生物技术通报 ›› 2021, Vol. 37 ›› Issue (6): 85-96.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1174
收稿日期:
2020-09-18
出版日期:
2021-06-26
发布日期:
2021-07-08
作者简介:
唐昊,男,博士,讲师,研究方向:竹木质纤维素降解;E-mail: 基金资助:
TANG Hao(), SUN Can, LI Yuan-qiu, LUO Chao-bing()
Received:
2020-09-18
Published:
2021-06-26
Online:
2021-07-08
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。
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1 纤维素降解菌PX19、PX20和PX21刚果红染色对比 图中数据表示平均值±标准误,不同字母表示不同菌株水解能力差异显著性(P<0.05,Duncan氏检验),下同
Fig.1 Comparison of Congo red staining of cellulose degrading bacterium PX19, PX20 and PX21 The average±standard error is shown in the figure, and different letters show significant difference in hydrolysis ability of different strains ( P < 0.05, Duncan’s test ). The same below
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 | - |
表1 R. ornithinolytica LL1的基因组特征
Table1 Genome characteristics of R. ornithinolytica LL1
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 |
表2 R. ornithinolytica LL1 COG注释
Table 2 COG annotation of R. ornithinolytica LL1
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 |
表3 碳水化合物代谢相关KEGG通路
Table 3 KEGG pathways related to carbohydrate metabolism
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 |
分类 Classification | 基因ID Gene ID | 功能预测 Function prediction |
---|---|---|
纤维素降解相关基因 Cellulose degradation related genes | gene2786 | GH9 (endoglucanase EC 3.2.1.4) |
gene0210 | GH8 (endoglucanase EC 3.2.1.4) | |
gene0218 | GH8 (endoglucanase EC 3.2.1.4) | |
gene0074 | GH4 (6-phospho-beta-glucosidase EC 3.2.1.86) | |
gene3342 | GH4 (6-phospho-beta-glucosidase EC 3.2.1.86) | |
gene1935 | GH4 (alpha-glucosidase EC 3.2.1.21) | |
gene1744 | GH3 (beta-glucosidase EC 3.2.1.21) | |
gene0827 | GH1 (beta-glucosidase EC 3.2.1.21) | |
gene0964 | GH1 (beta-glucosidase EC 3.2.1.21) | |
gene1208 | GH1 (beta-glucosidase EC 3.2.1.21) | |
gene1476 | GH1 (beta-glucosidase EC 3.2.1.21) | |
gene1736 | GH1 (beta-glucosidase EC 3.2.1.21) | |
gene1922 | GH1 (beta-glucosidase EC 3.2.1.21) | |
gene2493 | GH1 (beta-glucosidase EC 3.2.1.21) | |
gene2777 | GH1 (beta-glucosidase EC 3.2.1.21) | |
gene3805 | GH1 (beta-glucosidase EC 3.2.1.21) | |
gene1047 | GH32 (sucrose-6-phosphate hydrolase EC 2.4.1.-) | |
gene0301 | GH13 (α-glucosidase EC 3.2.1.20) | |
gene0164 | GH13 (α-glucosidase EC 3.2.1.20) | |
gene4341 | GH13 (α-glucosidase EC 3.2.1.20) | |
gene4934 | GH13 (α-glucosidase EC 3.2.1.20) | |
gene3011 | GH13 (α-glucosidase EC 3.2.1.20) | |
gene2026 | GH13 (α-glucosidase EC 3.2.1.20) | |
gene0300 | GH13 (α-glucosidase EC 3.2.1.20) | |
gene3799 | GH31 (α-glucosidase EC 3.2.1.20) | |
半纤维素降解相关基因 Hemicellulose degradation related genes | gene1732 | GH43 (xylan 1,4-beta-xylosidase EC:3.2.1.37) |
gene4854 | GH43 (xylan 1,4-beta-xylosidase EC:3.2.1.37) | |
gene4896 | GH43 (xylan 1,4-beta-xylosidase EC:3.2.1.37) | |
gene4257 | GH53 (arabinogalactan endo-1,4-beta-galactosidase EC:3.2.1.89) | |
gene4256 | GH42 (beta-galactosidase EC:3.2.1.23) | |
gene2601 | GH2 (beta-galactosidase EC:3.2.1.23) | |
木质素降解相关基因 Lignin degradation related genes | gene1352 | AA1 (laccase EC 1.10.3.2) |
