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

doi:10.13560/j.cnki.biotech.bull.1985.2022-0099

摘要/Abstract

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。

关键词: 脂肪酶, 琼氏不动杆菌, 全基因组测序, 基因功能注释, 比较基因组学

Abstract:

Acinetobacter junii WCO-9 is a high-yield lipase-producing strain isolated from oil-contaminated soil. The lipase produced by this strain has significantly better hydrolysis activity than that of the control strain ATCC 17908. In order to explore the efficient enzyme production mechanism of WCO-9 strain and to discover specific lipase functional genes，ONT（Oxford Nanopore Technologies）was used to complete the whole genome sequencing of WCO-9 strain，and the comparative genomic analysis of the same strain was carried out. The results showed that the genome size of lipase-producing WCO-9 strain was 3.19 Mb，GC content was 38.62%，and there were 2 929 coding genes，including 11 lipase-coding genes（1 specific gene）and 3 lipase molecular chaperones. The collinearity analysis also revealed that the WCO-9 strain was significantly different from the control strain ATCC 17908. The whole genome sequence analysis of WCO-9 strain will be of great significance for the study of high-efficiency enzyme production mechanism and the discovery of new lipase-specific genes，which provides a theoretical basis for the creation and industrial application of high-yield lipase engineering bacteria in the future.

Key words: lipase, Acinetobacter junii, whole genome sequencing, gene function annotation, comparative genome analysis

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.

图/表 11

图1 基于16S rRNA基因序列的系统发育树分析

Fig. 1 Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences

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

图3 菌株WCO-9基因组图谱

Fig. 3 Genome map of strain WCO-9

图4 产脂肪酶菌株WCO-9基因功能注释GO功能分类

Fig. 4 GO function classification map of lipase-producing strain WCO-9’s gene functions

图5 产脂肪酶菌株WCO-9基因功能注释KEGG代谢通路

Fig. 5 KEGG metabolic pathways annotated with the gene functions of lipase-producing strain WCO-9

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

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

图7 菌株WCO-9与菌株ATCC 17908基因组共线性分析

Fig. 7 Genomic collinearity analysis of strain WCO-9 and strain ATCC 17908

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

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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