Characterisation of a 3-hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necator

Hanko, Erik K.R.; Minton, Nigel P.; Malys, Naglis

Abstract

3-hydroxypropionic acid (3-HP) is an important platform chemical used as a precursor for production of added-value compounds such as acrylic acid. Metabolically engineered yeast, Escherichia coli, cyanobacteria and other microorganisms have been developed for the biosynthesis of 3-HP. Attempts to overproduce this compound in recombinant Pseudomonas denitrificans revealed that 3-HP is consumed by this microorganism using the catabolic enzymes encoded by genes hpdH, hbdH and mmsA. 3-HP-inducible systems controlling the expression of these genes have been predicted in proteobacteria and actinobacteria. In this study, we identify and characterise 3-HP-inducible promoters and their corresponding LysR-type transcriptional regulators from Pseudomonas putida KT2440. A newly-developed modular reporter system proved possible to demonstrate that PpMmsR/PmmsA and PpHpdR/PhpdH are orthogonal and highly inducible by 3-HP in E. coli (12.3- and 23.3-fold, respectively) and Cupriavidus necator (51.5- and 516.6-fold, respectively). Bioinformatics and mutagenesis analyses revealed a conserved 40-nucleotide sequence in the hpdH promoter, which plays a key role in HpdR-mediated transcription activation. We investigate the kinetics and dynamics of the PpHpdR/PhpdH switchable system in response to 3-HP and show that it is also induced by both enantiomers of 3-hydroxybutyrate. These findings pave the way for use of the 3-HP-inducible system in synthetic biology and biotechnology applications.

Citation

Hanko, E. K., Minton, N. P., & Malys, N. (in press). Characterisation of a 3-hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necator. Scientific Reports, 7, Article 1724. https://doi.org/10.1038/s41598-017-01850-w

• Journal Article Type: Article
• Acceptance Date: Apr 13, 2017
• Online Publication Date: May 11, 2017
• Deposit Date: May 18, 2017
• Publicly Available Date: May 18, 2017
• Journal: Scientific Reports
• Electronic ISSN: 2045-2322
• Publisher: Nature Publishing Group
• Peer Reviewed: Peer Reviewed
• Volume: 7
• Article Number: 1724
• DOI: https://doi.org/10.1038/s41598-017-01850-w
• Public URL: https://nottingham-repository.worktribe.com/output/860256
• Publisher URL: https://www.nature.com/articles/s41598-017-01850-w
• Contract Date: May 18, 2017