共表达分子伴侣提高漆酶在毕赤酵母中的分泌

Improving the Secretory Expression of Laccase in Pichia pastoris by Co-Expressing Chaperones

摘要:

通过共表达分子伴侣的策略提高漆酶在毕赤酵母中的产量。前期基于Pichia pastoris密码子偏好性，合成了来源于Cerrena sp. WR1的漆酶基因，克隆至pPIC9K，转化Pichia pastoris GS115，得到重组菌株PP-L。在PP-L中，分别过量表达蛋白质折叠（BIP、ERO1）、囊泡运输（SEC53、SEC1）、胁迫应激（HAC1、GCN4）相关的6种分子伴侣。结果显示，共表达这6种分子伴侣对分泌表达漆酶的菌株PP-L的正常生长没有影响；共表达BIP使重组菌胞外酶活提高359%，共表达ERO1胞外酶活反而降低22%；共表达其它4种分子伴侣对胞外酶活提高18%~53%。组合共表达BIP和HAC1的重组菌P1较PP-L胞外酶活提高了602%，酶活达到3 896 U/L。推测由于强化内质网中BIP水平进而提高了蛋白质折叠能力，从而提高其分泌效率。该研究结果将有助于发酵法生产漆酶的工业化。

Abstract:

In this study，the co-expression of chaperones was used to improve the production of laccase in Pichia pastoris. According to the codon bias of Pichia pastoris，the gene of laccase from Cerrena sp. WR1 was optimized and cloned into pPIC9K. The resulted plasmid was transformed into Pichia pastoris GS115，yielding the strain PP-L. In the PP-L，we co-expressed six chaperones，relating to protein folding（BIP and ERO1），transportation（SEC53 and SEC1） and stress response（HAC1 and GCN4）. The results indicated that the co-expressions of chaperones had no effect on the cell growth of strain PP-L. Expression of BIP increased the extracellular enzyme activities by 359% while ERO1 decreased that by 22%. The other four chaperones increased the extracellular activities by 18%~53%. In contrast to strain PP-L，simultaneous co-expression of BIP and HAC1（strain P1） increased 602% of the extracellular activities，reaching 3 896 U/L. It was suggested that the overexpression of BIP in endoplasmic reticulum improved the ability of protein folding，thus improving the secretion efficiency of laccase. The results obtained here may improve the industrialization of laccase.