Study on the Intracellular Glycosyl Donor and Structural Function of Cellobiose Synthase from <i>Rhizopus stolonifer</i>

doi:10.13523/j.cb.20180406

China Biotechnology

2018, Vol. 38

Issue (4): 38-45 DOI: 10.13523/j.cb.20180406

Study on the Intracellular Glycosyl Donor and Structural Function of Cellobiose Synthase from Rhizopus stolonifer

Ying-ying ZHANG¹,Bin TANG^1,²(

),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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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

Received: 06 December 2017 Published: 08 May 2018

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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.

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

Table 1 The sequences of primers for overlapping PCR

Table 2 The sequences of primers for site-directed mutagenesis

Table 3 The sequences of primers for RT-qPCR

Fig.1 Construction of △ggp and △ugp/△ggp mutants
(a) Construction of fusion gene ggp-ptrA by overlapping PCR M:5 000bp DNA marker; 1: ggp; 2: ggpA+ptrA; 3: ggp-ptrA; 4: ggpB+ptrA (b) PCR identification of the mutants M:10 000bp DNA marker; 1: ugp-hygB; 2: Genome DNA of △ugp/△ggp; 3: ggp-ptrA

Fig.2 Chromatographic detection of intracellular sugars

Fig.3 The transcription level of different genes
The data are presented as mean ± s.e.m. from three independent experiments. The statistical significance was calculated with the t-test, ^* P< 0.05

Fig.4 Interaction between CBS and its substrate molecular UDPG

Fig.5 HPLC profiles of the product synthesized by CBS and its mutants

Fig.6 Schematic diagram of the intracellular synthesis pathway of cellobiose


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