Spotlight on methanol-free protein production

products-servicesVALIDOGEN GmbH
July 27th 2015

VTU Technology has become the world leader in offering Pichia pastoris PAOX1 controlled production processes with unmatched product yields, accelerated development timelines and highest quality standards.VTU´s exclusive highly approved first-generation library of synthetic methanol-inducible PAOX1 promoter variants forms the core of the company´s cutting-edge in-house Pichia pastoris toolbox enabling high-level protein production and secretion of 10 to 20 g/L with remarkable peak productivities of 35 g/L.

The VTU library has been enhanced with groundbreaking and unique MeOH-free second-generation PAOX1 promoter variants facilitating strong expression without the need for methanol-induction.

The enhanced platform provides safer and more economically viable production processes, sophisticated high-throughput methods and streamlined procedures.

In a recent case study featuring expression of recombinant bacterial Phytase, this broad and versatile production platform hit the 20 g/L mark in methanol-free protein production.

PAOX1 – methanol-free

VTU´s highly proven Pichia pastoris expression toolbox is characterized by a large number of exclusive AOX1 promoter variants, allowing gene expression to be fine-tuned by selecting the perfect match of the respective promoter variant to a given target gene.

VTU´s 2nd generation AOX1 promoter variants are less susceptible to repression by common carbon sources like glucose and glycerol than the AOX1 wild type promoter or VTU´s 1st generation AOX1 promoter variants facilitating strong expression even with just glycerol or glucose as the sole carbon source. Elaborated fermentation and feeding strategies have been developed by VTU to further boost volumetric productivity by reducing the process time while maintaining final product concentrations clearly outperforming conventional promoter systems based on constitutive expression such as pGAP (Figure 1).


VTU´s unique methanol-free Pichia system offers strong expression along with further advantages such as:

  • Banishing toxic and explosive methanol from production process
  • Using glycerol or glucose as the sole carbon source
  • Significantly reduced heat formation and cooling demand at large scale
  • Reduced process time and cost of goods
  • Tunable AOX1 promoter library to comply with different regulatory properties
  • Boosted yields through fine-tuning of gene expression by increasing genetic diversity

Case Study: Methanol-free PAOX1 promoters used in expression of a recombinant bacterial phytase

The case study clearly demonstrates that while maintaining competitive development timelines the application of a higher number of promoter variants from VTU´s promoter libraries significantly boosted protein production in Pichia pastoris. Yield was boosted through fine-tuning of gene expression by increasing genetic diversity (Figure 2).

Fact and figures:

Expression: secreted

  • Methanol-induced expression using VTU´s 1st generation promoter library
  • Methanol-free expression using VTU´s 2nd generation promoter library

Screening size:

  • Small screening – 400 clones applying 2 PAOX1 promoter variants
  • Extended screening – 6000 clones applying 9 PAOX1 promoter variants


  • Bioreactor size: 0.75 L
  • Pichia strain cultivation using simple and cheap chemically defined media
  • Antibiotic markers used: None

The study furnished exceptionally high-yielding phytase secreting strains:

  • Methanol-induced: 22 g/L
  • Methanol-free: 20 g/L
  • 3.5-fold to 4.6-fold enhancement of protein yields from small to extended screening through increasing genetic diversity (increasing VTU´s promoter variants and clone numbers)
Spotlight on methanol-free protein production

Figure 1: Accumulations of recombinant target protein in bioreactors comparing:
(a) constitutive expression controlled by the GAP-promoter, (b) typical methanol-induced VTU expression, (c & d) MeOH-free processes both controlled by VTU AOX1 promoter variants but using different feeding strategies

Spotlight on methanol-free protein production

Figure 2: Methanol-induced and methanol-free phytase expression based on a small and an extended screening approach