gene2367 | AA1 (laccase EC 1.10.3.2) | |
gene2810 | AA2 (manganese peroxidase EC 1.11.1.13) | |
gene0833 | AA7 (FAD-binding protein) | |
gene0243 | AA3 (cellobiose dehydrogenase EC 1.1.99.18) | |
gene1369 | AA3 (cellobiose dehydrogenase EC 1.1.99.18) | |
gene2549 | AA3 (cellobiose dehydrogenase EC 1.1.99.18) | |
gene3520 | AA3 (cellobiose dehydrogenase EC 1.1.99.18) | |
gene3990 | AA3 (cellobiose dehydrogenase EC 1.1.99.18) | |
gene4124 | AA3 (cellobiose dehydrogenase EC 1.1.99.18) | |
gene4303 | AA3 (cellobiose dehydrogenase EC 1.1.99.18) |
表4 编码R. ornithinolytica LL1木质纤维素降解酶基因
Table 4 Genes encoding lignocellulose degradation in R. ornithinolytica LL1
分类 Classification | 基因ID Gene ID | 功能预测 Function prediction |
---|---|---|
纤维素降解相关基因 Cellulose degradation related genes | gene2786 | GH9 (endoglucanase EC 3.2.1.4) |
gene0210 | GH8 (endoglucanase EC 3.2.1.4) | |
gene0218 | GH8 (endoglucanase EC 3.2.1.4) | |
gene0074 | GH4 (6-phospho-beta-glucosidase EC 3.2.1.86) | |
gene3342 | GH4 (6-phospho-beta-glucosidase EC 3.2.1.86) | |
gene1935 | GH4 (alpha-glucosidase EC 3.2.1.21) | |
gene1744 | GH3 (beta-glucosidase EC 3.2.1.21) | |
gene0827 | GH1 (beta-glucosidase EC 3.2.1.21) | |
gene0964 | GH1 (beta-glucosidase EC 3.2.1.21) | |
gene1208 | GH1 (beta-glucosidase EC 3.2.1.21) | |
gene1476 | GH1 (beta-glucosidase EC 3.2.1.21) | |
gene1736 | GH1 (beta-glucosidase EC 3.2.1.21) | |
gene1922 | GH1 (beta-glucosidase EC 3.2.1.21) | |
gene2493 | GH1 (beta-glucosidase EC 3.2.1.21) | |
gene2777 | GH1 (beta-glucosidase EC 3.2.1.21) | |
gene3805 | GH1 (beta-glucosidase EC 3.2.1.21) | |
gene1047 | GH32 (sucrose-6-phosphate hydrolase EC 2.4.1.-) | |
gene0301 | GH13 (α-glucosidase EC 3.2.1.20) | |
gene0164 | GH13 (α-glucosidase EC 3.2.1.20) | |
gene4341 | GH13 (α-glucosidase EC 3.2.1.20) | |
gene4934 | GH13 (α-glucosidase EC 3.2.1.20) | |
gene3011 | GH13 (α-glucosidase EC 3.2.1.20) | |
gene2026 | GH13 (α-glucosidase EC 3.2.1.20) | |
gene0300 | GH13 (α-glucosidase EC 3.2.1.20) | |
gene3799 | GH31 (α-glucosidase EC 3.2.1.20) | |
半纤维素降解相关基因 Hemicellulose degradation related genes | gene1732 | GH43 (xylan 1,4-beta-xylosidase EC:3.2.1.37) |
gene4854 | GH43 (xylan 1,4-beta-xylosidase EC:3.2.1.37) | |
gene4896 | GH43 (xylan 1,4-beta-xylosidase EC:3.2.1.37) | |
gene4257 | GH53 (arabinogalactan endo-1,4-beta-galactosidase EC:3.2.1.89) | |
gene4256 | GH42 (beta-galactosidase EC:3.2.1.23) | |
gene2601 | GH2 (beta-galactosidase EC:3.2.1.23) | |
木质素降解相关基因 Lignin degradation related genes | gene1352 | AA1 (laccase EC 1.10.3.2) |
gene2367 | AA1 (laccase EC 1.10.3.2) | |
gene2810 | AA2 (manganese peroxidase EC 1.11.1.13) | |
gene0833 | AA7 (FAD-binding protein) | |
gene0243 | AA3 (cellobiose dehydrogenase EC 1.1.99.18) | |
gene1369 | AA3 (cellobiose dehydrogenase EC 1.1.99.18) | |
gene2549 | AA3 (cellobiose dehydrogenase EC 1.1.99.18) | |
gene3520 | AA3 (cellobiose dehydrogenase EC 1.1.99.18) | |
gene3990 | AA3 (cellobiose dehydrogenase EC 1.1.99.18) | |
gene4124 | AA3 (cellobiose dehydrogenase EC 1.1.99.18) | |
gene4303 | AA3 (cellobiose dehydrogenase EC 1.1.99.18) |
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