The vitamin‐sensitive promoter P_THI11 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 (P ) 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 was grown in the controlled environment of a bioreactor. The thiamine sensitivity of P was proven and specified in batch cultures containing different amounts of extracellular thiamine. Under non-repressing conditions P 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 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.

Rationale‐based selection of optimal operating strategies and gene dosage impact on recombinant protein production in Komagataella phaffii (Pichia pastoris)

“…Actin gene was used as housekeeping agent, but the primer sequence was that proposed by Landes et.al. (). The specific primers used are shown in Table S1.…”

Engineering of alcohol dehydrogenase 2 hybrid‐promoter architectures in Pichia pastoris to enhance recombinant protein expression on ethanol

“…Codon-optimized HSA gene with its native signal sequence was amplified from pPM2pN21_pAOX-HSA_opt plasmid (Landes et al, 2016) and cloned under the control of superior NEPVs, and for ERGÜN ET AL. comparison purpose under P ADH2 ; and then were digested with BamHI and KpnI, and ligated into BglII and KpnI digested pPICZα-A vector for generating pADH2-v::HSA plasmids. Codon-optimized HSA gene with its native signal sequence was amplified from pPM2pN21_pAOX-HSA_opt plasmid (Landes et al, 2016) and cloned under the control of superior NEPVs, and for ERGÜN ET AL. comparison purpose under P ADH2 ; and then were digested with BamHI and KpnI, and ligated into BglII and KpnI digested pPICZα-A vector for generating pADH2-v::HSA plasmids.…”

“…Sequences of the ADH2-promoter variants are provided in Method S2. Codon-optimized HSA gene with its native signal sequence was amplified from pPM2pN21_pAOX-HSA_opt plasmid (Landes et al, 2016) and cloned under the control of superior NEPVs, and for ERGÜN ET AL.…”

Cloning of PpKAR2 and PpPDI1 genes promoters from yeast Pichia pastoris, an evaluation of their activity and efficacy for the heterologous genes expression

“…Аналогичный подход показал эффективность и при работе с конститутивным промотором PGaP [7]. Одновременно с модификацией известных промоторов идет поиск новых промоторов, которые можно использовать для экспрессии гетерологичных генов [8][9][10][11]. На сегодняшний день увеличение ассортимента промоторов, пригодных для биотехнологии, а также изучение их регуляции является актуальной задачей.…”