纤维素降解菌Raoultella ornithinolytica LL1的筛选及基因组测序

doi:10.13560/j.cnki.biotech.bull.1985.2020-1174

摘要/Abstract

摘要：

纤维素作为一种资源丰富的天然聚合物,可被微生物降解为可溶性糖,因此筛选高效纤维素降解菌备受关注。本研究利用CMC选择培养基筛选出一株纤维素降解菌Raoultella ornithinolytica LL1,采用三代Pacbio Sequl和二代Illumina Novaseq平台进行基因组测序,并采用DNS法测定其纤维素酶活。R. ornithinolytica LL1基因组大小为5 584 354 bp,包含5 512个基因,其中NR、Swiss-Prot、Pfam、COG、GO、KEGG和CAZyme数据库中分别注释有5 511、4 419、4 584、4 745、3 610、3 336和128个基因,同时基因组注释有大量木质纤维素降解相关基因。R. ornithinolytica LL1具有较高的纤维素酶活性,其中β-葡萄糖苷酶活性最高,其次是内切葡聚糖酶,外切葡聚糖酶活性最低。本研究通过基因组测序及酶活测定揭示了R. ornithinolytica LL1降解纤维素的能力,为其降解纤维素及进一步纤维素转化奠定了基础。

关键词: Raoultella ornithinolytica LL1, 纤维素降解, 基因组测序, 纤维素酶

Abstract:

As a resource-rich natural polymer,cellulose can be degraded into soluble sugar by microorganism. Therefore,screening of efficient cellulose-degrading microorganism has attracted much attention. In the present study,CMC selective medium was used to screen a cellulose-degrading bacterium Raoultella ornithinolytica LL1.The genome sequence of R. ornithinolytica LL1 was conducted by Pacbio Sequl V3 and Illumina Novaseq platform. Furthermore,the cellulase activities of the strain were determined by DNS method. The results showed that the genome size of R. ornithinolytica LL1 was 5 584 354 bp and contained 5 512 genes,of which 5 511,4 419,4 584,4 745,3 610,3 336 and 128 genes were annotated in NR,Swiss-Prot,Pfam,COG,GO,KEGG and CAZyme databases. The genes related to lignocellulose degrading in the genome were also annotated. R. ornithinolytica LL1 presented high cellulase activities,among which β-glucosidase showed the highest activity,followed by endoglucanase and exoglucanase. In this study,the cellulose-degrading ability of R. ornithinolytica LL1 was revealed by genome sequencing and the determination of cellulase activity,which laid a foundation for further cellulose degradation and bioconversion.

Key words: Raoultella ornithinolytica LL1, cellulose degradation, genome sequencing, cellulase

唐昊, 孙灿, 李沅秋, 罗朝兵. 纤维素降解菌Raoultella ornithinolytica LL1的筛选及基因组测序[J]. 生物技术通报, 2021, 37(6): 85-96.

TANG Hao, SUN Can, LI Yuan-qiu, LUO Chao-bing. Screening and Genome Sequencing of Cellulytic Bacterium Raoultella ornithinolytica LL1[J]. Biotechnology Bulletin, 2021, 37(6): 85-96.

图/表 11

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特征 Characteristics	染色体 Chromosome	质粒 Plasmid
基因组大小Genome size/bp	5 440 254	144 100
GC含量GC content/%	55.23	52.06
基因区域GC含量 GC content in gene region/%	56.39	56.16
基因间区GC含量 GC content in gene spacer region/%	48.63	55.13
基因数量 Gene number	5 512	5 512
基因总长度Total gene length/bp	4 687 047	102 264
重复序列 Repetitive sequence	149	4
5S rRNA	9	-
16S rRNA	8	-
23S rRNA	8	-
tRNA	87	-
NR注释 NR annotation	5 511	-
Swiss-Prot注释 Swiss-Prot annotation	4 419	-
Pfam注释 Pfam annotation	4 584	-
COG注释 COG annotation	4 745	-
GO注释 Go annotation	3 610	-
KEGG注释 KEGG annotation	3 336	-

特征 Characteristics	染色体 Chromosome	质粒 Plasmid
基因组大小Genome size/bp	5 440 254	144 100
GC含量GC content/%	55.23	52.06
基因区域GC含量 GC content in gene region/%	56.39	56.16
基因间区GC含量 GC content in gene spacer region/%	48.63	55.13
基因数量 Gene number	5 512	5 512
基因总长度Total gene length/bp	4 687 047	102 264
重复序列 Repetitive sequence	149	4
5S rRNA	9	-
16S rRNA	8	-
23S rRNA	8	-
tRNA	87	-
NR注释 NR annotation	5 511	-
Swiss-Prot注释 Swiss-Prot annotation	4 419	-
Pfam注释 Pfam annotation	4 584	-
COG注释 COG annotation	4 745	-
GO注释 Go annotation	3 610	-
KEGG注释 KEGG annotation	3 336	-

