ARTP诱变钝顶螺旋藻突变体比较组学研究

doi:10.11949/0438-1157.20211340

化工学报 ›› 2021, Vol. 72 ›› Issue (12): 6298-6310.DOI: 10.11949/0438-1157.20211340

ARTP诱变钝顶螺旋藻突变体比较组学研究

苏楠¹(),吴亦楠¹(),陈韵亿¹,金丽华²,张翀¹,Aikawa Shimpei³,Hasunuma Tomohisa³,Kondo Akihiko³,邢新会^1,^4,⁵()

^1.清华大学化学工程系生物化工研究所，工业生物催化教育部重点实验室，合成与系统生物学中心，北京 100084
^2.北京电子科技职业技术学院生物技术系，北京 100029
^3.神户大学工程研究生院，化学科学与工程系，日本神户 657-8501
^4.清华大学深圳国际研究生院生物医药与健康工程研究院，广东深圳 518055
^5.深圳湾实验室医药健康技术与工程研究所，广东深圳 518055

收稿日期:2021-09-16 修回日期:2021-11-23 出版日期:2021-12-05 发布日期:2021-12-22
通讯作者: 邢新会
作者简介:苏楠（1983—），男，博士，sunan12345@126.com|吴亦楠（1992—），男，博士，wuyinan1992@qq.com
基金资助:
国家重点研发计划项目(2016YFD0102106);国家自然科学基金委国家重大科研仪器设备研制专项(21627812)

Comparative omics study of Spirulinaplatensis mutants based on ARTP mutagenesis breeding system

Nan SU¹(),Yinan WU¹(),Yinyee TAN¹,Lihua JIN²,Chong ZHANG¹,Aikawa SHIMPEI³,Hasunuma TOMOHISA³,Kondo AKIHIKO³,Xinhui XING^1,^4,⁵()

^1.Institute of Biochemical Engineering, Department of Chemical Engineering, Key Laboratory for Industrial Biocatalysis, Ministry of Education, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China
^2.Department of Biotechnology, Beijing Electronic Science and Technology Vocational College, Beijing 100029, China
^3.Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe 657-8501, Japan
^4.Institute of Biomedicine and Health Engineering, Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, Guangdong, China
^5.Medical and Health Technology and Engineering Institute, Shenzhen Bay Laboratory, Shenzhen 518055, Guangdong, China

Received:2021-09-16 Revised:2021-11-23 Online:2021-12-05 Published:2021-12-22
Contact: Xinhui XING

摘要/Abstract

摘要：

微藻作为地球上重要的生物资源，为水圈提供了大量的初级代谢产物，是合成生物学和生物制造研究和应用的重要底盘微生物。其中，钝顶螺旋藻(Spirulina platensis)具有多糖含量高、营养价值高、培养工艺成熟、应用范围广等特点，其诱变育种及比较组学研究可为微藻细胞工厂系统改造技术发展提供重要依据。本课题组前期通过常压室温等离子体(atmospheric and room temperature plasmas，ARTP)诱变方法获得了三株钝顶螺旋藻突变体。本研究在连续光照培养条件下，对三株突变体的重要生理特征进行了表征，发现突变株具有高絮凝表型，且重要化合物含量也与野生型具有一定的差异。进一步，本研究通过对其主要代谢产物的代谢组学分析和全基因组测序，对突变表型产生的机理进行了初步解析。

关键词: 钝顶螺旋藻, 絮凝, ARTP诱变, 代谢组学, 基因组比较组学, 生物能源, 生物工程, 生物技术

Abstract:

As an important biological resource on the earth, microalgae provides a large number of primary metabolites for the hydrosphere, and is an important chassis microorganism for the research and application of synthetic biology and biological manufacturing. Spirulina platensis is one of the cyanobacteria that has advantages of high polysaccharide content, high nutritional value, mature culture technology and wide application. The mutagenesis breeding and comparative omics studies can provide important basis for the development of microalgae cell-factory system modification. With atmospheric and room temperature plasmas (ARTP) mutagenesis method, we obtained three S. platensis mutants. By continuous light culture in this study, we characterized the mutants' important physiological characteristics. It was found that the mutants have much higher flocculation intensity than wild strain and differences in important metabolites content. Furthermore, through the mutants' metabolomic analysis and whole genome sequencing, we preliminarily analyzed the mechanism related to mutant phenotype.

Key words: Spirulina platensis, flocculation, ARTP mutagenesis, metabolomics, comparative genomics analysis, bioenergy, biological engineering, biotechnology

中图分类号:

Q 819

苏楠, 吴亦楠, 陈韵亿, 金丽华, 张翀, Aikawa Shimpei, Hasunuma Tomohisa, Kondo Akihiko, 邢新会. ARTP诱变钝顶螺旋藻突变体比较组学研究[J]. 化工学报, 2021, 72(12): 6298-6310.

