Optimization Of Secretory Expression Of Glucose Oxidase In Pichia Pastoris

Glucose oxidase(Glucose oxidase,GOD)is widely used in food,chemistry,medicine,biotechnology and other fields.Traditionally,Aspergillus niger or Penicillium has been used for GOD production.However,the productivity of GOD is low,and it is difficult to separate and purify GOD due to its intracellular distribution.Here,to achieve a high and efficient GOD production in Pichia pastoris,several metabolic engineering strategies were implemented to construct recombinant strains,and their performance was investigated in the 5-L and 50-L scale bioreactor,thus providing the foundation for industrial production and application of recombinant GOD.The original nucleotide sequence of Asperigillus niger GOD gene was optimized according to the codon bias of P.pastoris,synthesized and used to construct the GOD secretion expression vector under the control of AOX1 promoter.Then,the recombinant strain G/GOD was constructed by transforming the expression vector into P.pastoris.Furthermore,the recombinant strain G/GODM containing multicopy GOD integrants was obtained through G418 gradient resistance screen,showing a secretion ratio of 74.6%and the extracellular GOD specific activity of 5843.24 U/g·DCW in shake flasks.On the basis of G/GODM,additional co-expression of vgb gene and folding factors,as well as the enhancement of central carbon metabolism,was tried to further improve GOD production.Co-expression of vgb gene improved cell growth in the late phase of shake flask culture,but decreased the GOD production.With the co-expression of PDI1,PDI3 and HAC1,the extracellular GOD activity were improved by 33.8%,11.3%and 39.3%,and the corresponding secretion ratio was increased by 3.3%,2.6%and 9.1%,respectively.Extracellular GOD activity in strains co-expressing sol3(in the PP pathway)and mdh1(in the TCA cycle)was enhanced by 7.3%and 17.5%,respectively.Among all these engineered strains,the highest extracellular specific GOD activity(71.72 U/mL,9022.07 U/g·DCW)was obtained in the recombinant strain G/GMH1 containing an additional HAC1 gene.The performance of the recombinant G/GOD,G/GODM,G/GMP1 and G/GMH1 strain was evaluated in the 5-L bioreactor.The highest GOD production in G/GMH1 was confirmed in the 5-L bioreactor to be 953.53 U/mL and 7030.37 U/g·DCW,respectively.We found that relatively high methanol concentration(952.07 U/mL)and high OUR level(180 mmol/L·h)were beneficial for cell growth and GOD production in G/GMH1.In a 50-L bioreactor,the extracellular GOD activity was improved to 1150.45 U/mL in G/GMH1,indicating its bright prospect for industrial application.