Promoter Modification and Fermentation Condition Optimization Enhance the Expression of Human Type III Collagen in Pichia pastoris

• 1、School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122

Abstract：Collagen is the most abundant component of human cytoskeleton and extracellular matrix. As a biomaterial, it has good biological properties, such as good biocompatibility, low immunity and biodegradability. The heterologous expression of collagen by microorganisms can not only avoid the hidden dangers of traditional extraction methods and low water solubility, but also break through the restrictions on the length of collagen in chemical synthesis methods, making the design of collagen sequence more flexible. In this paper, a truncated human type III collagen N-HC3240 gene was synthesized. Through the strategy of promoter modification, the effects of promoters AOX1, DAS1, DAS2, FLD1, FDH1, THI4 and THI11 on the expression of N-HC3240 in Pichia pastoris were studied. The results showed that AOX1 promoter was the best promoter for N-HC3240 expression. And the expression of N-HC3240 using AOX1 promoter was optimized by dissolved oxygen, initial induced bacterial concentration, induced pH and methanol concentration. Finally, the yield of N-HC3240 in shaking flask reached 0.28 g/L, which was 2.5 times of that before optimization. The correct expression of N-HC3240 was identified by mass spectrometry. This study provides a basis for the heterologous and high-level expression of human type III collagen in Pichia pastoris.

Keywords： Collagen Pichia pastoris Heterologous expression Promotor Fermentation optimization