High-level Expression Of Glucose Oxidase In Pichia Pastoris

Glucose oxidase?Glucose oxidase,EC 1.1.3.4?,referred to as GOD,is one aerobic dehydrogenase,it can only effectively oxidize?-D-glucose to gluconic acid and effectively remove oxygen.It has been developed as an important industrial enzyme and widely used in medicine,food,fermentation,chemical,biological and other fields.Glucose oxidase mainly exists in many kinds of animal,plant and fungus and bacteria.However,the content of GOD in plants and animals is low and there are some limitations,and the amount of GOD produced by bacteria is also relatively small,so it is difficult to reach the ideal yield.At present,the main production strains of GOD in industry are Aspergillus Niger and Penicillium Niger,but the production of GOD by Aspergillus Niger and Penicillium Niger is low,and the extraction and purification process is complicated.Therefore,using genetic engineering to construct more excellent and high-yielding GOD production strains has been the research direction of researchers.In order to obtain high yield and high activity glucose oxidase expression strain,we optimized the codon of GOD double mutation T132S/T56V according to the preference of codon in Pichia pastoris.A 45 bp signal peptide h2-factor was inserted into the front of the optimized gene sequence.Then we got a optimized glucose oxidase gene sequence named GOD-M which was obtained by artificial gene synthesis.We cloned the sequence into the expression vector pGAPk got Recombinant expression plasmid pGAPk-h₂-GOD-M of Pichia pastoris The recombinant plasmid was transformed into Pichia pastoris GS115 by electroporation.The recombinant Pichia pastoris strain was screened based on the enzymatic properties of GOD,and the expression of GOD gene was verified.In order to find a more efficient GOD expression vector,started with the promoter we modified the recombinant vector pGAPk-h₂-GOD-M as follows:1.Replaced the promoter pGAP with pGCW14 in plasmid pGAPk-h₂-GOD-M,and got the recombinant vector pGCW14k-h₂-GOD-M,numbered G1;2.Replaced the promoter pGAP with pAOX1 in plasmid pGAPk-h₂-GOD-M,and the recombinant vector pAOX1k-h₂-GOD-M,numbered A1;3.Modified the promoter pGCW14 by site-specific mutagenesis in plasmid pGCW14k-h₂-GOD-M,and got two mutant vector numbered MG1 and MG2;4.Modified a partial sequence of the promoter pAOX1 in plasmid pAOX1k-h₂-GOD-M,and got three mutanted weotors with differert promoter numbered MA1,MA2 and MA3 respectively.5.Exchanged the 5'UTR sequence of promoter pGCW14 and pAOX1 with each other,and got two recombinant promoter,numbered GU_A and AU_G respectively.The recombinant expression vector above was transformed into Pichia pastoris GS115 and fermented to determine the GOD enzyme activity in the supernatant of fermentation,so as to compare the efficiency of hvarious promoters.We have successfully screened out the constitutive promoters pGCW14U_A and pGCW14G+20A/C-467T and the inducible promoter pAOX1-DL*,which can express GOD more efficiently,which is expected to have a certain prospect in the research and application of Pichia pastoris.