基于转录组学挖掘兽疫链球菌内源表达元件及高产透明质酸应用

doi:10.13560/j.cnki.biotech.bull.1985.2024-0541

摘要/Abstract

摘要：

【目的】构建兽疫链球菌内源性表达元件文库，并探究其在提高透明质酸产量中的应用。【方法】通过对兽疫链球菌发酵指数期和平台期进行转录组测序分析，初步筛选32种高、中、低3种强度的启动子与RBS组合（表达元件PR）；进一步利用绿色荧光蛋白基因转录水平及荧光强度来验证PR元件的强度；最后，利用筛选到的较强元件PR31过表达透明质酸合成关键基因hasA, hasB, hasC, hasD, hasE，并采用2 L发酵罐评价强表达元件在提高透明质酸产量的效果。【结果】上述基因的相对转录水平分别提高至原来的8.17、7.32、3.72、39.48、9倍。其中，过表达hasA及hasD后，透明质酸产量相对于野生型分别提高了43％和31％，达到5.654 g/L和5.283 g/L。【结论】成功构建了兽疫链球菌内源表达元件文库，可用于透明质酸合成途径强化及竞争途径弱化等代谢工程改造。

关键词: 兽疫链球菌, 转录组测序, 启动子, 核糖体结合位点, 透明质酸

Abstract:

【Objective】 This work is aimed at establishing a library of endogenous expression elements of Streptococcus epizooticus，and investigating the application of it in increasing hyaluronic acid yields.【Method】 The 32 kinds of high, medium and low strength promoter and RBS combination（expression element, PR）were preliminarily screened, through transcriptomics analysis of S. zooepidemicus in the exponential or stationary stage of fermentation. Then, the strength of the 32 PRs was verified via detecting the transcriptional level of gfp gene and fluorescence intensity of GFP. Eventually, the key hyaluronic acid synthesis gene hasA, hasB, hasC, hasD, and hasE was overexpressed respectively with the selected strong expression element PR31, and the impact of the strong PR on enhancing hyaluronic acid production was assessed through a 2 L fermentation.【Result】 The relative transcription of the aforementioned genes were elevated to 8.17, 7.32, 3.72, 39.48 and 9-fold. Among them, the overexpression of hasA and hasD increased the hyaluronic acid yield by 43% and 31%, respectively, compared to the wild type, reaching 5.654 g/L and 5.283 g/L.【Conclusion】 A library of endogenous expression elements of S. zooepidemicus had been constructed successfully, which can be employed for metabolic engineering, such as reinforcing the hyaluronic acid synthesis pathway or weakening competitive pathways.

Key words: Streptococcus zooepidemicus, transcriptomics analysis, promoter, ribosome binding site, hyaluronic acid

赵锐, 狄靖宜, 张广通, 刘浩, 高伟霞. 基于转录组学挖掘兽疫链球菌内源表达元件及高产透明质酸应用[J]. 生物技术通报, 2024, 40(10): 296-304.

ZHAO Rui, DI Jing-yi, ZHANG Guang-tong, LIU Hao, GAO Wei-xia. Screening Endogenous Expression Elements in Streptococcus zooepidemicus via Transcriptomics Analysis and Applications for High Yield of Hyaluronic Acid[J]. Biotechnology Bulletin, 2024, 40(10): 296-304.

图/表 7

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表达元件Expression element	表达元件控制的基因功能Gene functions controlled by expression elements	FPKM	大小Size/bp
PR0	cp25（常见的用于兽疫链球菌的异源表达元件）^[26]	-	59
PR1	3-phosphoshikimate 1-carboxyvinyltransferase	65.45	130
PR2	Conserved hypothetical protein	65.68	423
PR3	tRNA-specific 2-thiouridylase MnmA	65.97	283
PR4	Maltodextrin transport system permease protein MalF	89.14	299
PR5	Alkaline phosphatase synthesis transcriptional regulatory protein PhoP	97.38	149
PR6	Sugar uptake protein	105.48	500
PR7	16S rRNA m（2）G 1207 methyltransferase	117.68	233
PR8	Glutamate dehydrogenase	137.6	500
PR9	UDP-N-acetylmuramoyl-L-alanyl-D-glutamate synthetase	430.41	152
PR10	Putative cell-cycle regulation histidine triad protein HIT	436.44	472
PR11	L-lactate dehydrogenase	561.89	191
PR12	ABC transporter ATP-binding protein	644.25	120
PR13	S-adenosylmethionine:tRNA ribosyltransferase-isomerase	708.09	268
PR14	Methylated-DNA--protein-cysteine methyltransferase	709.89	492
PR15	30S ribosomal protein S4	855.87	500
PR16	50S ribosomal protein L28	1029.4	500
PR17	Cell division protein	1152.84	211
PR18	50S ribosomal protein L32	1734.26	269
PR19	NADH oxidase	1980.71	249
PR20	30S ribosomal protein S13	2550.44	159
PR21	Fructose-bisphosphate aldolase	4387.94	200
PR22	6-phosphofructokinase	4644.17	79
PR23	phosphoglycerate kinase	4912.63	200
PR24	C3-bisphosphoglycerate-dependent phosphoglycerate mutase GpmA	4918.12	122
PR25	UDP-glucose 6-dehydrogenase HasB	6662.7	253
PR26	Hyaluronan synthase HasA	7891.36	396
PR27	Glyceraldehyde-3-phosphate dehydrogenase	9036.98	112
PR28	Cold shock protein	10105.49	206
PR29	Conserved hypothetical protein	11533.49	500
PR30	Hypothetical protein	12563.03	340
PR31	Szp protein	13899.81	384
PR32	Phosphopyruvate hydratase	20029.66	213

