A novel 3-hydroxypropionic acid-inducible promoter regulated by the LysR-type transcriptional activator protein MmsR of Pseudomonas denitrificans

Nam Hoai Nguyen,Sunghoon Park,Satish Kumar Ainala,Shengfang Zhou

doi:10.1038/s41598-019-41785-y

Abstract

MmsR (33.3 kDa) is a putative LysR-type transcriptional activator of Pseudomonas denitrificans. With the help of 3-hydroxypropionic acid (3-HP), an important platform chemical, MmsR positively regulates the expression of mmsA, which encodes methylmalonylsemialdehyde dehydrogenase, the enzyme involved in valine degradation. In the present study, the cellular function of MmsR and its binding to the regulatory DNA sequence of mmsA expression were investigated both in vivo and in vitro. Transcription of the mmsA was enhanced >140-fold in the presence of 3-HP. In the MmsR-responsive promoter region, two operators showing dyad symmetry, designated O1 and O2 and centered at the −79 and −28 positions, respectively, were present upstream of the mmsA transcription start site. An electrophoretic mobility shift assay indicated that MmsR binds to both operator sites for transcription activation, probably in cooperative manner. When either O1 or O2 or both regions were mutated, the inducibility by the MmsR-3-HP complex was significantly reduced or completely removed, indicating that both sites are required for transcription activation. A 3-HP sensor was developed by connecting the activation of MmsR to a green fluorescent readout. A more than 50-fold induction by 25 mM 3-HP was observed.

Highlights

3-Hydroxypropionic acid (3-HP) is a commercially important platform chemical that can be converted to acrylic acid, malonic acid, acrylamide, 1,3-propanediol and other valuable chemicals
L-val and 3-HIB were chosen because mmsA, whose transcription was regulated by MmsR, encodes methylmalonylsemialdehyde dehydrogenase, an enzyme involved in L-val degradation
P. denitrificans was cultured on minimal medium with and without each compound to be tested, and transcription of mmsA was measured by quantitative RT-PCR (Fig. 1b)

Summary

Introduction

3-Hydroxypropionic acid (3-HP) is a commercially important platform chemical that can be converted to acrylic acid, malonic acid, acrylamide, 1,3-propanediol and other valuable chemicals. We found that the transcription of the mmsA gene encoding the MmsA enzyme was up-regulated at a high level (>100-fold) but only in the presence of 3-HP8. This suggests that there exists positive regulation on the expression of the enzyme, where 3-HP acts as an inducer. 3-HP) and its high induction efficiency (over 100-fold), the promoter should be useful for the expression of pathway enzymes for 3-HP synthesis or development of 3-HP-responsive biosensors. This study aimed to elucidate the 3-HP-inducible gene regulation necessary for the expression of mmsA at the cellular and biochemical levels. This study should be useful for developing efficient 3-HP synthetic pathways and 3-HP sensors in many microorganisms

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Conclusion