Expression ofAspergillus nigerglucose oxidase in yeastPichia pastorisSMD1168

Abstract

The aim of this study was to establish a system for high expression of Aspergillus niger glucose oxidase (GOD) with glyceraldehyde-3-phosphate dehydrogenase gene promoter (pGAP) in Pichia SMD1168. The GOD gene from A. niger accc30161 was inserted into pGAPZaA plasmid carrying a pGAP promoter and was expressed in yeast Pichia pastoris. The expression vector, pGAPZaA-GOD, was validated by colony polymerase chain reaction (PCR), agarose gel electrophoresis, restriction enzyme analysis and sequencing methods. The pGAPZaA-GOD vector was transformed into yeast P. pastoris SMD1168 by electroporation and the positive strain was validated by PCR. The expression and enzyme activity of the recombinant GOD were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and enzymatic detection. The recombinant GOD in yeast was purified by ion exchange chromatography, and its biochemical properties (dynamics, thermal and pH stability) were analysed. The obtained results showed that the expression vector, pGAPZaA-GOD, was successfully constructed to express A. niger accc30161 GOD protein. After transformation, the pGAPZaA-GOD DNA fragment had been integrated into the P. pastoris SMD1168-GOD genome. High expression of GOD was achieved in SMD1168-GOD, and the enzyme activity of GOD reached 107.18 U/mL in the supernatant of the culture medium at 30 C and pH 6. The activity of recombinant GOD protein in yeast was 1.35-fold higher than the commercial A. niger GOD, and its affinity to glucose, thermal stability and pH stability are similar to commercial GOD. Using pGAP promoter, A. niger GOD protein is efficiently expressed in recombinant P. pastoris SMD1168-GOD with defective protease.

Improvement Strategies, Cost Effective Production, and Potential Applications of Fungal Glucose Oxidase (GOD): Current Updates

“…A. niger GOD can be produced by S. cerevisiae at 9 g/L. Additionally, the P. pastoris yeast expression system was successfully used to produce active GOD originating from A. niger or from P. variable P16 that was not over glycosylated (Guo et al, 2010 ; Qiu et al, 2016 ). Naturally, A. niger or Penicillium spp.…”

Hypersecretory production of glucose oxidase in Pichia pastoris through combinatorial engineering of protein properties, synthesis, and secretion

“…Previous studies mainly focused on the overexpression of heterologous GOD genes with strong promoters and increasing GOD gene copy numbers in P. pastoris (Belyad et al, 2018;Gao et al, 2012;Qiu et al, 2016). Furthermore, efforts have been made to improve GOD production by co-expressing genes involved in the protein folding machinery and abnormal folding stress responses, such as Hac1p (Yu et al, 2020), SEC.…”

“…In contrast, Pichia pastoris enables the elimination of hyperglycosylation and secretion of heterologous proteins (Schwarzhans et al, 2017) and has been engineered for the secretory expression of GOD. Previous studies mainly focused on the overexpression of heterologous GOD genes with strong promoters and increasing GOD gene copy numbers in P. pastoris (Belyad et al, 2018; Gao et al, 2012; Qiu et al, 2016). Furthermore, efforts have been made to improve GOD production by co‐expressing genes involved in the protein folding machinery and abnormal folding stress responses, such as Hac1p (Yu et al, 2020), SEC.…”

Expression of Aspergillus niger glucose oxidase in Pichia pastoris and its antimicrobial activity against Agrobacterium and Escherichia coli

“…GODs are produced by molds such as Aspergillus niger and Penicillium ( Frederick et al, 1990 ; Meng et al, 2014 ; Qiu et al, 2016 ; Farshad, Amin & Catherine, 2018 ). Many literatures reported that GOD could inhibit the growth of microbes in foods or food preparation media ( Tiina & Sandholm, 1989 ; Yoo & Rand, 1995 ; Li et al, 2019 ).…”