Methanol-free Protein Expression in Pichia pastoris
Enhanced safety and streamlined efficiency without compromising performance
Methanol-free protein production with UNLOCK PICHIA® - Intensified processes, enhanced process safety
Our UNLOCK PICHIA® methanol-free protein expression system utilizes a specialized subset of our library of AOX1 promoter variants, eliminating the need for methanol as inducer while maintaining the beneficial characteristics of the wild-type AOX1 promoter. This significantly reduces the metabolic burden associated with a change of carbon source, as well as minimizing oxygen consumption and heat evolution. Unlike constitutive promoters, this system enables strong, time-controlled initiation of recombinant protein production.
Pairing these strains with the expertise of our fermentation team has resulted in the development of optimized methanol-free cultivation protocols, designed for robustness and scalability. This methanol-free technology is already in commercial use in bioreactors up to 100,000L. For applications where methanol-use is to be avoided, this technology provides a safe, efficient, and scalable alternative.
Benefits of VALIDOGEN's methanol-free Pichia expression system
- UNLOCK PICHIA® methanol-free processes leverage specially engineered AOX1 promoters for high-performance protein expression
- Methanol-free induction preserves the key advantage of the wild-type AOX1 promoter such as strong and tightly controlled protein expression while enhancing process safety
- Glycerol (or glucose) used throughout the process means there is no metabolic burden from carbon source switching, ensuring smoother cell growth and protein production
- This technology is adaptable to different cultivation strategies, including short cultivations (~60h) to enable process intensification, and optimized standard cultivations (~110 h) to maximize product titers
Case Study: Methanol-free bivalent VHH expression in Pichia pastoris
A recent study highlighted the development of methanol-free processes for the efficient production of a bivalent VHH. Through process optimization, we achieved product yields of 12 g/L in just 64 hours and 19 g/L within VALIDOGEN's standard cultivation time of 111 hours. These processes demonstrated exceptionally high space-time yields (STY).
In situations where short batch cultivations are advantageous, such as limitations in downstream capabilities, bacterial-like fermentation times in our optimized short culture process (~60h) can deliver high product yields with remarkably high volumetric productivity - case study I . In contrast, when maximized batch yield is the ultimate goal, our optimized feeding strategies can result in significant titer increases under a standard time cultivation (~110h). MeOH-free AOX1 promotor variants from our UNLOCK PICHIA® toolbox present a unique solution to achieve high protein expression levels in the absence of methanol, under both short and standard cultivation times.
Read the full case studies here.
White Paper: High level methanol-free enzyme production in Pichia pastoris
VALIDOGEN’s Pichia pastoris protein production toolbox and screening know-how was harnessed in an enzyme expression study, and resulted in the secreted production of a phytase variant at 22 g/L supernatant in a methanol-induced process, and 20 g/L under methanol-free conditions. These are the highest yields of yeast-derived recombinant phytase reported to date.
This study demonstrates that the favorable features of AOX1-driven expression, such as tight transcriptional control and high yield, are equally attainable using VALIDOGEN’s methanol-free AOX1 promoter variants. These findings underscore the robustness and efficiency of VALIDOGEN’s methanol-free technology platform.
Read the full study in this white paper.