Identification of the Toxic Mechanisms Employed by Lignin Derivatives and Its Application to Improve n-Butanol Production within Clostridium beijerinckii

Abstract

The main focus of this study is the transcriptomic analysis of bacterial stains exposed to lignin hydrolysate-related compounds to determine the effects of them on global gene expression thereby evaluating their stresses, and the use of the results from the analysis to make novel strains. To achieve this, some works were performed: transcriptomic analysis with a model organism and its use for biosensor construction, and another transcriptomic analysis using a fermentative strain and construction of lignin-derived phenolic compound tolerant strain. Initially, global transcriptional response of E. coli BL21 (DE3) exposed to ferulic acid was investigated by microarray. When E. coli BL21 (DE3) was cultured in presence of 0.25 or 0.5 g/L ferulic acid, several gene sets showing significant change were identified. It was also shown that several individual genes were strongly up-regulated, including marA, inaA, aaeA and aaeB. Thus, these 4 genes, along with htpG, were selected as biomarker genes for phenolics toxicity. Five biomarkers were applied to monitor the response of E. coli exposed to various phenolic acid and aldehyde, which can be generated from lignin, and spruce hydrolysate, where the potential of those five genes was demonstrated as biomarkers to determine toxicity of lignin-derived chemicals. Afterwards, based upon the analysis, transcriptional fusions with the lux operon were constructed using the promoter of inaA, aaeXAB, and yhcN, giving pSP4, pDMA3, pDMY1, respectively, in collaboration with Ajay Kalanjana Monnappa. First, pSP4 was characterized within E. coli BL21 (DE3) with several compounds, suggesting that phenolics were major inducers other than acetic acid and furfural. The Plasmid pSP4 inside different hosts was also tested, including mar regulon mutants, and compared with pDMA3 and pDMY1. Furthermore, these bioreporter strains were applied to monitor the degradation of several aromatic compounds by T. aromatica or A. baylyi, and to analyze spruce and rice straw hydrolysates, which demonstrates that the bioreporters constructed are possible to analyze lignocellulosic biomass hydrolysates for the presence of inhibitory phenolic compounds. In addition, a novel technology for improving response of bioreporter strains was investigated by using serum complement. The effect of this method was proven using genotoxin detectable bioreporter, pRec3. Finally, the effects of ferulic acid on growth, solvent production, and transcriptome of C. beijerinckii NCIMB 8052 were investigated. C. beijerinckii growth rate and butanol production was decreased by ferulic acid at 0.5 g/L. Through microarray analysis with 3 different exposure duration, several groups of the genes were found to be highly up-regulated or down-regulated, including efflux system, heat shock proteins, and two-component system. Among the genes showing significant induction responding to ferulic acid, groE operon genes was overexpressed within C. beijerinckii to have this strain tolerant to lignin hydrolysate-related compounds, which was characterized in this study.