生物技术通报 ›› 2022, Vol. 38 ›› Issue (10): 216-225.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0099
张泽颖1,3(), 范清锋1,3, 邓云峰1,3, 韦廷舟1,3, 周正富2, 周建1,3, 王劲2, 江世杰1,3()
收稿日期:
2021-01-21
出版日期:
2022-10-26
发布日期:
2022-11-11
作者简介:
张泽颖,女,研究方向:微生物酶工程;E-mail:基金资助:
ZHANG Ze-ying1,3(), FAN Qing-feng1,3, DENG Yun-feng1,3, WEI Ting-zhou1,3, ZHOU Zheng-fu2, ZHOU Jian1,3, WANG Jin2, JIANG Shi-jie1,3()
Received:
2021-01-21
Published:
2022-10-26
Online:
2022-11-11
摘要:
琼氏不动杆菌WCO-9是从油污污染的土壤中分离的一株高产脂肪酶菌株,所产脂肪酶具有显著优于对照同种菌株ATCC 17908的水解活性。为探究WCO-9菌株高效产酶机制,挖掘新型特异脂肪酶功能基因,采用Oxford Nanopore技术完成WCO-9菌株的全基因组测序,并对同属菌株进行比较基因组学分析。结果显示,脂肪酶产生菌WCO-9的基因组大小为3.19 Mb,GC含量38.62%,含有2 929个编码基因,其中包含11个脂肪酶编码基因(1个特异基因)和3个脂肪酶分子伴侣基因;共线性分析也说明WCO-9菌株基因组与对照菌株ATCC 17908具有显著差异。WCO-9菌株全基因组序列分析,对该菌的高效产酶机制研究及新型脂肪酶特异基因挖掘具有重要意义,为后期高产脂肪酶工程菌的创制及工业化应用提供理论基础。
张泽颖, 范清锋, 邓云峰, 韦廷舟, 周正富, 周建, 王劲, 江世杰. 一株高产脂肪酶菌株WCO-9全基因组测序及比较基因组分析[J]. 生物技术通报, 2022, 38(10): 216-225.
ZHANG Ze-ying, FAN Qing-feng, DENG Yun-feng, WEI Ting-zhou, ZHOU Zheng-fu, ZHOU Jian, WANG Jin, JIANG Shi-jie. Whole Genome Sequencing and Comparative Genomic Analysis of a High-yield Lipase-producing Strain WCO-9[J]. Biotechnology Bulletin, 2022, 38(10): 216-225.
菌株Strain | 脂肪酶活力Lipase activity/(U·L-1) |
---|---|
WCO-9 | 2 833.33±166.67A |
ATCC 17908 | 24.44±8.39B |
表1 菌株WCO-9所产脂肪酶对底物橄榄油的水解能力分析
Table 1 Analysis of the hydrolysis ability of lipase from WCO-9 strain on the substrate olive oil by GB/T 23535-2009 method
菌株Strain | 脂肪酶活力Lipase activity/(U·L-1) |
---|---|
WCO-9 | 2 833.33±166.67A |
ATCC 17908 | 24.44±8.39B |
图 2 WCO-9菌株产脂肪酶活性分析 A:罗丹明B-油脂同化平板法分析WCO-9菌株产脂肪酶能力;B:WCO-9菌株脂肪酶水解不同对硝基苯酯的活性差异,***P<0.01
Fig. 2 Lipase activity analysis of WCO-9 strain A: Analysis of lipase-producing ability of WCO-9 strain by rhodamine B-oil assimilation plate method; B: Differences in hydrolytic activity of lipase from WCO-9 strain towards various ρ-NP esters, ***P<0.01
编号No. | 通路Pathway ID | 功能描述Description | 基因编号Gene ID |
---|---|---|---|
1 | ko00071 | 脂肪酸降解 Fatty acid degradation | GE000138、GE000223、GE000309、GE000310、GE000604、GE000605、GE000616、GE001181、GE001341、GE001407、GE001474、GE001518、GE001530、GE001747、GE001825、GE001884、GE001964 |
2 | ko00561 | 甘油酯代谢 Glycerolipid metabolism | GE000861、GE000871、GE001701、GE001702、GE002495、GE002594 |
3 | ko00564 | 甘油磷脂代谢 Glycerophospholipid metabolism | GE000081、GE000318、GE000487、GE000575、GE000725、GE000800、GE000863、GE000871、GE001133、GE001134、GE001196、GE001398、GE001531、GE001566、GE002494、GE002495、GE002594、GE002873、GE002885 |
