Complete genome sequencing and diversity analysis of lipolytic enzymes in Stenotrophomonas maltophilia OUC_Est10
Hao Dong¹, Junpeng Rui², Jianan Sun¹, Xiangzhen Li², Xiangzhao Mao¹
1. College of Food Science and Engineering, Ocean University of China, Qingdao 266003, Shandong Province, China;
2. Key Laboratory of Environmental and Applied Microbiology, Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, Sichuan Province, China
Received: 3 May 2017; Revised: 5 August 2017; Published online: 15 August 2017
Foundation item: Supported by the National Natural Science Foundation of China (31501516), by the Major Special Science and Technology Projects in Shandong Province (2016YYSP016), by the Applied Basic Research Program of Qingdao (16-5-1-18-jch) and by the Fundamental Research Funds for the Central Universities (201564018)
Corresponding author: Xiangzhao Mao, Tel:+86-532-82031360, Fax:+86-532-82032272, E-mail:xzhmao@ouc.edu.cn.

Abstract: Objective The aim of this study was to study the diversity of lipolytic enzymes in Stenotrophomonas maltophilia OUC_Est10.Methods Ion exchange chromatography, genome sequencing and heterologous expression were used to study the diversity of lipolytic enzymes in Stenotrophomonas maltophilia OUC_Est10.Results Stenotrophomonas maltophilia OUC_Est10 could secret a wide range of lipolytic enzymes (lipases and esterases) as revealed by ion exchange chromatography. The complete genome is of 4668743 bp in length, with an average GC content of 66.25%. Genome annotation indicated the presence of 33 candidate genes whose products possess the predicted lipolytic enzyme activities. Analysis of catalytic features was carried out by expressing five putative lipolytic enzyme genes, and lipolytic enzymes in OUC_Est10 had different catalytic properties.Conclusion We proved that Stenotrophomonas maltophilia OUC_Est10 was a good candidate to produce diverse lipolytic enzymes, with potential applications in various fields.
Key words: Stenotrophomonas maltophiliaseparationlipolytic enzymecomplete genome sequencecatalytic properties
嗜麦芽窄食单胞菌OUC_Est10全基因组测序及脂类水解酶多样性分析
董浩¹, 芮俊鹏², 孙建安¹, 李香真², 毛相朝¹
1. 中国海洋大学食品科学与工程学院, 山东 青岛 266003;
2. 中国科学院成都生物研究所, 环境与应用微生物重点实验室, 环境微生物四川省重点实验室, 四川 成都 610041
收稿日期：2017-05-03；修改日期：2017-08-05；网络出版日期：2017-08-15
基金项目：国家自然科学基金（31501516）；山东省重大特殊科技项目（2016YYSP016）；青岛市应用基础研究项目（16-5-1-18-jch）；中央高校基金（201564018）
作者简介：毛相朝，博士，中国海洋大学食品科学与工程学院教授，国家现代农业产业技术体系岗位科学家、教育部霍英东青年教师奖获得者。从事海洋水产资源生物加工的理论与技术研究，注重应用酶工程、代谢工程和发酵工程等生化工程技术进行海洋生物资源的高值化绿色全利用和海洋食品、功效物质的生物制造。主持国家自然科学基金3项、国家虾蟹产业技术体系项目1项、山东省重点研发计划项目1项以及其他省部级课题十几项。以第一作者或通讯作者在Biotechnology Advances、Journal of Cleaner Production、Molecular Nutrition and Food Research、Applied and Environmental Microbiology、Journal of Functional Foods、Journal of Agricultural and Food Chemistry等期刊发表SCI文章30余篇。获授权发明专利16项，计算机软件著作权1项。以第一完成人荣获教育部技术发明二等奖、海洋工程科学技术二等奖和青岛市科技进步一等奖各1项；2016年，荣获第十五届教育部霍英东青年教师奖、山东省自然科学学术创新奖和山东省优秀博士后等荣誉称号。.
通讯作者：毛相朝, Tel:+86-532-82031360, Fax:+86-532-82032272, E-mail:xzhmao@ouc.edu.cn.

