Secretory production of xylanase in Corynebacterium glutamicum using its endogenous elements.

Wei Zhang; Xiuxia Liu; Yankun Yang; Zhonghu Bai

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Secretory production of xylanase in Corynebacterium glutamicum using its endogenous elements.

Author: Wei ZHANG ¹ ; Xiuxia LIU ¹ ; Yankun YANG ¹ ; Zhonghu BAI ¹
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1. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Key Laboratory of Industrial Biotechnology, Ministry of Education, National Engineering Laboratory of Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Publication Type:Journal Article
Keywords: Corynebacterium glutamicum; bicistronic; secretory production; xylanase
MeSH: Corynebacterium glutamicum; Promoter Regions, Genetic; Protein Sorting Signals; Protein Transport
From: Chinese Journal of Biotechnology 2019;35(3):425-434
CountryChina
Language:Chinese
Abstract: We constructed bicistronic expression system containing AH6 promoter, 5' UTR and its fore 38 bp sequence from Corynebacterium glutamicum, followed by a conserved Shine-Dalgarno (SD) sequence for xylanase expression. The two major secretory pathways signal peptide in C. glutamicum, Tat (CgR0949) and Sec (CspB) dependent signal peptide were added before xylanase for its secretion. Fed-batch cultivation was done in a 5 L jar for high-level xylanase secretion. The enzyme properties of the purified xylanase were then studied, including the effect of temperature and pH on its activity. The xylanase could be secreted into the culture supernatant when the Sec-dependent signal peptide CspB was used, but none was detected when CgR0949 was used. The secretory production level of xylanase in a flask was 486.2 U/mL and become 1 648.7 U/mL when in a 5 L jar, which was 3.4 fold as in the flask. The optimal pH and temperature of xylanase were pH 4.5 and 45 ℃, respectively. Its activity was 80% of initial activity after pretreatment at 4 ℃ for 24 h at pH 4-11, 95% after incubation below 50 ℃ for 15 min, and 20% when the temperature above 60 ℃. The xylanase could be efficiently secreted into the culture medium by C. glutamicum using its own genetic elements, and the secretion level could be improved through large-scale fed-batch cultivation. This bicistronic expression system can provide a useful tool for heterologous proteins secretion in C. glutamicum. In addition, the catalyze activity of xylanase could be further improved by enzyme properties study.