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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (4): 38-45    DOI: 10.13523/j.cb.20180406
研究报告     
匍枝根霉纤维二糖合成酶胞内糖基供体的初探及结构功能研究
张莹莹1,汤斌1,2(),堵国成1
1 江南大学生物工程学院 工业生物技术教育部重点实验室 无锡 214122
2 安徽工程大学生物与化学工程学院 芜湖 241000
Study on the Intracellular Glycosyl Donor and Structural Function of Cellobiose Synthase from Rhizopus stolonifer
Ying-ying ZHANG1,Bin TANG1,2(),Guo-cheng DU1
1 School of Biotechnology, Jiangnan University, Wuxi 214122, China
2 College of Biochemical Engineering, Anhui Polytechnic University, Wuhu 241000, China
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摘要：

纤维二糖可有效诱导丝状真菌产纤维素酶,前期研究表明匍枝根霉Rhizopus stolonifer TP-02具有纤维二糖合成酶(CBS),可以尿苷二磷酸葡萄糖(UDPG)为糖基供体合成纤维二糖,从而开启纤维素酶的自诱导合成途径。为研究R. stolonifer中纤维二糖的胞内合成途径,通过重叠PCR在GDP-葡糖焦磷酸化酶基因ggp中引入硫胺吡啶抗性基因ptrA,分别转化原菌TP-02和△ugp突变株,构建△ggp和△ugp/△ggp突变株。利用液质联用(LC-MS)检测突变株的胞内糖组分,发现ggp的缺失对胞内纤维二糖合成的影响较弱,但同时缺失ugp则将直接导致二糖合成受阻。RT-qPCR结果显示△ggp突变株中纤维素酶基因转录水平较原株TP-02下调20%左右,而△ugp/△ggp突变株中被测基因的转录水平则出现了高达80%左右的下调。同时对突变株纤维素酶表达水平进行研究,发现△ugp/△ggp突变株中几乎检测不到纤维素酶活力。结果显示,UDPG为R. stolonifer胞内合成纤维二糖的主要糖基供体,而GDPG可能是UDPG的替代物,在UDPG不足时维持胞内二糖合成。此外,利用生物信息学方法对CBS结构功能深入分析,经丙氨酸扫描确定其合成纤维二糖的关键作用残基为Asp210和Asp300,为后续进一步研究及理性改造提供方向和理论依据。
关键词： 纤维二糖;  纤维素酶;  匍枝根霉;  合成机制    
Abstract:

Cellobiose can effectively induce the production of cellulase by filamentous fungi. Our previous studies showed that Rhizopus stolonifer TP-02 has a cellobiose synthase (CBS) that can synthesize cellobiose by utilizing uridine diphosphate glucose (UDPG) as the glycosyl donor, thereby opening the self-induced synthesis pathway of cellulase from glucose. To study the intracellular biosynthesis pathway of cellobiose, the pyrithiamine resistance gene ptrA was inserted into the GDP-glucose pyrophosphorylase gene ggp by overlapping PCR. The fused gene ggp-ptrA was respectively transformed into TP-02 and △ugp for constructing the △ggp and △ugp/△ggp mutants. LC-MS was used to analyze the intracellular sugar components of mutants. The results showed that the lack of ggp has a weak effect on the synthesis of intracellular cellobiose, while the lack of ugp directly inhibits the synthesis of disaccharides. The result of RT-qPCR showed that the transcription level of cellulase genes in △ggp mutant are 20% lower than that of the original strain, while the tested gene in △ugp/△ggp are down-regulated by 80%. Furthermore, the expression levels of cellulase were also studied. However, the FPA activity of △ugp/△ggp was not detected. These results showed that UDPG is the major glycosyl donor for intracellular synthesis of cellobiose in R. stolonifer, whereas GDPG may be the substitute for UDPG, maintaining the synthesis of disaccharides in the absence of UDPG. In addition, bioinformatics methods were used to analyze the structure and function of CBS. Through alanine scanning the Asp210 and Asp300 were confirmed as the key residues of CBS to synthesize the cellobiose, providing a direction and theoretical basis for further research and rational transformation.
Key words: Cellobiose    Cellulase    Rhizopus stolonifer    Synthesis mechanism
收稿日期: 2017-12-06 出版日期: 2018-05-08
ZTFLH:  Q78  
基金资助: * 国家自然科学基金资助项目(31270135)
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引用本文:

