Galacto-oligosaccharide hydrolysis by genetically-engineered alpha-galactosidase-producing Pseudomonas chlororaphis strains

Various Pseudomonas chlororaphis strains have been shown to produce rhamnolipid, poly(hydroxyalkanoate), and antifungal compounds for plants. Ability to metabolize galacto-oligosaccharides would allow P. chlororaphis to use soy molasses as a low-cost fermentation feedstock. In this study, genetically engineered P. chlororaphis strains expressing a Streptomyces coelicolor α-galactosidase (α-gal) were constructed. In recombinant P. chlororaphis [chr::AG], the α-gal was integrated into the chromosome. P. chlororaphis [pBS-dAG], however, contains a truncated α-gal (coding for the N-terminal catalytic domain of the enzyme) on an expression vector. Real-time RT-qPCR showed 1,438-fold higher α-gal gene expression in [pBS-dAG] than [chr::AG]. In agreement with qPCR study, the results of an enzyme assay using p-nitrophenyl-α-galactopyranoside (p-NP-α-Gal) as a chromogenic substrate also showed that the cell extracts of [pBS-dAG] contained ca. 8-times higher p-NP-α-Gal-hydrolyzing activity than that of [chr::AG]. The cell extracts of [pBS-dAG] were also demonstrated to hydrolyze raffinose (32.7 ± 4.1% of the initial amount remained in the reaction mixture) > melibiose (65.4 ± 7.9%) > stachyose (72.8 ± 11.9%). The incubation of an EDTA-permeabilized (1.5 μM, 28 °C, 200 rpm shaking, 20 min) P. chlororaphis [pBS-dAG] whole-cell preparation with 0.5% (w/v) raffinose in a Medium E* for 7 days resulted in the reduction of the carbon source to 0.14% (w/v), or 28% relative to the initially added amount, and the biomass reached a value of 0.46 g CDW (cell dry weight)/l. In contrast, EDTA-permeabilized wild-type P. chlororaphis did not hydrolyze the 0.5% (w/v) raffinose in the medium, and the final biomass yield was 0.26 g CDW/l.