Nan SU, Yinan WU, Yinyee TAN, Lihua JIN, Chong ZHANG, Aikawa SHIMPEI, Hasunuma TOMOHISA, Kondo AKIHIKO, Xinhui XING. Comparative omics study of Spirulinaplatensis mutants based on ARTP mutagenesis breeding system[J]. CIESC Journal, 2021, 72(12): 6298-6310.

图/表 16

图1 野生和三株突变藻株在连续光照培养条件下的生长曲线

Fig.1 Growth curves of wild-type S. platensis and the three mutants under the continuous light irradiation culture

图2 突变体藻株在生长过程中的絮凝度变化: (a) 各藻株摇床培养24 h后，静置5 min时的照片; (b) 各藻株摇床7 d培养过程中的絮凝度变化; (c) 经24 h摇床培养的藻株，静置培养不同时间后的絮凝度

Fig.2 Change of flocculation over culture time. (a) Pictures of S. platensis strain cultures after 24 h incubation in the shaker; (b) The change of flocculation of strain cultures during 7 d of incubation in the shaker; (c) The change of flocculation of strain cultures (harvested after 24 h of incubation in the shaker) after stationary sedimentation in tubes for different time periods

图3 各藻株在生长过程中的叶绿素含量测定

Fig.3 Change of chlorophyll concentrations over time

图4 各藻株在生长过程中的总碳水化合物含量测定

Fig.4 Change of total carbohydrate concentrations over time

图5 各藻株在生长过程中的葡萄糖含量测定

Fig.5 Change of glucose concentrations over culture time

图6 各藻株胞内主要代谢产物含量测定

Fig.6 Change of the concentrations of import metabolites over time

图7 测定中间代谢产物在各代谢途径中的分布红色标记中间产物为突变体与野生型含量有较显著差异的代谢产物;黄色标记中间产物为突变体与野生型含量没有显著差异的代谢产物Ac-CoA —乙酰辅酶A(acetyl-CoA); ADP-GLC —ADP-葡萄糖(ADP-glucose); CA —柠檬酸盐(citrate); E4P —赤藻糖(erythrose-4-phosphate); F6P —果糖-6-磷酸(fructose-6-phosphate); FUM —延胡索酸(fumarate); GAP —甘油醛-3-磷酸(glyceraldehyde-3-phosphate); G1P —葡萄糖-1-磷酸(glucose-1-phosphate); G3P —甘油-3-磷酸(glycerol-3-phosphate); G6P —葡萄糖-6-磷酸(glucose-6-phosphate); ICTT —异柠檬酸(isocitrate); LPA —溶血磷脂酸(lysobisphosphatidic acids); α-KG —α-酮戊二酸(2-ketoglutarate); MAA —苹果酸盐(malate); OAA —草酰乙酸盐(oxaloacetate); PEP —磷酸烯醇丙酮酸盐(phosphoenolpyruvate); PYR —丙酮酸(pyruvic acid); 3PGA —3-磷酸甘油酸(3-phosphoglycerate); R5P —核糖-5-磷酸(ribose-5-phosphate); Ru5P —核酮糖-5-磷酸(ribulose-5-phosphate); RuBP —核酮糖-1,5-二磷酸(ribulose-1,5-bisphosphate); S7P —景天庚糖-7-磷酸(sedoheptulose-7-phosphate); SBP —景天庚糖-1,7-二磷酸(sedoheptulose-1,7-bisphosphate); SSA —琥珀酸辅酶A(succinate-CoA); SUCC —琥珀酸(succinate); UDP-GLC—UDP-葡萄糖(UDP-glucose); X5P —木酮糖-5-磷酸(xylulose-5-phosphate)

Fig.7 Location of detected important metabolites in strain metabolic networks

表1 突变体突变基因GO聚类分析

Table 1 Gene Ontology clustering analysis of mutated strains

聚类分析	3-A10	3-B2	4-B3
生物学过程相关 (BP)	910	913	910
细胞组分相关 (CC)	103	101	101
分子功能相关 (MF)	668	672	667

图A1 螺旋藻藻株培养条件示意图/培养照片

Fig.A1 Culture conditions of Spirulina platensis

图A2 钝顶螺旋藻细胞干重与OD560标准曲线

Fig.A2 Standard curve between dry cell weight and OD560

图A3 总碳水化合物测定标准曲线

Fig.A3 Standard curve of carbohydrate determination

图A4 3-A10较WT有显著（0.1%阈值）差异的GO分布图

Fig.A4 GO distribution diagram of significant difference in gene sequence between 3-A10 and WT

图A5 3-B2较WT有显著（0.1%阈值）差异的GO分布图

Fig.A5 GO distribution diagram of significant difference in gene sequence between 3-B2 and WT