摘要：目的 本研究的目的是研究嗜麦芽窄食单胞菌OUC_Est10中脂类水解酶的多样性。方法 使用离子交换层析、全基因组测序和异源表达三种方法研究嗜麦芽窄食单胞菌OUC_Est10中脂类水解酶的多样性。结果 离子交换层析结果显示嗜麦芽窄食单胞菌OUC_Est10可以分泌多种脂类水解酶。通过全基因组测序，我们给出了该菌的全基因组序列，该基因组大小为4668743 bp，GC含量为66.25%。通过详细的基因组序列分析，我们从该基因组中找到33个可能具有脂类水解酶活性的假定基因。通过异源表达OUC_Est10中的5个假定脂类水解酶基因，来研究其催化特性的多样性，结果显示这些脂类水解酶具有不同的催化特性。结论 我们证明了嗜麦芽窄食单胞菌OUC_Est10拥有多样的脂类水解酶，这暗示了它在不同领域中的应用潜力。
关键词：嗜麦芽窄食单胞菌分离脂类水解酶全基因组测序催化特性
Lipolytic enzymes, including esterases (3.1.1.1) and lipases (3.1.1.3), are members of the α/β hydrolase superfamily, which contain a catalytic triad (Ser-His-Asp/Glu)^[1]. With the development of industrial biotechnology, lipolytic enzymes are important catalysts in the biological manufacturing processes, such as food, bioenergy, detergent, pharmaceutical and advanced chemical manufacturing^[2-3]. Lipolytic enzymes can catalyze the cleavage and formation of ester bonds, and the catalytic mechanisms for esterases and lipases are similar^[4]. In organic media, both esterases and lipases catalyze various reactions, such as esterification, transesterification, aminolysis and interesterification^[5]. The increasing demand for novel biocatalysts has prompted the development of new methods which are used to screen for new genes, such as genome sequencing.
Stenotrophomonas maltophilia is a Gram-negative bacterium which, in our previous research, has been demonstrated to secrete esterases and lipases to prepare free astaxanthin efficiently^[6]. This result suggests that S. maltophilia OUC_Est10 is a good microbial resource for novel lipolytic enzymes. However, there are only few genome sequences available for S. maltophilia^[7-8]. They are all focused on the drug resistance of S. maltophilia, and to the best of our knowledge, there has been no study on the diversity of lipolytic enzymes. Since lipolytic enzymes have wide application in industry, it is necessary to investigate the diversity of relevant genes in the genome of S. maltophilia in order to discover more encoding genes for the biocatalysts.
In this study, the lipolytic enzymes of S. maltophilia OUC_Est10 (China General Microbiological Culture Collection Center (CGMCC), 10672) were induced in the fermentation medium (KH₂PO₄, 0.025%; MgSO₄·7H₂O, 0.025%; FeSO₄·7H₂O, 0.001%; beef extract, 0.1%; peptone, 1.0%; cholesterol oleate, 0.1%; Tween-80, 1.0%; H₂O, 100 mL; pH 7.0). Esterase activity and lipase activity were determined spectrophotometrically at 405 nm. Fermentation liquid of OUC_Est10 was centrifuged at 5439×g for 10 min, and the supernatant was subjected to ammonium sulfate precipitation. The resulting crude enzyme was put onto a DEAE-Sepharose Fast Flow column, which was previously equilibrated with buffer A (20 mmol/L Tris-HCl, pH 8.0). The unbound proteins were washed with buffer A until the absorbance at 280 nm reached the baseline. Furthermore, the bound proteins were eluted by a gradient of 0.1–0.7 mol/L NaCl in buffer A (Figure 1). The fractions resulted in a peak were analyzed in terms of esterase activity (p-nitrophenyl butyrate (pNPB) as substrate) and lipase activity (p-nitrophenyl palmitate (pNPP) as substrate).

Figure 1 Ion exchange chromatography map. The elution buffer was 20 mmol/L Tris-HCl buffer (pH 7.0) with NaCl concentration: peak 1, 0 mol/L; peak 2, 0.1 mol/L; peak 3, 0.2 mol/L; peak 4, 0.3 mol/L; peak 5, 0.4 mol/L; peak 6, 0.5 mol/L; peak 7, 0.6 mol/L; peak 8, 0.7 mol/L.