4 | ko01212 | 脂肪酸代谢 Fatty acid metabolism | GE000138、GE000223、GE000309、GE000310、GE000573、GE000587、GE000604、GE000605、GE000616、GE001181、GE001182、GE001359、GE001360、GE001404、GE001474、GE001769、GE001820、GE001825、GE002165、GE002197、GE002198、GE002362、GE002463、GE002920 |
表2 产脂肪酶菌株WCO-9基因组脂肪代谢降解通路及相关基因
Table 2 Fat metabolism and degradation pathways in lipase-producing strain WCO-9 genome and their related genes
编号No. | 通路Pathway ID | 功能描述Description | 基因编号Gene ID |
---|---|---|---|
1 | ko00071 | 脂肪酸降解 Fatty acid degradation | GE000138、GE000223、GE000309、GE000310、GE000604、GE000605、GE000616、GE001181、GE001341、GE001407、GE001474、GE001518、GE001530、GE001747、GE001825、GE001884、GE001964 |
2 | ko00561 | 甘油酯代谢 Glycerolipid metabolism | GE000861、GE000871、GE001701、GE001702、GE002495、GE002594 |
3 | ko00564 | 甘油磷脂代谢 Glycerophospholipid metabolism | GE000081、GE000318、GE000487、GE000575、GE000725、GE000800、GE000863、GE000871、GE001133、GE001134、GE001196、GE001398、GE001531、GE001566、GE002494、GE002495、GE002594、GE002873、GE002885 |
4 | ko01212 | 脂肪酸代谢 Fatty acid metabolism | GE000138、GE000223、GE000309、GE000310、GE000573、GE000587、GE000604、GE000605、GE000616、GE001181、GE001182、GE001359、GE001360、GE001404、GE001474、GE001769、GE001820、GE001825、GE002165、GE002197、GE002198、GE002362、GE002463、GE002920 |
图6 产脂肪酶菌株WCO-9 COG功能分类 1:RNA加工和修饰;2:染色质结构和动力学;3:能源生产和转换;4:细胞周期控制、细胞分裂、染色体分割;5:氨基酸转运与代谢;6:核苷酸转运和代谢;7:碳水化合物运输和代谢;8:辅酶转运和代谢;9:脂质转运与代谢;10:翻译、核糖体结构和生物起源;11:转录;12:复制、重组和修复;13:细胞壁/膜/被膜生物发生;14:细胞运动;15:翻译后修饰、蛋白质周转、分子伴侣;16:无机离子转运与代谢;17:次级代谢物生物合成、运输和分解代谢;18:一般功能预测;19:功能未知;20:信号转导机制;21:细胞内运输、分泌和囊泡运输;22:防御机制;23:胞外结构;24:核结构;25:细胞骨架
Fig. 6 COG function classification chart of lipase-produc-ing strain WCO-9 1:RNA processing and modification. 2:Chromatin structure and dynamics. 3:Energy production and conversion. 4:Cell cycle control,cell division,chromosome partitioning. 5:Amino acid transport and metabolism. 6:Nucleotide transport and metabolism. 7:Carbohydrate transport and metabolism. 8:Coenzyme transport and metabolism. 9:Lipid transport and metabolism. 10:Translation,ribosomal structure and biogenesis. 11:Transcription. 12:Replication,recombination and repair. 13:Cell wall/membrane/envelope biogenesis. 14:Cell motility. 15:Posttranslational modification,protein turnover,chaperones. 16:Inorganic ion transport and metabolism. 17:Secondary metabolites biosynthesis,transport and catabolism. 18:General function prediction only. 19:Function unknown. 20:Signal transduction mechanisms. 21:Intracellular trafficking,secretion,and vesicular transport. 22:Defense mechanisms. 23:Extracellular structures. 24:Nuclear structure. 25:Cytoskeleton