表达元件Expression element	表达元件控制的基因功能Gene functions controlled by expression elements	FPKM	大小Size/bp
PR0	cp25（常见的用于兽疫链球菌的异源表达元件）^[26]	-	59
PR1	3-phosphoshikimate 1-carboxyvinyltransferase	65.45	130
PR2	Conserved hypothetical protein	65.68	423
PR3	tRNA-specific 2-thiouridylase MnmA	65.97	283
PR4	Maltodextrin transport system permease protein MalF	89.14	299
PR5	Alkaline phosphatase synthesis transcriptional regulatory protein PhoP	97.38	149
PR6	Sugar uptake protein	105.48	500
PR7	16S rRNA m（2）G 1207 methyltransferase	117.68	233
PR8	Glutamate dehydrogenase	137.6	500
PR9	UDP-N-acetylmuramoyl-L-alanyl-D-glutamate synthetase	430.41	152
PR10	Putative cell-cycle regulation histidine triad protein HIT	436.44	472
PR11	L-lactate dehydrogenase	561.89	191
PR12	ABC transporter ATP-binding protein	644.25	120
PR13	S-adenosylmethionine:tRNA ribosyltransferase-isomerase	708.09	268
PR14	Methylated-DNA--protein-cysteine methyltransferase	709.89	492
PR15	30S ribosomal protein S4	855.87	500
PR16	50S ribosomal protein L28	1029.4	500
PR17	Cell division protein	1152.84	211
PR18	50S ribosomal protein L32	1734.26	269
PR19	NADH oxidase	1980.71	249
PR20	30S ribosomal protein S13	2550.44	159
PR21	Fructose-bisphosphate aldolase	4387.94	200
PR22	6-phosphofructokinase	4644.17	79
PR23	phosphoglycerate kinase	4912.63	200
PR24	C3-bisphosphoglycerate-dependent phosphoglycerate mutase GpmA	4918.12	122
PR25	UDP-glucose 6-dehydrogenase HasB	6662.7	253
PR26	Hyaluronan synthase HasA	7891.36	396
PR27	Glyceraldehyde-3-phosphate dehydrogenase	9036.98	112
PR28	Cold shock protein	10105.49	206
PR29	Conserved hypothetical protein	11533.49	500
PR30	Hypothetical protein	12563.03	340
PR31	Szp protein	13899.81	384
PR32	Phosphopyruvate hydratase	20029.66	213

培养基 Medium	组成型强表达元件 Constitutive strongly expressive elements	指数期强表达元件 Strongly expressed elements in exponential phase	稳定期强表达元件 Strongly expressed elements in stable phase
THY	PR15、PR17、PR26、PR30、PR32	PR8、PR11、PR16、PR18、PR19、PR21、PR24、PR29	PR12、PR31
FSB	PR8、PR16、PR24、PR26、PR28、PR30、PR31	PR5、PR13	PR6、PR12、PR21、PR22、PR23、PR32

培养基 Medium	组成型强表达元件 Constitutive strongly expressive elements	指数期强表达元件 Strongly expressed elements in exponential phase	稳定期强表达元件 Strongly expressed elements in stable phase
THY	PR15、PR17、PR26、PR30、PR32	PR8、PR11、PR16、PR18、PR19、PR21、PR24、PR29	PR12、PR31
FSB	PR8、PR16、PR24、PR26、PR28、PR30、PR31	PR5、PR13	PR6、PR12、PR21、PR22、PR23、PR32