Study On Strengthening The Dissolved Oxygen In Proinsulin Expression By Pichia Pastoris

Background: Diabetes is one of the three major killers that threat human’s health inmodern society, and so far insulin is still one of the most effective anti-diabetic drugs. Recentwith the development of biotechnology, recombinant human insulin has shared most of theinsulin market. Currently, E.coli and yeast are commonly used as expression systems forrecombinant human insulin. Pichia pastoris has many advantages and attracted muchattention as a heterologous expression system for protein drugs. However, dissolved oxygen isalways the most important limiting factor during the heterologous expression by Pichiapastoris. The expression of target proteins will be significantly impaired by low concentrationof dissolved oxygen during fermentation.Objective: Zhuhai United Laboratories Co., Ltd. uses genetically engineered Pichiapastoris to express proinsulin. In the late fermentation process (approximately80hours afterfermentation), dissolved oxygen is in a low level, even zero. It severely hinders the expressionof proinsulin and also restricts the further scaling up. This study focus on improving dissolvedoxygen during the late fermentation.Methods: This project aims to study on strengthening the dissolved oxygen during thefermentation by Pichia pastoris, which is constructed to express proinsulin by Zhuhai UnitedLaboratories Co., Ltd. The strategy is to modify mechanical parts of fermentor. In20Lfermenter, a baffle is added in, the shape of agitator paddle is changed from curved paddlesto semi-circle paddles and the dimension of agitator paddle is enlargeed to one half offermenter diameter for dissolved oxygen improvement.Results: After mechanicalmodification, the concentration of dissolved oxygen increasedfrom nearly zero to10%~20%in later fermentation. With the improvement of dissolvedoxygen, the specific growth rate and the yield for substrate have increased significantly. Thespecific production rate increased from2.62×10-5g/h to4.07×10-5g/h, while the protein yieldincreased from0.6857g/g to0.8539g/g. The concentration of target protein raise from nomore than1g/L to the highest of1.5g/L. The total protein was obtained from5.12g to7.6g in20L fermentor, which is50%up.Conclusion: After mechanical modification in20L fermentor, which include modifyingthe shape of agitator paddle (from curved paddles to semi-circle paddles) and enlarging thedimension of agitator paddle to one half of fermenter diameter, the restriction of dissolvedoxygen during the fermentation by Pichia pastoris was solved effectively and thusconcentration of target protein significantly increased. This study has built a solidfoundation for industrial scale-up of the recombinant human insulin production in ZhuhaiUnited Laboratories Co., Ltd.