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The result showed that esterase activities and lipase activities were both determined in 8 peaks (Table 1), and it was finally proven that OUC_Est10 could secrete a wide range of lipolytic enzymes. S. maltophilia OUC_Est10 was isolated from the slaughterhouse soil in Qingdao, China. The slaughterhouse soil was rich in lipids, which might explain the wide range of lipolytic enzymes in OUC_Est10. The feature of multiple lipolytic enzymes in S. maltophilia OUC_Est10 contributes to the efficient hydrolysis of lipids in the slaughterhouse soil.
Table 1. Separation of lipolytic enzymes in the fermentation liquid of OUC_Est10

Peak	c (NaCl)/(mol/L)	Total protein/mg	Total activity/U			Specific activity/(U/mg)
			pNPB	pNPP		pNPB	pNPP
1	0	25.310	148.058	7.535		5.850	0.298
2	0.1	20.278	8.681	10.726		0.428	0.529
3	0.2	9.105	8.183	15.582		0.899	1.711
4	0.3	5.205	2.977	3.769		0.572	0.724
5	0.4	6.612	3.711	4.966		0.561	0.751
6	0.5	6.200	3.588	6.118		0.579	0.987
7	0.6	4.040	2.856	4.989		0.707	1.235
8	0.7	1.390	1.265	1.689		0.910	1.215

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To investigate the diversity of lipolytic enzyme genes, the genomic DNA of OUC_Est10 was extracted using Puregene Yeast/Bact. Kit B (QIAGEN, Maryland, USA) and sent to Tianjin Biochip Corporation (Tianjin, China) to sequence. The whole genomic DNA was sequenced on the single-molecule real-time (SRMT) sequencing platform PacBio RS Ⅱ (Pacific Biosciences, USA). The genomic sequence was obtained after the reads were de novo assembled using the RS Hierarchical Genome Assembly Process (HGAP) assembly protocol version 3.0 in SMRT Analysis version 2.3.0 (Pacific Biosciences, USA). The protein coding sequences (CDSs) were predicted using Glimmer 3.0^[9]. The procedures of tRNA and rRNA prediction were conducted using tRNAscan-SE^[10] and RNAmmer^[11], respectively. Functional annotation and metabolic pathway analysis were performed on the Integrated Microbial Genomes-Expert Review (IMG-ER) pipeline^[12].
The features of the complete genome sequence of S. maltophilia OUC_Est10 are listed in Table 2. The complete genome sequence consists of a single chromosome of 4668743 bp with a GC content of 66.25% (Figure 2), 3315 genes were identified in OUC_Est10, and the average number of genes in sequenced S. maltophilia was classified to functional categories according to clusters of orthologous genes (COG) designation (Table 3). Through genome searching, it was found that S. maltophilia was rich in lipolytic enzyme genes, which was one of the factors contributing to the virulence in S. maltophilia^[7]. After detailed analysis, 33 proteins with putative lipolytic enzyme activities are found in the genome sequence of OUC_Est10 (Table 4), and the locations of these genes were also marked in Figure 2. This indicates that OUC_Est10 may be a good candidate used for hydrolyzing lipids.
Table 2. Genome features of Stenotrophomonas maltophilia OUC_Est10

Feature	Value
Genome size/bp	4668743
G+C content/%	66.25
Protein coding genes (CDS)	4189
rRNA (5S, 16S, 23S)	13
tRNA	73
Miscellaneous RNA	38

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Figure 2 Circular diagram of the main features of OUC_Est10.

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Table 3. Number of genes of functional categories

Functional category	Average of S. maltophilia	OUC_Est10
General function prediction only	265.2±25.7	276
Transcription	241.2±27.9	261
Cell wall/membrane/envelope biogenesis	211.4±20.9	224
Signal transduction mechanisms	203.9±21.9	220
Amino acid transport and metabolism	207.6±18.3	218
Translation, ribosomal structure and biogenesis	207.3±15.3	218
Function unknown	198.6±20.9	214
Inorganic ion transport and metabolism	185.4±22.8	201
Energy production and conversion	184.9±15.6	190
Posttranslational modification, protein turnover, chaperones	147.1±12.7	155
Coenzyme transport and metabolism	146.4±14.7	152
Carbohydrate transport and metabolism	144.4±14.2	148
Lipid transport and metabolism	136.5±12.4	144
Cell motility	110.1±9.6	116
Defense mechanisms	97.0±11.2	111
Replication, recombination and repair	107.2±14.9	105
Secondary metabolites biosynthesis, transport and catabolism	80.3±7.7	82
Intracellular trafficking, secretion, and vesicular transport	77.5±10.2	73
Nucleotide transport and metabolism	67.8±5.1	71
Mobilome: prophages, transposons	30.2±16.7	51
Extracellular structures	47.2±6.6	48
Cell cycle control, cell division, chromosome partitioning	32.9±4.2	34
RNA processing and modification	1.0±0.4	1
Chromatin structure and dynamics	1.0±0.2	1
Cytoskeleton	1.0±0.2	1
Total genes	3133.0±283.4	3315

