Mmsr-Mediated 3-Hydroxypropionic Acid-Inducible Gene Expression System of Pseudomonas Denitrificans

Ainala, S. K., Pusan National University
Somasundar, A., Noroo Holdings Co., Ltd
Park, S., Pusan National University

3-Hydroxypropionic acid (3-HP), an important platform chemical that boats a variety of industrial applications, can be biologically produced from glycerol by two enzymes consisting of coenzyme B12-dependent glycerol dehydratase involving dehydration of glycerol to 3-hydroxypropionaldehyde (3-HPA) and aldehyde dehydrogenase for oxidation of 3-HPA to 3-HP. To increase the yield of 3-HP production, it is essential to optimize this pathway through a gene expression system which can be inducible by 3-HP and control the expression of these two enzymes in high and balanced manner. Genome and transcriptome analyses showed that Pseudomonas denitrificans can natively synthesize coenzyme B12, an essential for activation of glycerol dehydratase and has a 3-HP inducible system for genes related to valine degradation, mmsA and hbdH-4, which encode methylmalonylsemialdehyde dehydrogenase and 3-hydroxyisobutyl dehydrogenase, respectively. As a result, it showed 3-HP functions as an inducer relying on the action of transcriptional regulatory protein, MmsR. The underlying mechanism of MmsR-mediated 3-HP inducible system was studied in vivo and in vitro. In the presence of 3-HP, MmsR regulated the transcription of mmsA and hbdH-4 genes by higher than 140-fold, indicating that two promoters controlling these genes, Pcm and Pc4, are strong for gene expression in P. denitrificans. The transcription activation was depleted when mmsR was deleted from the chromosome, but was recovered by complementation of mmsR from a plasmid. The mmsR was cloned in Escherichia coli and the recombinant MmsR was purified. The result exhibited MmsR was monomeric at the concentrations below 65 nM whereas oligomeric at higher concentrations. Electrophoretic mobility shift assay indicated that MmsR could bind to two adjacent operator sites located in the divergently overlapping promoter region present between mmsR and mmsA. The two operators, designated O1 and O2, contained sequences of dyad symmetry centered at position –58 and –9, respectively, upstream of the mmsA transcription start site. When both O1 and O2 were present, binding affinity of MmsR to the operator regions was higher than either O1 or O2 alone, the result suggests that the binding is cooperative. MmsR could bind to the operator regions in the absence of 3-HP, indicating that 3-HP is not compulsory for binding of MmsR to the operator regions; however, the transcription of downstream genes requires 3-HP as an inducer. This inducible system is novel and robust for expressing important enzymes in 3-HP production, excretion pathways and enhances the 3-HP titer in P. denitrificans.