Heterologous Expression, Molecular Modification, And Fermentation Of Glucose Oxidase From Aspergillus Niger

Glucose oxidase (EC1.1.3.4, GOD) is a flavoprotein. GOD catalyzes the oxidation ofβ-D-glucose to gluconic acid using molecular oxygen as an electron acceptor, with theproduction concomitant of hydrogen peroxide. GOD is widely used in the chemistry, medicine,food, beverage, medical diagnosis, and environmental fields. GOD has found plenty ofcommercial applications across multiple industry fields, including glucose biosensor fordiabetes monitoring and improvement of color and shelf life from food and beverages. Hence,GOD owns a very well market prospect. However, the main problems encountered during theindustrial production and application of GOD, such as low productivity of GOD fermentation,low efficiency of extraction, and unstable stability. Therefore, it is still necessary to workfurther on the enzyme modification and heterologous expression of GOD.In this study, GOD is selected as the research object and a series of works are carried out.The main results are listed as follows:1. By qualitative and quantitative methods, a wild fungus displaying glucose oxidaseactivity was screened from environmental samples. The wild fungus was named asAspergillus niger BBE11721by molecular identification and phylogenetic analysis.A coding gene of A. niger BBE11721glucose oxidase was cloned by PCR. The openreading frame was made up of1818bp and encoded606amino acids with atheoretical molecular mass of65.6kDa. Furthermore, the GOD encoding gene wastransformed into P. pastoris GS115, and successfully expressed by the recombinant P.pastoris GS115(pPIC9K/GOD). After purification, the characterization ofrecombinant GOD was performed. The optimum pH and temperature were6.0and35oC, and the kcat, KmA,and kcat/KmAwere456.65s-1,38.95mM, and11.72mM-1s-1,respectively.2. Based on the analysis of comparable residues among different GODs from A. niger, P.amagasakiense, and P. variabile,14potential key residues were selected for directedmutation. The kcat/KmAvalues of D92Q, G131D, G539A and M578V were increasedby1.4-,1.2-,1.2-, and1.9-fold, respectively. Based on the results of single directedmutants, the above mentioned4positive mutants were selected for the furthercompositive mutation. After this step, the characterization of these compositivemutants was performed. The results showed that the performances ofD92Q-G539A-M578V were increased the most, the kcat/KmAvalues was increased by2.4-fold.3. After analyzing the structure of A. niger catalase (CAT), two active domains of CATwere selected for fusion expression at N-and C-terminal domain of GOD. Afterfusion expression, the KmAvalues of mutants GCCB and GCCD were decreased. Thekcat/KmAvalues of GCCB were increased compared to that before fusion. The thermalstability of GCCB at60oC was increased by1.8-fold compared to that before fusion.Moreover, fusion the domain B of CAT at the C-terminal domain of GOD increasedthe anti-oxidation stability by1.7-fold. 4. By integrating the enhancement of UPR transcription pathway at different cultivationtemperatures, the effects on the extracellular enzyme concentration and specificproductivity of heterologous GOD were investigated. Compared to control strain,PP-G-HAC1produced larger total quantities of enzymes, resulting in a34%higherenzyme yield of161U mL-1in shake flasks. The highest GOD activity andextracellular concentration at28°C in PP-G-HAC1were1008U mL-1and14.43g L-1, resulting in a3.12-fold increase. Comparative analysis of transcriptional levelsof genes involved in protein synthesis and secretion indicated that the relief ofprotein folding/glycosylation stress and general stress in cellular machinery may bethe main reason for the improvement on the production of GOD.5. In this study, we improved the yield of GOD by manipulating genes involved inprotein folding machinery and abnormal folding stress responses. First, genes withfolding and secretion functions were used to modulate the folding rate of GOD in theER and its secretion level in the cytoplasm. Next, the potential benefits of the ERADelements were determined. Cellular resistance to ER derived stress was thenstrengthened by overexpressing the stress response gene GCN4. A modulecombination strategy, which co-expressed the SEC53, CNE1and GCN4genes, wasemployed to construct the Pichia Pastoris strain S17. This increased the yield ofGOD to21.81g L-1, with an activity of1972.9U mL-1, which were2.53-and5.11-fold higher, respectively, than the control strain. After analysis of theintracellular ROS level and cell viability, the results indicated that the modularengineer strategy could enhance the resistance ability of host against extra ROSstress. Furthermore, with the aim to achieve the production of GOD on industrialscale, we investigated the scale up experiment in a5M3fermentor. The constructstrain S17resulted in a GOD activity of1305.7U mL-1, which was increased by2.4-fold compared to the control strain. The results indicated that S17was moresuitable for industrial-scale production.