类型Type | 基因组大小Genome size /bp | GC含量GC content /% | 编码基因数目Coding genes | GenBank登录号GenBank INSDC ID |
---|---|---|---|---|
WCO-9 | 3 193 903 | 38.47 | 2 943 | CP090890.1 |
Acinetobacter junii ATCC 17908 | 3 357 744 | 38.90 | 3 185 | APPX00000000.1 |
Acinetobacter baumannii ATCC 19606 | 3 927 723 | 39.18 | 3 681 | CP058289.1 |
Acinetobacter chinensis WCHAc010005 | 3 600 635 | 42.48 | 3 434 | CP032134.1 |
表3 菌株WCO-9与3株不动杆菌全基因组序列基本特征比较
Table 3 Comparison of basic characteristics of whole genome sequences of strain WCO-9 and 3 strains of Acinetobacter
类型Type | 基因组大小Genome size /bp | GC含量GC content /% | 编码基因数目Coding genes | GenBank登录号GenBank INSDC ID |
---|---|---|---|---|
WCO-9 | 3 193 903 | 38.47 | 2 943 | CP090890.1 |
Acinetobacter junii ATCC 17908 | 3 357 744 | 38.90 | 3 185 | APPX00000000.1 |
Acinetobacter baumannii ATCC 19606 | 3 927 723 | 39.18 | 3 681 | CP058289.1 |
Acinetobacter chinensis WCHAc010005 | 3 600 635 | 42.48 | 3 434 | CP032134.1 |
WCO-9基因编号 Gene ID of WCO-9 | 功能注释 Function annotation | 注释来源数据库 Source of function | GenBank 中序列一致性 Sequence identity in GenBank/% | 同源菌株 Homologous source strain | ATCC 17908中同源蛋白编号 Homologous protein ID of ATCC 17908 | ATCC 17908中同源蛋白序列一致性Sequence identity with homologous protein ID of ATCC 17908/% |
---|---|---|---|---|---|---|
GE000005 | 脂肪酶2 Lipase 2 | Swissprot、TrEMBL | 100 | Acinetobacter junii NIPH 182 | WP_004965292.1 | 99.44 |
GE000880 | 脂肪酶1 Lipase 1 | Swissprot、TrEMBL | 100 | Acinetobacter junii NBRC 110497 | WP_004963343.1 | 99.41 |
GE000882 | GDSL脂肪酶 Lipase_GDSL | Pfam | 100 | Acinetobacter junii KCTC 42611 | ENV66375.1 | 99.52 |
GE001015 | 甘油三酯脂肪酶活性 Triglyceride lipase activity | GO | 99.78 | Acinetobacter junii YR7 | ENV66472.1 | 84.53 |
GE001547 | GDSL脂肪酶2 Lipase GDSL 2 | Pfam | 99.16 | Acinetobacter junii KCTC 42611 | WP_004962046.1 | 96.22 |
GE001701 | 三酰甘油脂肪酶 Triacylglycerol lipase | KEGG | 100 | Acinetobacter junii NIPH 182 | ENV65669.1 | 42.90 |
GE001702 | 三酰甘油脂肪酶 Triacylglycerol lipase | KEGG | 100 | Acinetobacter junii NIPH 182 | WP_035333945.1 | 40.29 |
GE001718 | 甘油三酯脂肪酶 Triglyceride lipase activity | GO | 100 | Acinetobacter junii TUM15553 | WP_004962461.1 | 98.78 |
GE001892 | 甘油三酯脂肪酶 Triglyceride lipase activity | GO | 100 | Acinetobacter junii SH205 | WP_004953257.1 | 99.80 |
GE001931 | 甘油三酯脂肪酶 Triglyceride lipase activity | GO | 90.76 | Acinetobacter sp. SAT183 | — | — |
