Enhanced alpha-galactosidase expression in pseudomonas chlororaphis

Authors: Solaiman, Daniel; Ashby, Richard - Rick; Cross, Nicole

Submitted to: American Oil Chemists' Society Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 4/11/2017
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Pseudomonas chlororaphis is a non-pathogenic bacterium useful for fermentative production of biopolymer (i.e., poly(hydroxyalkanoates); PHA) and biosurfactant (i.e., rhamnolipid; RhL). In order to enable P. chlororaphis to better fermentatively utilize the residual soy sugars in soy molasses – a low-cost byproduct of soybean processing, we introduced a previously cloned alpha-galactosidase gene of Streptomyces coelicolor (a-gal(Sc)) into the bacteria. Two approaches were employed to introduce a-gal(Sc) into P. chlororaphis. In one approach, the a-gal(Sc) gene was inserted into the chromosomal DNA of P. chlororaphis (chr::gal), and the site of insertion was then mapped to ascertain its location. In the other approach, the a-gal(Sc) gene was spliced into an expression vector (i.e., pBS29-P2) to yield pBS29P2-gal which was subsequently electroporated into P. chlororaphis. Expression levels of the heterologous a-gal(Sc) in the two types of genetically modified P. chlororaphis were compared using real-time RT-qPCR technique. The results showed that both types of recombinant strains were able to transcribe the cloned a-gal(Sc) gene into its mRNA. Furthermore, the transcription level of a-gal(Sc) was many folds higher in the cells containing pBS29P2-gal plasmid in comparison to that in the cells harboring the chromosomally integrated a-gal(Sc). This research thus yielded two active biocatalysts for future research to compare their genetic stability, a-Gal enzyme activity, and cell growth and PHA/RhL production on soy sugars.