纤维二糖水解酶N-糖基化对其在草酸青霉中的分泌和酶活影响<sup>*</sup>

doi:10.13523/j.cb.2101002

中国生物工程杂志

2021, Vol. 41

Issue (4): 18-29 DOI: 10.13523/j.cb.2101002

研究报告

纤维二糖水解酶N-糖基化对其在草酸青霉中的分泌和酶活影响^*

林艳梅,罗湘,李瑞杰,秦秀林(

),冯家勋

广西大学生命科学与技术学院亚热带农业生物资源保护与利用国家重点实验室广西微生物与酶工程技术研究中心南宁 530004

Probing the Role of N-glycosylation on the Catalytic Domain in the Activity and Secretion of Fungal Cellobiohydrolase

LIN Yan-mei,LUO Xiang,LI Rui-jie,QIN Xiu-lin(

),FENG Jia-xun

College of Life Science and Technology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi Research Center for Microbial and Enzyme Engineering Technology, Guangxi University, Nanning 530004, China

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摘要：

目的:纤维素酶水解天然纤维素产生易被微生物利用的葡萄糖是进行生物炼制的关键。丝状真菌分泌的纤维素酶大多数是经过糖基化修饰的,研究丝状真菌纤维二糖水解酶(Cel7A)的催化功能域N-糖基化修饰对其分泌及酶活的影响,有助于优化纤维素酶的表达。方法:利用定点突变将草酸青霉和深绿木霉Cel7A催化功能域的N-糖基化位点去除,构建突变体PoCel7A^*和TaCel7A^*。以草酸青霉为宿主构建分泌表达PoCel7A^*、TaCel7A和TaCel7A^*的重组菌,检测N-糖基化去除对Cel7A分泌和酶活力的影响。结果:PoCel7A催化功能域的N-糖基化去除对其蛋白分泌和酶活力无影响。TaCel7A催化功能域的N-糖基化去除不影响其蛋白分泌;但突变体的pNPCase、FPase和Avicelase酶活力分别下降了21.2%,15.2%和17.6%。去除Cel7A催化功能域N-糖基化,加强了细胞内UPR响应。外源蛋白TaCel7A和TaCel7A^*的表达也加强了胞内UPR响应。结论:不仅可以为丝状真菌Cel7A的酶工程改造提供理性设计思路,而且为进一步了解糖基化在纤维素酶降解纤维素过程中的作用及机理奠定一定基础。

关键词： 纤维素酶; 纤维二糖水解酶; N-糖基化; 丝状真菌

Abstract:

Objective: Cellulases are responsible for the turnover of plant cell wall polysaccharides in the biosphere, and thus form the foundation of enzyme engineering efforts in biofuels research and industrial processes. Many of these carbohydrate-active enzymes from filamentous fungi contain both N-glycans and O-glycans, which in turn can unpredictably affect activity and secretion. Understanding the roles of glycosylation in the function of cellulase is important for further improvement of the enzyme technology for biomass conversion. Methods: The N-glycans defective mutants PoCel7A^* and TaCel7A^*, removal of the N-glycans on the catalytic domain of cellobiohydrolases from Penicillium oxalicum (PoCel7A) and Trichoderma atroviride (TaCel7A), were constructed using site directed mutagenesis. The extracellular protein concentration and enzymatic activity of the recombinant strains, expressing of PoCel7A ^*, TaCel7A or TaCel7A^*, were determined to investigate whether the removal of N-glycosylation affects the activity and secretion of cellobiohydrolases. Results: The mutant PoCel7A^* homologous expression in P. oxalicum, the removal of Asn137 glycosylation site at PoCel7A hardly affected the activity and secretion of Cel7A. However, after the Asn287 glycosylation site of TaCel7A was removed (TaCel7A ^*) and heterologous expression in P. oxalicum, the pNPCase, FPase and Avicelase activity of mutant decreased by 21.2%, 15.2% and 17.6%, respectively. Furthermore, compared to the parental strain, the expression levels of the UPR marker genes pdi1, bip1 and hac1 were significantly induced in recombinant strains, indicating increased demand for protein folding and transport capacity in recombinant strains. Conclusion: These results indicate that differential roles of N-glycan modifications in contributing to the function of Cel7A and highlight the potential of improving the activity and secretion of Cel7A by tuning proper interactions between glycans and functional residues.

Key words: Cellulase Cellobiohydrolase N-glycosylation Filamentous fungi

收稿日期: 2020-12-31 出版日期: 2021-04-30

ZTFLH:

Q819

基金资助: *国家自然科学基金(31300076);广西自然科学基金资助项目(2019GXNSFAA245001);广西自然科学基金资助项目(2018GXNSFAA281005);广西自然科学基金资助项目(2017GXNSFAA198136)

通讯作者: 秦秀林 E-mail: xiulinqin@gxu.edu.cn

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引用本文:

林艳梅,罗湘,李瑞杰,秦秀林,冯家勋. 纤维二糖水解酶N-糖基化对其在草酸青霉中的分泌和酶活影响^*[J]. 中国生物工程杂志, 2021, 41(4): 18-29.

