一株木质纤维素降解菌的筛选及其全基因组分析

Screening and genomic analysis of a lignocellulose degrading bacterium

摘要:

[目的] 筛选和鉴定有木质纤维素降解能力的1株细菌,测定其相关酶活力并进行全基因组分析,为构建木质纤维素降解工程菌提供依据。[方法] 采用3种木质素类似物(天青-B;酚红;愈创木酚)的脱色/染色法,从腐木和被枝叶覆盖的土壤中分离和筛选出1株具有较强木质纤维素降解能力的细菌。通过16S rRNA基因和全基因组序列分析对该菌进行种属鉴定。使用紫外分光光度法测定其锰过氧化物酶(MnP)、漆酶(Lac)、羧甲基纤维素酶(CMCase)以及滤纸酶(FPA)活力,了解该菌相关酶活力大小在一定时间内的变化趋势。使用Illumina Miseq和454 GS Junior测序平台获取该菌的全基因组序列,将其全基因组序列经过注释的基因蛋白质序列提交COG和KEGG数据库进行BLASTp比对分析,确定该菌潜在的重要酶类和代谢途径,并对部分注释基因进行定量RT-PCR验证。[结果] 筛选得到1株优势菌株S12,该菌经鉴定后命名为解鸟氨酸拉乌尔菌(Raoultella ornithinolytica)。在液体CMC-Na培养基中发酵28 h,菌体生长达到稳定期,纤维素降解相关酶活力也在此时达到峰值。生物信息学分析结果表明,菌株S12具有木质素降解通路中重要酶类的编码基因,如过氧化物酶、Fe-Mn型超氧化物歧化酶、邻苯二酚1,2-双加氧酶和原儿茶酸-3,4-双加氧酶等,这些基因在以碱性木质素为碳源的培养条件下表达量不同程度地高于以葡萄糖为碳源的培养条件。另外,菌株S12具备完整的纤维素降解和乙醇生成通路。[结论] 本研究首次揭示了Raoultella ornithinolytica S12具备有效的木质纤维素降解性能,这对于推动木质纤维素应用产业的发展具有重要意义。

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.