GE002660 | 甘油三酯脂肪酶 Triglyceride lipase activity | GO | 99.38 | Acinetobacter junii NIPH 182 | ENV65669.1 | 78.72 |
GE000502 | 脂肪酶伴侣 Lipase chaperone | Pfam | 99.67 | Acinetobacter junii SB132 | — | — |
GE002659 | 脂肪酶伴侣 Lipase chaperone | Pfam、Swissprot、 TrEMBL | 98.56 | Acinetobacter junii YR7 | WP_004964537.1 | 63.98 |
GE001700 | 脂肪酶伴侣 Lipase chaperone | Pfam、Swissprot、 TrEMBL | 100 | Acinetobacter junii lzh-X15 | WP_004964537.1 | 32.56 |
表4 脂肪酶基因与NCBI数据库中基因序列相似度
Table 4 Sequence similarity between lipase genes and genes in NCBI database
WCO-9基因编号 Gene ID of WCO-9 | 功能注释 Function annotation | 注释来源数据库 Source of function | GenBank 中序列一致性 Sequence identity in GenBank/% | 同源菌株 Homologous source strain | ATCC 17908中同源蛋白编号 Homologous protein ID of ATCC 17908 | ATCC 17908中同源蛋白序列一致性Sequence identity with homologous protein ID of ATCC 17908/% |
---|---|---|---|---|---|---|
GE000005 | 脂肪酶2 Lipase 2 | Swissprot、TrEMBL | 100 | Acinetobacter junii NIPH 182 | WP_004965292.1 | 99.44 |
GE000880 | 脂肪酶1 Lipase 1 | Swissprot、TrEMBL | 100 | Acinetobacter junii NBRC 110497 | WP_004963343.1 | 99.41 |
GE000882 | GDSL脂肪酶 Lipase_GDSL | Pfam | 100 | Acinetobacter junii KCTC 42611 | ENV66375.1 | 99.52 |
GE001015 | 甘油三酯脂肪酶活性 Triglyceride lipase activity | GO | 99.78 | Acinetobacter junii YR7 | ENV66472.1 | 84.53 |
GE001547 | GDSL脂肪酶2 Lipase GDSL 2 | Pfam | 99.16 | Acinetobacter junii KCTC 42611 | WP_004962046.1 | 96.22 |
GE001701 | 三酰甘油脂肪酶 Triacylglycerol lipase | KEGG | 100 | Acinetobacter junii NIPH 182 | ENV65669.1 | 42.90 |
GE001702 | 三酰甘油脂肪酶 Triacylglycerol lipase | KEGG | 100 | Acinetobacter junii NIPH 182 | WP_035333945.1 | 40.29 |
GE001718 | 甘油三酯脂肪酶 Triglyceride lipase activity | GO | 100 | Acinetobacter junii TUM15553 | WP_004962461.1 | 98.78 |
GE001892 | 甘油三酯脂肪酶 Triglyceride lipase activity | GO | 100 | Acinetobacter junii SH205 | WP_004953257.1 | 99.80 |
GE001931 | 甘油三酯脂肪酶 Triglyceride lipase activity | GO | 90.76 | Acinetobacter sp. SAT183 | — | — |
GE002660 | 甘油三酯脂肪酶 Triglyceride lipase activity | GO | 99.38 | Acinetobacter junii NIPH 182 | ENV65669.1 | 78.72 |
GE000502 | 脂肪酶伴侣 Lipase chaperone | Pfam | 99.67 | Acinetobacter junii SB132 | — | — |
GE002659 | 脂肪酶伴侣 Lipase chaperone | Pfam、Swissprot、 TrEMBL | 98.56 | Acinetobacter junii YR7 | WP_004964537.1 | 63.98 |
GE001700 | 脂肪酶伴侣 Lipase chaperone | Pfam、Swissprot、 TrEMBL | 100 | Acinetobacter junii lzh-X15 | WP_004964537.1 | 32.56 |
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