Screening and genomic analysis of a lignocellulose degrading bacterium

Abstract:

[Objective] This study is aimed to screen and identify a bacterium with the ability to degrade lignocellulose, to perform its genomic analysis, and to determine its related enzymatic activities. [Methods] Using a bleaching/dyeing method with three kinds of lignin analogues (Azure-B; Phenol red; Guaiacol), we separated and screened a bacterium strain, with a strong ability to degrade lignocellulose, from soil enriched by decaying wood and leaves. We identified the species of this bacterium according to its 16S rRNA gene and core gene sequence analysis. In order to understand the trend of enzymatic activities within a certain period, we used ultraviolet spectrophotometry on manganese peroxidase (MnP), laccase (Lac), carboxymethyl cellulose (CMCase) and filter paper (FPA). The whole genome was sequenced by Illumina MiSeq and 454 GS Junior platforms. The protein sequences were annotated from the whole genome and compared with COG and KEGG databases through BLASTp to determine several potential lignocellulose-degrading enzymes and pathways. Some of the annotated genes were further verified by realtime RT-PCR. [Results] We obtained strain S12 which was identified as Raoultella ornithinolytica. The bacterium grew to stationary phase after being incubated in CMC-Na liquid medium for 28 h, at which its cellulose degradation related enzymatic activities reached to peak values. Bioinformatic analysis results showed that strain S12 has some significant genes that encode enzymes working in the lignin degradation pathway, such as peroxidase, Fe-Mn superoxide dismutase, catechol 1,2-dioxygenase, protocatechuate 3, 4-dioxygenase, etc. The expression levels of these genes were higher when strain S12 was grown in a medium with lignin as the unique carbon source than in a medium with glucose as the unique carbon source. Also, strain S12 has a complete cellulose degradation and ethanol generation pathway. [Conclusion] Raoultella ornithinolytica S12 has the ability to degrade lignocellulose effectively, which is significant in promoting the development of the lignocellulose application industry.