Detection of toxic lignin hydrolysate-related compounds using an inaA::luxCDABE fusion strain

Abstract

Real-time quantitative PCR analyses of Escherichia coli str. BL21(DE3) exposed to 0.5 g/L ferulic and coumaric acid showed that the inaA gene was significantly induced (7.7- and 3.6-fold higher, respectively). Consequently, a transcriptional fusion of the inaA promoter with the luxCDABE operon was constructed and characterized with several compounds identified within hydrolysates. Tests demonstrated that the phenolics were major inducers, while acetic acid and furfural had only a minor or no effect on the inaA expression respectively. Additional tests with mutant E. coli strains found that a marA partially abolished the response while a marB knock-out led to a 2-3-fold higher basal level expression as evidenced by the bioluminescent levels of the cultures. However, a significant induction was seen even in the marA mutant, suggesting some other control mechanism is involved in regulating inaA expression during an exposure to the hydrolysate compounds. Finally, E. coli str. BL21(DE3)/pSP4 was used to analyze a spruce hydrolysate sample. Real-time quantitative PCR showed a 2.8-fold induction of the inaA expression level while the bioluminescence from the exposed culture was 22-fold higher than the control, demonstrating the possible application of this reporter strain to analyze hydrolysates for the presence of fermentation-inhibiting phenolics.