The vitamin-sensitive promoter PTHI11 enables pre-defined autonomous induction of recombinant protein production in Pichia pastoris.

Abstract

The methylotrophic yeast Pichia pastoris is widely used for production of recombinant proteins. Here we characterize a vitamin-sensitive regulatory sequence, which can be controlled independently of the main culture medium compounds such as carbon, nitrogen, or phosphor source. The THI11 promoter (PTHI11 ) sequence derives from a gene involved in biosynthesis of thiamine. For characterization, a P. pastoris strain expressing recombinant human serum albumin under control of PTHI11 was grown in the controlled environment of a bioreactor. The thiamine sensitivity of PTHI11 was proven and specified in batch cultures containing different amounts of extracellular thiamine. Under non-repressing conditions PTHI11 offers a constitutive expression pattern with growth rate dependent product formation. Furthermore, promoter activity and thus product formation can be repressed for a desired period of time by supplementing the culture with a pre-defined amount of exogenous thiamine. Once a threshold of biomass is reached, PTHI11 driven expression starts autonomously without external intervention. Based on these findings a tailor-made process strategy was developed and experimentally verified. Additionally, we compared the THI11 promoter with the commonly used GAP promoter. In conclusion, the THI11 promoter is a versatile and easy to control regulatory sequence which enables the realization of novel protein production strategies. Biotechnol. Bioeng. 2016;113: 2633-2643. © 2016 Wiley Periodicals, Inc.