Technology

Methanol-free platform

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

Technology

Pichia
pastoris Methanol-free
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