Background: Furfural and 5-hydroxymethylfurfural (HMF) are the two major inhibitor compounds generated from lignocellulose pretreatment, especially for dilute acid, steam explosion, neutral hot water pretreatment methods. The two inhibitors severely inhibit the cell growth and metabolism of fermenting strains in the consequent bioconversion step. The biodetoxification strain Amorphotheca resinae ZN1 has demonstrated its extraordinary capacity of fast and complete degradation of furfural and HMF into corresponding alcohol and acid forms. The elucidation of degradation metabolism of A. resinae ZN1 at molecular level will facilitate the detoxification of the pretreated lignocellulose biomass and provide the metabolic pathway information for more powerful biodetoxification strain development.
Results: Amorphotheca resinae ZN1 was able to use furfural or HMF as the sole carbon source for cell growth. During the detoxification process, A. resinae ZN1 firstly reduced furfural or HMF into furfuryl alcohol or HMF alcohol, and then oxidized into furoic acid or HMF acid through furan aldehyde as the intermediate at low concentration level. The cell mass measurement suggested that furfural was more toxic to A. resinae ZN1 than HMF. In order to identify the degradation mechanism of A. resinae ZN1, transcription levels of 137 putative genes involved in the degradation of furfural and HMF in A. resinae ZN1 were investigated using the real-time quantitative PCR (qRT-PCR) method under the stress of furfural and HMF, as well as the stress of their secondary metabolites, furfuryl alcohol and HMF alcohol. Two Zn-dependent alcohol dehydrogenase genes and five AKR/ARI genes were found to be responsible for the furfural and HMF conversion to their corresponding alcohols. For the conversion of the two furan alcohols to the corresponding acids, three propanol-preferring alcohol dehydrogenase genes, one NAD(P)(+)-depending aldehyde dehydrogenase gene, or two oxidase genes with free oxygen as the substrate were identified under aerobic condition.
Conclusions: The genes responsible for the furfural and HMF degradation to the corresponding alcohols and acids in A. resinae ZN1 were identified based on the analysis of the genome annotation, the gene transcription data and the inhibitor conversion results. These genetic resources provided the important information for understanding the mechanism of furfural and HMF degradation and modification of high tolerant strains used for biorefinery processing.
Keywords: 5-Hydromethylfurfural (HMF); Amorphotheca resinae ZN1; Biodetoxification; Furfural; Transcription level; qRT-PCR.