APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY Discovering a novel D-xylonate-responsive promoter: the P yjhI -driven genetic switch towards better 1,2,4-butanetriol production Angelo B. Bañares 1 & Kris Niño G. Valdehuesa 1 & Kristine Rose M. Ramos 1 & Grace M. Nisola 1 & Won-Keun Lee 2 & Wook-Jin Chung 1 Received: 11 July 2019 /Revised: 11 July 2019 /Accepted: 31 July 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019 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 P yjhI to have low background activity and wide response range to D-xylonate. A P yjhI -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. Keywords D-Xylonate . Dahms pathway . yjhI promoter . Genetic switch . 1,2,4-Butanetriol Introduction The Dahms and Weimberg pathways have been gaining atten- tion for D-xylose metabolism during the recent years. Unlike the traditional D-xylose isomerase or reductase pathways, the Dahms and Weimberg pathways begin with the oxidation of D-xylose to D-xylonate. Hence, both pathways are collectively called the D-xylose oxidative pathways (XOP) (Valdehuesa et al. 2018). D-Xylonate then undergoes two to four more reactions to form pyruvate or 2-ketoglutarate (Dahms 1974; Weimberg 1961). The XOP has been found in strains belong- ing to the Archaea and Bacteria domains (Brouns et al. 2006; Stephens et al. 2007) and was engineered in several industrial strains (Cabulong et al. 2018a; Radek et al. 2014; Salusjärvi et al. 2017). Interestingly, recruiting the Dahms pathway in Escherichia coli only requires the expression of a non-native xylose dehydrogenase (EC 1.1.1.179) to catalyze the oxidation of D-xylose to form D-xylonate, which E. coli apparently has the capability to assimilate (Liu et al. 2013). The inherent D-xylonate assimilative route in E. coli in- volves two steps: D-xylonate dehydration and a keto-acid al- dol cleavage reaction (Fig. 1a). These enzymes appear to be unique to the K-12 strain and its derivatives, with the respec- tive genes clustered in yag or yjh regions of the genome (Fig. 1b). From this pre-existing set of genes, the full XOP Angelo B. Bañares and Kris Niño G. Valdehuesa contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00253-019-10073-0) contains supplementary material, which is available to authorized users. * Won-Keun Lee wklee@mju.ac.kr * Wook-Jin Chung wjc0828@gmail.com 1 Department of Energy Science and Technology (DEST), Energy and Environment Fusion Technology Center (E2FTC), Myongji University, Myongji-ro 116, Cheoin-gu, Yongin 17058, Gyeonggi-do, Republic of Korea 2 Division of Bioscience and Bioinformatics, Myongji University, Myongji-ro 116, Cheoin-gu, Yongin 17058, Gyeonggi-do, Republic of Korea Applied Microbiology and Biotechnology https://doi.org/10.1007/s00253-019-10073-0