张莹莹,汤斌,堵国成. 匍枝根霉纤维二糖合成酶胞内糖基供体的初探及结构功能研究[J]. 中国生物工程杂志, 2018, 38(4): 38-45.

Ying-ying ZHANG,Bin TANG,Guo-cheng DU. Study on the Intracellular Glycosyl Donor and Structural Function of Cellobiose Synthase from Rhizopus stolonifer. China Biotechnology, 2018, 38(4): 38-45.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180406    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I4/38

Primers Sequences (5'-3')
ggp-F 5'-ATGGATAAAAGTGAAGAAATTCTTG-3'
ggpPtrA-F1 5'-GAAAGCCCCTACTGGCTCATATGCATGTCTCCTCCAGCTGCCATCTAC-3'
ggpPtrA-F2 5'-GTAGATGGCAGCTGGAGGAGACATGCATATGAGCCAGTAGGGGCTTTC-3'
ggpPtrA-R1 5'-CGTCAGCGCGAGTGTGCTGAGTAATTTAGAAATAGCATGTTTGATGAG-3'
ggpPtrA-R2 5'-CTCATCAAACATGCTATTTCTAAATTACTCAGCACACTCGCGCTGACG-3'
ggp-R 5'-TTATAGGTTAGCATTATGCGTTTC-3'
表1  重叠PCR引物序列
Primers Sequences (5'-3')*
D210A-F 5'-GCCATCTTCGCCGCAGACTCC-3'
D210A-R 5'-GGAGTCTGCGGCGAAGATGGC-3'
S298A-F 5'-CAGCTTCGCCGAGGATTGGC-3'
S298A-R 5'-GCCAATCCTCGGCGAAGCTG-3'
D300A-F 5'-CTTCTCCGAGGCCTGGCAGC-3'
D300A-R 5'-GCTGCCAGGCCTCGGAGAAG-3'
E336A-F 5'-CACATTGCAGCCCGCAACCG-3'
E336A-R 5'-CGGTTGCGGGCTGCAATGTG-3'
W340A-F 5'-GCAACCGGGCCCACATTGGC-3'
W340A-R 5'-GCCAATGTGGGCCCGGTTGC-3'
表2  定点突变引物序列
Gene Primers Sequences (5'-3')
gpdA GPDA-F 5'-TACCGCTGCCCAGAACATC-3'
GPDA-R 5'-GGAGTGGCTGTCACCGTTC-3'
eg EG2-F 5'-TTATTGGGTTTGTTGTCAGGC-3'
EG2-R 5'-GTGCTTTGAATTGATTGCTCC-3'
bg BG3-F 5'-CGAGGACATTGCCTTGCTGA-3'
BG3-R 5'-GTTTGTGGAGGGAATAGTGGG-3'
cbh1 CBH1-F 5'-CTTATTGTGGAGGCGGTTGC-3'
CBH1-R 5'-CAGGTGGTATCGGTGGAGC-3'
cbh2 CBH2-F 5'-CCTGGCTATCCCATCCCTC-3'
CBH2-R 5'-CGTTCTGGGCTTTGATGTCG-3'
表3  RT-qPCR各引物序列
图1  突变株△ggp和△ugp/△ggp的构建
图2  色谱检测胞内糖组分
图3  各个基因的转录水平
图4  CBS与底物分子UDPG的互作
图5  CBS及其突变体合成产物的HPLC测定
图6  纤维二糖胞内合成途径示意图
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