USE OF PHASINS TO ENHANCE HETEROLOGOUS ENZYME PRODUCTION

MEZZINA MARIELA PAULA; PETTINARI MARÍA JULIA; ALVAREZ DANIELA SOLEDAD

Congreso; Congreso SAIB-SAMIGE 2020; 2020

SAMIGE

Phasin PhaP from Azotobacter sp. FA-8 is a polyhydroxyalkanoate (PHA) granule-associated protein that not only plays an important structural role in polymer accumulation, but has also been shown to have an unexpected protective effect in non-PHA synthesizing Escherichia coli. This protective effect was observed under both normal and stress conditions, resulting in increased growth and higher resistance to both heat shock, superoxide stress by paraquat, and ionic and neutral osmotic stress. Moreover, PhaP has both in vitro and in vivo chaperone activity, and was shown to enhance bacterial fitness in the presence of biofuels, such as ethanol and butanol, and to other chemicals, such as 1,3-propanediol. The effect of PhaP was also studied in solvent-producing strains, in which PhaP was observed to increase ethanol and 1,3-propanediol titers.In view of these findings, we studied the ability of PhaP to improve heterologous enzyme production in recombinant E. coli. Two β-galactosidases from Bifidobacterium breve DSM 20213 were overexpressed in a lacZ mutant strain, with or without a PhaP-expressing plasmid. The presence of PhaP was shown to significantly increase β-galactosidase activity of both enzymes. These results could be attributed to its chaperone-like properties and expand the possible applications for this protein.In order to test whether the properties described for PhaP are common among this group of proteins, we employed the β-galactosidase co-expression experiment with phasins belonging to different bacteria. Although phasins do not constitute a highly conserved group of proteins, several protein motifs have been defined and four types of phasin families have been distinguished based on sequence similarity. We selected six phasins for our essay, belonging to three different phasin families: PhbP from Pseudomonas extremaustralis, PhaP1 from Cupriavidus necator, PhaF and PhaI from P. putida, PhaP from Azotobacter chroococcum and PhaP from Bacillus megaterium. We conducted an in silico structural comparison between these proteins and observed that, despite their low sequence similarity, they share some common features, such as a preponderant α-helix composition and the presence of amphipathic helices. We have cloned the selected phasins into the expression vectors chosen for the β-galactosidase assay. When the assay was tested with PhbP from P. extremaustralis, expression of this phasin did not result in significant differences in enzyme activity, even though PhbP had been shown to exhibit in vitro chaperone activity in experiments performed previously. Further experiments are needed to find out whether these contrasting results are due to the differences between the phasins or to expression problems. Future tests involving the other phasins selected are expected to shed light on this, and to help determine both the potential of phasins in recombinant protein expression and which phasin structures are involved in this phenomenon.