
(Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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