Discovering a novel d-xylonate-responsive promoter: the PyjhI-driven genetic switch towards better 1,2,4-butanetriol production
The capability of Escherichia coli to catabolize d-xylonate is a crucial component for building and optimizing the Dahms pathway. It relies on the inherent dehydratase and keto-acid aldolase activities of E. coli. Although the biochemical characteristics of these enzymes are known, their inherent expression regulation remains unclear. This knowledge is vital for the optimization of d-xylonate assimilation, especially in addressing the problem of d-xylonate accumulation, which hampers both cell growth and target product formation. In this report, molecular biology techniques and synthetic biology tools were combined to build a simple genetic switch controller for d-xylonate. First, quantitative and relative expression analysis of the gene clusters involved in d-xylonate catabolism were performed, revealing two d-xylonate-inducible operons, yagEF and yjhIHG. The 5′-flanking DNA sequence of these operons were then subjected to reporter gene assays which showed PyjhI to have low background activity and wide response range to d-xylonate. A PyjhI-driven synthetic genetic switch was then constructed containing feedback control to autoregulate d-xylonate accumulation and to activate the expression of the genes for 1,2,4-butanetriol (BTO) production. The genetic switch effectively reduced d-xylonate accumulation, which led to 31% BTO molar yield, the highest for direct microbial fermentation systems thus far. This genetic switch can be further modified and employed in the production of other compounds from d-xylose through the xylose oxidative pathway.
Keywordsd-Xylonate Dahms pathway yjhI promoter Genetic switch 1,2,4-Butanetriol
This work was supported by Korea Research Fellowship Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2015H1D3A1062172 and 2016R1C1B1013252) and by the Ministry of Education (2018R1D1A1B07043993).
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Conflict of interest
The authors declare that they have no conflict of interest.
Compliance with ethical standards
This article does not contain any studies with human participants performed by any of the authors.
- Brouns SJJ, Walther J, Snijders APL, van de Werken HJG, Willemen HLDM, Worm P, de Vos MGJ, Andersson A, Lundgren M, Mazon HFM, van den Heuvel RHH, Nilsson P, Salmon L, de Vos WM, Wright PC, Bernander R, van der Oost J (2006) Identification of the missing links in prokaryotic pentose oxidation pathways: evidence for enzyme recruitment. J Biol Chem 281:27378–27388. https://doi.org/10.1074/jbc.M605549200 CrossRefGoogle Scholar
- Cabulong RB, Lee W-K, Bañares AB, Ramos KRM, Nisola GM, Valdehuesa KNG, Chung W-J (2018a) Engineering Escherichia coli for glycolic acid production from D-xylose through the Dahms pathway and glyoxylate bypass. Appl Microbiol Biotechnol 102:2179–2189. https://doi.org/10.1007/s00253-018-8744-8 CrossRefGoogle Scholar
- Cabulong RB, Valdehuesa KNG, Bañares AB, Ramos KRM, Nisola GM, Lee W-K, Chung W-J (2018b) Improved cell growth and biosynthesis of glycolic acid by overexpression of membrane-bound pyridine nucleotide transhydrogenase. J Ind Microbiol Biotechnol 46:159–169. https://doi.org/10.1007/s10295-018-2117-2 CrossRefGoogle Scholar
- Gao Q, Wang X, Hu S, Xu N, Jiang M, Ma C, Yang J, Xu S, Chen K, Ouyang P (2019) High-yield production of D-1,2,4-butanetriol from lignocellulose-derived xylose by using a synthetic enzyme cascade in a cell-free system. J Biotechnol 292:76–83. https://doi.org/10.1016/j.jbiotec.2019.01.004 CrossRefGoogle Scholar
- Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
- Radek A, Krumbach K, Gätgens J, Wendisch VF, Wiechert W, Bott M, Noack S, Marienhagen J (2014) Engineering of Corynebacterium glutamicum for minimized carbon loss during utilization of D-xylose containing substrates. J Biotechnol 192:156–160. https://doi.org/10.1016/j.jbiotec.2014.09.026 CrossRefGoogle Scholar
- Solovyev VV, Salamov A (2011) Automatic annotation of microbial genomes and metagenomic sequences. In: Li RW (ed) Metagenomics and its applications in agriculture, biomedicine and environmental studies. Nova Science Publishers, New York, pp 61–78Google Scholar
- Valdehuesa KNG, Lee W-K, Ramos KRM, Cabulong RB, Choi J, Liu H, Nisola GM, Chung W-J (2015) Identification of aldehyde reductase catalyzing the terminal step for conversion of xylose to butanetriol in engineered Escherichia coli. Bioprocess Biosyst Eng 38:1761–1772. https://doi.org/10.1007/s00449-015-1417-4 CrossRefGoogle Scholar
- Valdehuesa KNG, Ramos KRM, Nisola GM, Bañares AB, Cabulong RB, Lee W-K, Liu H, Chung W-J (2018) Everyone loves an underdog: metabolic engineering of the xylose oxidative pathway in recombinant microorganisms. Appl Microbiol Biotechnol 102:7703–7716. https://doi.org/10.1007/s00253-018-9186-z CrossRefGoogle Scholar
- Weimberg R (1961) Pentose oxidation by Pseudomonas fragi. J Biol Chem 236:629–635Google Scholar