图A6 4-B3较WT有显著（0.1%阈值）差异的GO分布图

Fig.A6 GO distribution diagram of significant difference in gene sequence between 4-B3 and WT

表A1 螺旋藻各藻株基因组测序突变数量统计

Table A1 Statistics on the number of mutations in genome sequencing of Spirulina platensis

序列差异性	对照	3-A10	3-B2	4-B3
单碱基突变数量	241873	243818	241020	244320
过滤野生型基因后的数量	—	4063	2532	4536
同义单碱基突变	—	1040	681	1229
错义单碱基突变	—	1591	1059	1823
进化率	—	1.53	1.55	1.48
序列插入突变数量	345	353	330	362
过滤野生型基因后的数量	—	134	101	123
错义插入突变	—	82	56	81

表A2 突变体突变基因所属代谢网络分析

Table A2 Metabolic network analysis of mutant genes

代谢途径	突变体			代谢途径	突变体			代谢途径	突变体
代谢途径	3-A10	3-B2	4-B3	代谢途径	3-A10	3-B2	4-B3	代谢途径	3-A10	3-B2	4-B3
糖酵解	9	9	9	苯丙氨酸代谢	2	2	2	乙醛酸、二羧酸代谢	4	4	4
三羧酸循环	2	2	2	色氨酸代谢	1	1	1	丙酸代谢	1	1	1
磷酸戊糖途径	7	6	7	苯丙氨酸、酪氨酸、色氨酸合成	10	10	10	苯乙烯降解	1	1	1
果糖、甘露糖代谢	5	5	5	苯丙氨酸、酪氨酸、色氨酸合成	10	10	10	叶酸合成	8	8	8
半乳糖代谢	4	4	4	新生毒素生物合成	1	1	1	甲烷代谢	6	6	6
脂肪酸合成	3	3	3	β-丙氨酸代谢	2	2	2	碳固定及光合作用	8	8	8
氧化磷酸化	5	5	5	牛磺酸代谢	1	1	1	原核生物碳固定途径	6	5	5
光合作用	2	2	2	有机含硒化合物代谢	2	2	2	硫胺素代谢	3	3	3
嘌呤代谢	17	17	17	氨基甲酸代谢	2	2	2	核黄素代谢	7	7	7
丙氨酸、天冬氨酸、谷氨酸代谢	5	5	4	D-谷氨酰胺、D-谷氨酸代谢	1	1	1	维生素B₆代谢	1	1	1
丙氨酸、天冬氨酸、谷氨酸代谢	5	5	4	D-谷氨酰胺、D-谷氨酸代谢	1	1	1	烟酸盐、烟酰胺代谢	3	3	3
四环素合成	1	1	1	D-丙氨酸代谢	1	1	1	泛酸盐及辅酶a生物合成	8	8	8
黄曲霉毒素合成	1	1	1	谷胱甘肽代谢	7	7	7	泛酸盐及辅酶a生物合成	8	8	8
甘氨酸、丝氨酸、苏氨酸代谢	4	4	4	淀粉、蔗糖代谢	7	7	7	生物素代谢	1	1	1
甘氨酸、丝氨酸、苏氨酸代谢	4	4	4	氨基糖、核苷酸糖代谢	6	6	6	叶酸生物合成	3	3	3
半胱氨酸、蛋氨酸代谢	6	5	6	链霉素生物合成	5	5	5	阿特拉津降解	1	1	1
缬氨酸、亮氨酸、异亮氨酸生物合成	2	2	2	聚酮糖单元生物合成	2	2	2	卟啉、叶绿素代谢	15	15	15
缬氨酸、亮氨酸、异亮氨酸生物合成	2	2	2	丁松香和新霉素生物合成	2	2	2	萜类化合物生物合成	3	3	3
赖氨酸生物合成	2	2	2	丁松香和新霉素生物合成	2	2	2	萜类化合物生物合成	3	3	3
精氨酸、脯氨酸代谢	8	8	8	脂多糖生物合成	1	1	0	氮代谢	7	7	7
组氨酸代谢	4	4	4	肽聚糖生物合成	3	3	3	糖代谢	6	6	6
氨基苯甲酸降解	2		2	甘油酯代谢	2	2	2	苯丙素生物合成	1	1	1
药物代谢相关酶类	2	2	2	磷酸肌醇代谢	2	2	2	氨酰基-tRNA生物合成	6	6	6
磷脂酰肌醇信号系统	1	1	1	甘油磷脂代谢	4	4	4	氨酰基-tRNA生物合成	6	6	6
丙酮酸代谢	7	7	7	鞘脂类代谢	2	2	2	细胞色素P450代谢	1	1	1
鞘糖脂生物合成	1	1	1	鞘糖脂生物合成乳糖	1	1	1

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ARTP诱变钝顶螺旋藻突变体比较组学研究

Comparative omics study of Spirulinaplatensis mutants based on ARTP mutagenesis breeding system

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