The vitamin‐sensitive promoter P THI11 enables pre‐defined autonomous induction of recombinant protein production in Pichia pastoris

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 (P THI11 ) sequence derives from a gene involved in biosynthesis of thiamine. For characterization, a P. pastoris strain expressing recombinant human serum albumin under control of P THI11 was grown in the controlled environment of a bioreactor. The thiamine sensitivity of P THI11 was proven and specified in batch cultures containing different amounts of extracellular thiamine. Under non‐repressing conditions P THI11 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, P THI11 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.