序号 Serial number	COG类型 COG type	基因数目 Number of genes	相对比例 Relative ratio/%
1	未知功能 Unknown function	1 217	25.65
2	氨基酸转运与代谢 Amino acid transport and metabolism	464	9.78
3	碳水化合物转运和代谢 Carbohydrate transport and metabolism	454	9.57
4	转录 Transcription	419	8.83
5	无机离子转运与代谢 Transport and metabolism of inorganic ions	359	7.57
6	复制、重组和修复 Replication, recombination and repair	313	6.60
7	能源生产与转换 Energy production and conversion	274	5.77
8	细胞壁/膜/包膜生物发生 Cell wall / membrane / capsule biogenesis	249	5.25
9	翻译、核糖体结构与生物发生 Translation, ribosomal structure and biogenesis	189	3.98
10	翻译后修饰、蛋白质转运、分子伴侣 Post-translational modifications, protein transport, molecular chaperones	151	3.18
11	信号转导机制 Signaling mechanism	146	3.08
12	辅酶转运与代谢 Coenzyme transport and metabolism	141	2.97
13	脂质转运与代谢 Lipid transport and metabolism	94	1.98
14	核苷酸转运和代谢 Nucleotide transport and metabolism	90	1.90
15	细胞内转运、分泌和囊泡转运 Intracellular transport, secretion and vesicle transport	86	1.81
16	次生代谢物生物合成、运输和分解代谢 Biosynthesis, transport and catabolism of secondary metabolites	73	1.54
17	防御机制 Defense mechanism	55	1.16
18	细胞周期控制、细胞分裂和染色体分离 Cell cycle control, cell division and chromosome separation	37	0.78
19	细胞运动性 Cell mobility	18	0.38
20	RNA加工与修饰 RNA processing and modification	1	0.02
21	染色质结构与动力学 Chromatin structure and kinetics	1	0.02

序号 Serial number	COG类型 COG type	基因数目 Number of genes	相对比例 Relative ratio/%
1	未知功能 Unknown function	1 217	25.65
2	氨基酸转运与代谢 Amino acid transport and metabolism	464	9.78
3	碳水化合物转运和代谢 Carbohydrate transport and metabolism	454	9.57
4	转录 Transcription	419	8.83
5	无机离子转运与代谢 Transport and metabolism of inorganic ions	359	7.57
6	复制、重组和修复 Replication, recombination and repair	313	6.60
7	能源生产与转换 Energy production and conversion	274	5.77
8	细胞壁/膜/包膜生物发生 Cell wall / membrane / capsule biogenesis	249	5.25
9	翻译、核糖体结构与生物发生 Translation, ribosomal structure and biogenesis	189	3.98
10	翻译后修饰、蛋白质转运、分子伴侣 Post-translational modifications, protein transport, molecular chaperones	151	3.18
11	信号转导机制 Signaling mechanism	146	3.08
12	辅酶转运与代谢 Coenzyme transport and metabolism	141	2.97
13	脂质转运与代谢 Lipid transport and metabolism	94	1.98
14	核苷酸转运和代谢 Nucleotide transport and metabolism	90	1.90
15	细胞内转运、分泌和囊泡转运 Intracellular transport, secretion and vesicle transport	86	1.81
16	次生代谢物生物合成、运输和分解代谢 Biosynthesis, transport and catabolism of secondary metabolites	73	1.54
17	防御机制 Defense mechanism	55	1.16
18	细胞周期控制、细胞分裂和染色体分离 Cell cycle control, cell division and chromosome separation	37	0.78
19	细胞运动性 Cell mobility	18	0.38
20	RNA加工与修饰 RNA processing and modification	1	0.02
21	染色质结构与动力学 Chromatin structure and kinetics	1	0.02

序号 Serial number	通路ID Pathway ID	通路 Pathways	基因数目 Number of genes	相对比例 Relative proportion/%
1	ko00500	淀粉和蔗糖代谢 Starch and sucrose metabolism	67	2.01
2	ko00010	糖酵解/糖异生 Glycolysis/gluconeogenesis	62	1.86
3	ko00620	丙酮酸代谢 Pyruvate metabolism	58	1.74
4	ko00030	戊糖磷酸途径 Pentose phosphate pathway	48	1.44
5	ko00520	氨基糖和核苷酸糖代谢 Amino and nucleotide sugar metabolism	47	1.41
6	ko00051	果糖和甘露糖代谢 Fructose and mannose metabolism	43	1.29
7	ko00650	丁酸代谢 Butyric acid metabolism	40	1.20
8	ko00040	戊糖、葡萄糖醛酸转换 Conversion of pentose to glucuronic acid	36	1.08
9	ko00640	丙酸代谢 Propionic acid metabolism	33	0.99
10	ko00630	乙醛酸和二羧酸代谢 Glyoxylic acid and dicarboxylic acid metabolism	33	0.99
11	ko00020	三羧酸循环 Tricarboxylic acid cycle	33	0.99
12	ko00053	抗坏血酸和醛酸代谢 Ascorbic acid and aldehyde acid metabolism	32	0.96
13	ko00052	半乳糖代谢 Galactose metabolism	26	0.78
14	ko00660	C5支链二元酸代谢 C5 branched diacid metabolism	13	0.39
15	ko00562	磷酸肌醇代谢Phosphoinositide metabolism	11	0.33