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Table 4. Genes with predicted lipolytic enzyme activities

No.	Length/bp	Function
LEn1	435	Esterase YdiI
LEn2	891	Pimeloyl-ACP ME carboxylesterase
LEn3	813	Pimeloyl-ACP ME carboxylesterase
LEn4	1260	Putative esterase
LEn5	927	Pimeloyl-ACP ME carboxylesterase
LEn6	645	Lipase_GDSL_2
LEn7	702	Lipase_GDSL_2
LEn8	942	Pimeloyl-ACP ME carboxylesterase
LEn9	795	Pimeloyl-ACP ME carboxylesterase
LEn10	837	carboxylesterase
LEn11	696	Pimeloyl-ACP ME carboxylesterase
LEn12	1308	Lipase_GDSL_2
LEn13	1029	Esterase
LEn14	954	Pimeloyl-ACP ME carboxylesterase
LEn15	1038	Fermentation-respiration switch protein FrsA
LEn16	1005	Esterase, PHB depolymerase family
LEn17	879	Pimeloyl-ACP ME carboxylesterase
LEn18	1188	Lipase_GDSL_2
LEn19	960	Pimeloyl-ACP ME carboxylesterase
LEn20	825	Pimeloyl-ACP ME carboxylesterase
LEn21	1911	Predicted acyl esterase
LEn22	1329	Lipase (class 3)
LEn23	789	Lipase_GDSL_2
LEn24	939	Esterase
LEn25	843	Esterase
LEn26	1203	Secretory lipase
LEn27	1854	Outer membrane lipase/esterase
LEn28	618	Lipase_GDSL_2
LEn29	831	Esterase
LEn30	660	carboxylesterase
LEn31	780	Pimeloyl-ACP ME esterase
LEn32	702	Pimeloyl-ACP ME carboxylesterase
LEn33	855	Pimeloyl-ACP ME carboxylesterase
ACP represents acyl-carrier protein; ME represents methyl ester.

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To prove the diversity and the potential use of lipolytic enzymes in S. maltophilia OUC_Est10, 16 pairs of primers were designed. The result of nucleic acid electrophoresis is shown in Supplementary material Figure S1. After being ligated to the pET-28a (+) vector, LEn4, LEn6, LEn12, LEn27 and LEn30 were successfully expressed in BL21 (DE3). Substrate specificity determination was carried out using p-nitrophenyl (pNP) esters with different acyl chain length. The results showed that they had different preferences for the length of fatty acid. LEn4, LEn12 and LEn27 had a preference for short-chain fatty acids, while LEn6 and LEn30 had a preference for medium-chain fatty acids (data not shown). It was worth noting that LEn27 was able to hydrolyze pNP esters with acyl chain length from 4 to 16, which indicated that LEn27 could be widely used for hydrolysis or synthesis of esters with different acyl chain lengths.
The organic solvents tolerance experiment showed that LEn4, LEn6, LEn12 and LEn27 had good organic solvent resistant properties, while LEn30 was highly denatured by organic solvents. LEn4, LEn6, LEn12 and LEn27 could all be used to synthesize ethyl esters (such as cinnamyl acetate) in a non-aqueous system (Figure 3). The determination of cinnamyl acetate was carried out using an HP-5 capillary column (30 m×0.25 mm×0.25 mm).

Figure 3 GC analysis of cinnamyl alcohol and cinnamyl acetate. Peak 1, the substrate (cinnamyl alcohol); Peak 2, the product (cinnamyl acetate).

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These experiments indicated that lipolytic enzymes in OUC_Est10 had varied catalytic properties, which could broaden the application range of OUC_Est10. Our further studies will focus on expressing these putative lipolytic enzymes, and they will be used in many fields according to their catalytic properties.
Nucleotide sequence accession number The complete genome sequence of S. maltophilia OUC_Est10 is available at the IMG database under the accession number Ga0114270 and the GenBank database under the accession number CP015612.

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