葡萄糖氧化酶在毕赤酵母中的高效分泌表达

Efficient Secretory Expression of Glucose Oxidase in Pichia pastoris

为了获得高效分泌表达葡萄糖氧化酶(GOD)的毕赤酵母,采用黑曲霉来源的GOD基因序列,按照毕赤酵母的密码子偏好性进行优化,构建AOX1启动子诱导的分泌表达载体pPIC9K-GOD和重组菌G/GOD,通过提高遗传霉素G418浓度筛选到第1代高拷贝高产量重组菌G/GODM,单位细胞干重胞外产酶水平可达5843.2 U/g,为G/GOD最高产量的8.2倍。在此基础上,进一步构建第2代高产菌,分别考察了共表达不同辅助折叠因子和强化中心碳代谢途径基因对GOD产量的影响。结果表明,共表达辅助折叠因子PDI1、PDI2和HAC1,可使GOD的胞外产酶产量分别提高32.7%、8.9%和54.4%;强化磷酸戊糖途径的SOL3基因和三羧酸循环的MDH1基因后,GOD的胞外产量提高了6.3%和11.6%。在50 L反应器中,共表达HAC1的高产菌G/GMH1胞外GOD单位菌体酶活(单位细胞干重)可达6656.6 U/g。

Glucose oxidase(GOD)is widely used in food,chemistry,medicine,biotechnology and other industrial applications.In this study,the gene GOD from Aspergillus niger was optimized according to the codon bias of pichia pastoris(P.pastoris),then it was used to construct the GOD secretory expression vector pPIC9K-GOD and the corresponding recombinant P.pastoris strain G/GOD.Subsequently,the higher concentration of geneticin(G418)was used to select the multicopy genomic integration strain G/GODM,and the extracellular GOD specific activity was improved to 5843.2 U/g,which was 8.2 times as high as that of the single copy strain G/GOD.On the basis of the first generation high producing strain,additional co-expression of folding factors,as well as the enhancement of central carbon metabolism,were used to construct the second generation strain.Co-expression of the protein folding factors PDI1,PDI2 and HAC1 improved the extracellular GOD activity by 32.7%,8.9%and 54.4%,respectively.With the co-expression of the pentose phosphate pathway gene SOL3 and the tricarboxylic acid cycle gene MDH1,the extracellular GOD activity was enhanced by 6.3%and 11.6%,respectively.The best strain G/GMH1 was selected for the bioreactor fermentation to achieve 6656.6 U/g of the extracellular GOD activity in a 50 L bioreactor,indicating its value for industrial application.