L-glutamate oxidase (GLOD) and L-amino acid oxidase (AAO) were reported to be capable of convert L-glutamic acid to α-aketoglutaric acid (α-KG). These two enzymes gene have been successfully expressed by using pHBM905BDM in Pichia pastoris to produce α-aketoglutaric acid from L-glutamic acid in our previous studies. Here these two enzymes were displayed on P. pastoris to achieve the conversion. We constructed multi-copy expression plasmids using plasmid pHBM905BDM. By using this plasmid, multi-copy strains were constructed and named as PGLOD(1–3)-AGα1 and PAAO(1–3)-AGα1, respectively. The following results showed that expression of GLOD(1–3)-AGα1 and AAO(1–3)-AGα1 in multi-copy strains increased as designed and strain PGLOD3-AGα1 and PAAO3-AGα1 was chosen for high-density fermentation and enzyme activity experiments. By using a multi-copy expression approach and high-density fermentation, we achieved a GLOD expression yield of 688.5 U/g dry cell weight and AAO expression yield of 626.7 U/g dry cell weight. By using displayed GLOD, the average production rate of L-glutamic acid to α-KG was 6.22 g/L/h and the highest α-KG titer (124.5 g/L) was converted from 135 g/L L-glutamic acid. By using displayed AAO, the average production rate of L-glutamic acid to α-KG was 5.78 g/L/h and the highest α-KG titer (115.6 g/L) was converted from 135 g/L L-glutamic acid. It showed that displaying enzymes on P. pastoris are suitable for use in industrial applications.

据报道，L-谷氨酸氧化酶（GLOD）和L-氨基酸氧化酶（AAO）能够将L-谷氨酸转化为α-酮戊二酸（α-KG）。在我们先前的研究中，通过在巴斯德毕赤酵母中使用pHBM905BDM成功表达了这两种酶基因，从而从L-谷氨酸生产α-酮戊二酸。这两种酶都显示在巴斯德毕赤酵母上实现转换。我们使用质粒pHBM905BDM构建了多拷贝表达质粒。通过使用该质粒，构建了多拷贝菌株并将其分别命名为PGLOD（1-3）-AGα1和PAAO（1-3）-AGα1。以下结果表明，GLOD（1-3）-AGα1和AAO（1-3）-AGα1在多拷贝菌株中的表达按设计增加，并且选择了菌株PGLOD3-AGα1和PAAO3-AGα1用于高密度发酵和酶活动实验。通过使用多拷贝表达方法和高密度发酵，我们获得了688.5 U / g干细胞重量的GLOD表达产量和626.7 U / g干细胞重量的AAO表达产量。通过使用显示的GLOD，L-谷氨酸向α-KG的平均生产率为6.22g / L / h，并且最高的α-KG效价（124.5g / L）由135g / L L-谷氨酸转化。通过使用显示的AAO，L-谷氨酸向α-KG的平均生产率为5.78 g / L / h，最高的α-KG效价（115.6 g / L）由135 g / L L-谷氨酸转化而来。结果表明在巴斯德毕赤酵母适合用于工业应用。