LIN Yan-mei,LUO Xiang,LI Rui-jie,QIN Xiu-lin,FENG Jia-xun. Probing the Role of N-glycosylation on the Catalytic Domain in the Activity and Secretion of Fungal Cellobiohydrolase. China Biotechnology, 2021, 41(4): 18-29.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2101002 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I4/18

引物名称	引物序列(5'- 3')	用途
PoCel_Fu	ATGAAGGGTTCCATCTCCTACC	Amplification of the Pocel7A^*
PoCel_Ru	TTACAGGCATGGGAGTAGTACTC
PoN137F	ACCTGCTGGAGGACGACACCACTTACCAGAAGTTCAACCTCCTGAACCAG	For site directed mutagenesis of Pocel7A
PoN137R	GGTAAGTGGTGTCGTCCTCCAGCAGGTAGAGACGAGAACCGATGT
TaCel_cF	ATGTATCAGAAACTAGCGG	Amplification of the Tacel7A cDNA
TaCel_cR	TTACAAGCACTGAGAGTAGTATTG
TaCel_Fu	ATGTATCAGAAACTAGCGGCAA	Amplification of the Tacel7A^*
TaCel_Ru	TTACAAGCACTGAGAGTAGTATTGG
TaN287F	CCATACCGCTTGGGCGACCACACTTTCTATGGCC	For site directed mutagenesis of Tacel7A
TaN287R	CCATAGAAAGTGTGGTCGCCCAAGCGGTATGGGT
5'PoCel_F1	GTCCGAAGAAAGGGTACAGC	Amplification of the 5' homologous region of Pocel7A
5'PoCel_R1	ATGCTCCTTCAATATCATCTTCTTGTGATGGATTGGATCAAAGATC
3'PoCel_F	TAATCCTTCTTTCTAGAGTCTTGAGTGGTCGTCGAGGTCCTGCTCG	Amplification of the 3' homologous region of Pocel7A
3'PoCel_R	AACTCCACCGCCACCGTCTC
G418_F1	GATCTTTGATCCAATCCATCACAAGAAGATGATATTGAAGGAGC	Amplification of the kan expression cassette
G418_R1	TCGACGACCACTCAAGACTCTAGAAAGAAGGATTACCTCT
5'PoCel_F2	GTCCGAAGAAAGGGTACAGC	Amplification of the 5' flanking region of cel7A expression cassette
5'PoCel_R2	AGGAGATGGAACCCTTCATTGTGATGGATTGGATCAAAGATCA
G418_F2	TAAAGAAGATGATATTGAAGGAG	Amplification of the kan
G418_R2	ACCTCGACGACCACTCAAGACTCTAGAAAGAAGGATTAC
G418_F3	TACTACTCTCAGTGCTTGTAAAGAAGATGATATTGAAGGAG
TaCel_F	TCTTTGATCCAATCCATCACAATGAAGGGTTCCATCTCCTACC	Amplification of the Tacel7A
TaCel_R	TCCTTCAATATCATCTTCTTTACAAGCACTGAGAGTAGTATTG
PoCel_F	TCTTTGATCCAATCCATCACAATGAAGGGTTCCATCTCCTAC	Amplification of the mutant Pocel7A^*
PoCel_R	TCCTTCAATATCATCTTCTTTACAGGCACTGGGAGTAGTAC
FuCel_F	AATCCTGCTCCAAGGTCGTT	Amplification of the cel7A expression cassette
FuCel_R	CGTGTCATGAGATTCCAACCTT
Vcel7AF	TGAGTTTCGCTGCTCCCAAG	For checking correct integration
VGR	ATCGATGCTTCGGTAGAATAGG
VGF	CTCAAGCCTACAGGACACAC
Vcel7AR	CTCAGGATGGTGTTTCAAGA
actin-qF	CTCCATCCAGGCCGTTC	qRT-PCR for quantification of actin
actin-qR	CATGAGGTAGTCGGTCAAG
bip1-qF	CCTGACGAGGCTGTTGCTTTC	qRT-PCR for quantification of bip1
bip1-qR	TGACGGAGTTACGGGGGATG
pdi1-qF	GCCCTCCATCGTTCTCTACAAG	qRT-PCR for quantification of pdi1
pdi1-qR	CTCGGCGAAGATGTAGGCAA
hac1-qF	ATAGTATCACCAGCCAGTCGC	qRT-PCR for quantification of hac1
hac1-qR	GGGATGAACTTTACCAATGCC

表1 引物序列

图1 利用SWISS-MODEL同源建模分析P. oxalicum HP7-1的PoCel7A三维结构及其N-糖基化位点突变

图2 利用SWISS-MODEL同源建模分析T. atroviride HP35-3的TaCel7A三维结构及其N-糖基化位点突变

图3 Pocel7A基因缺失的草酸青霉重组菌ΔPocel7A构建

图4 Cel7A及其突变体表达重组菌构建

图5 重组菌胞外蛋白浓度

图6 Cel7A催化功能域N-糖基化位点缺失对纤维素酶酶活力影响

图7 重组菌中UPR相关基因转录水平

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