Abstract
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.
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Funding
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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Bañares, A.B., Valdehuesa, K.N.G., Ramos, K.R.M. et al. Discovering a novel d-xylonate-responsive promoter: the PyjhI-driven genetic switch towards better 1,2,4-butanetriol production. Appl Microbiol Biotechnol 103, 8063–8074 (2019). https://doi.org/10.1007/s00253-019-10073-0
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DOI: https://doi.org/10.1007/s00253-019-10073-0