Technology

Pichia pastoris​ - Methanol-free platform​ - General toolbox​

Pichia pastoris​

Pichia pastoris (renamed Komagataella phaffii) is a well-established and regulatory-approved host organism for industrial protein and enzyme production. The ability to efficiently secrete proteins facilitates downstream processing and reduces production costs.
Pichia pastoris​

Established for industrial protein and enzyme production

  • High cell densities in inexpensive defined medium
  • Stable strains by genomic integration
  • Efficient tools for genetic modification
  • Well scalable fermentation and downstream processes

Approved by regulatory bodies (EMA/EFSA, FDA, etc.)

  • GRAS status (generally recognized as safe)​
  • Free of endotoxins and viruses

Efficient secretion of recombinant proteins​

  • Simplified downstream processing (no cell disruption)
  • Very low impurities from host cell proteins ​

Essential features of higher eukaryotic hosts​

  • Co-and post-translational modifications​
  • Engineered strains with modified glycosylation patterns ​

Methanol undesired in large scale manufacturing​

Most frequently well-established strong methanol inducible promoters like AOX1 are used to achieve the protein production rates required for commercially viable processes. However, the flammability and toxicity of methanol causes challenges in scale-up as well as safety concerns in food and related applications.

Methanol poses challenges​

  • Flammability: scale-up more complicated and expensive (safety measures) ​
  • Toxicity: regulatory restrictions and customer awareness​
  • Volatility: evaporation complicates process control; environmental pollution ​
  • Cooling capacity: high oxygen demand and heat generation of methanol metabolism

This creates a strong demand for alternative methanol free strategies with competitive productivity. Derepressed promoters for which transcription occurs upon carbon starvation have shown to be alternatives. Yet, many of them do not reach the methanol-induced level when used derepressed.​

Methanol-free platform

The unique myBIOS expression platform offers methanol-free inducible expression which equals or outperforms productivity of methanol-induced expression.
Methanol-free platform

Engineered for Methanol-Free (EMF)​

At myBIOS we provide novel inducible processes for enhanced protein production using low or no methanol. We have developed two independent systems building on non-toxic inducers, inducible promoters and engineered strains. To outperform methanol induced processes we perform coordinated optimization of promoter/inducer, host strain and fermentation conditions.

  • P1 dual: strong induction with methanol and alternative inducer.
  • P2 tight: regulated and induced such as the well-known AOX1 promoter but with an alternative inducer.

Non-toxic inducers

Using a non-toxic inducer the same expression levels as with methanol induction are achieved. Levels of derepressed expression are largely surpassed.

Data is from 5 L bioreactor cultivations using the P1 dual system. The same strain was studied under 3 conditions, i.e., under carbon source starvation, with methanol or with our alternative inducer.

Non-toxic inducers

Dual system

Dual system

The P1 dual promoter can be induced with methanol or an alternative inducer. With alternative inducer similar expression levels as with methanol AOX1 expression are achieved. The expression with P1 dual can be further improved by adding low amounts (1/10) of methanol to the alternative inducer.

Engineered strains

We engineered our host strains to further improve expression levels with our alternative inducer.

Engineered strains

General Toolbox

The independent myBIOS Pichia pastoris expression toolbox is built on >15 years of experience in Pichia pastoris strain development. The core of the platform is our methanol-free expression technology which combines promoter, strain and process developments.

To maximize productivity the expression strategy must be tailored to the protein of interest. We established a broad expression toolbox (host strains, secretion signals, fusion proteins, chaperones, etc.) and high-throughput analytics for cost and time efficient strain optimization. In addition, we perform tailor made custom knock-outs and create co-expression strains to further increase protein titres or modify protein modifications.

The myBIOS expression toolbox is also designed to fulfil quality, safety and regulatory requirements. For example, strains can be generated without antibiotic resistance markers.

Various promoters

  • Methanol induced
  • Methanol free (P1 dual): induction with methanol or alternative inducer
  • Methanol free (P2 tight): alternative inducer, no leaky expression

Markers

  • Antibiotic
  • Antibiotic recycled (removed from genome)
  • Auxotrophic

Host strain collection

  • Wild type strains: CBS2612, CBS7435
  • Phenotype strains
  • Auxotrophic strains
  • Protease deficient strains

Customized knock out strains

Secretion signals

Chaperone library

Fusion proteins