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

Authors: Solaiman, Daniel; Ashby, Richard - Rick; ANEJA, KAWALPREET - Former ARS Employee; Cross, Nicole; Liu, Yanhong

Submitted to: Biocatalysis and Agricultural Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/19/2017
Publication Date: 12/21/2017
Citation: Solaiman, D., Ashby, R.D., Aneja, K., Crocker, N.V., Liu, Y. 2017. Galacto-oligosaccharide hydrolysis by genetically-engineered alpha-galactosidase-producing Pseudomonas chlororaphis strains. Biocatalysis and Agricultural Biotechnology. 12:213-218. https://doi.org/10.1016/j.bcab.2017.12.008.
DOI: https://doi.org/10.1016/j.bcab.2017.12.008

Interpretive Summary: Pseudomonas chlororaphis (Pc) is a microorganism that can produce useful bioproducts such as surfactant, bioplastic, and antifungal chemicals. Soy molasses (SM) on the other hand is an inexpensive industrial byproduct containing mostly inedible sugars. In the presence of an enzyme called alpha-galactosidase (AG), the sugars in SM can be broken down into components for use by bacteria to grow and make bioproducts. In this study, a gene (a-gal) that codes for AG was introduced into Pc. A systematic study showed that the gene was properly used in Pc to make active enzyme that was further demonstrated to degrade the types of sugars found in SM. More specifically, we demonstrated that the genetically engineered (GE-) Pc could break down raffinose (one type of the sugars found in SM) to support cell growth. Further improvement of this GE-Pc should make possible the use of the low-cost SM to economically produce those useful bioproducts.

Technical Abstract: Various Pseudomonas chlororaphis strains have been shown to produce rhamnolipid (a biosurfactant), poly(hydroxyalkanoate) (PHA; a biopolymer), and/or antifungal compounds for plants. An ability to metabolize galacto-oligosaccharides in soy molasses would allow P. chlororaphis to use the byproduct as a low-cost fermentation feedstock. In this study, two (2) genetically engineered P. chlororaphis strains expressing a Streptomyces coelicolor a-galactosidase (a-gal) were constructed. In recombinant P. chlororaphis [chr::AG], the a-gal gene was integrated into the chromosome. P. chlororaphis [pBS-dAG], on the other hand, contains a truncated a-gal (coding for the N-terminal catalytic domain of the enzyme) on an expression vector. Real-time RT-qPCR results showed 1,438-fold higher a-gal gene expression in [pBS-dAG] in comparison to [chr::AG]. In agreement with the qPCR study, the results of an in vitro enzyme assay using p-nitrophenyl-a-galactopyranoside (p-NP-a-Gal) as a chromogenic substrate also showed that the cell extracts of [pBS-dAG] contained ca. 8-times higher p-NP-a-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 (65.4 ± 7.9%). The incubation of an EDTA-permeabilized (1.5 uM, 28 degrees 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.