Introducing a novel fluorescent marker to assess antimicrobial peptide production in Pichia pastoris

  1. 15th Jan, 2024

Ingenza is proud to announce that our molecular biology team recently co-authored a groundbreaking research paper published in Applied Microbiology International.1 The study addressed current challenges in producing recombinant proteins in the methylotrophic yeast Pichia pastoris.

P. pastoris is a well-established host organism for recombinant protein expression. However, identifying a high yielding manufacturing strain following transformation can be challenging, requiring the screening of hundreds – if not thousands – of individual clones. Existing fluorescent reporters – such as GFP – tend to be restricted by their large size, maturation requirements and narrow temperature range. Our molecular biologists looked to overcome these limitations by searching for a rapid and high throughput screening method to assess recombinant protein expression levels in P. pastoris.

The team investigated the use of improved light-oxygen-voltage (iLOV) protein2 as a fluorescent marker to identify high yielding P. pastoris strains easily and rapidly. iLOV is a flavin-based fluorescent protein that contains a LOV-sensing domain with a non-covalently bound flavin mononucleotide (FMN) as its chromophore. This powerful fluorescent marker is smaller than existing reporter molecules, imposing a lower cellular burden. It also works well across a wide range of temperatures and pH levels, and matures faster than commonly used fluorescent proteins.

Dr Leonardo Magneschi, Head of Molecular Biology at Ingenza, commented: “Our research revealed the ability of iLOV to streamline the screening of engineered yeast strains for the expression of challenging antimicrobial targets, such as the first-in-class epidermicin NI01 bacteriocin, which shows great promise as a therapeutic agent. Using iLOV in combination with ultra-high throughput fluorescence activated cell sorting (FACS) enabled efficient and precise selection of the most productive and stable recombinant yeast strains for NI01 manufacturing. The chosen fluorescent tagging strategy also allows real-time monitoring of NI01 expression levels during upstream and downstream process development, and the tag can be removed scarlessly at low cost following purification. The publication of this paper highlights Ingenza’s continuous innovation and ongoing dedication to novel bioengineering solutions that are accelerating biologics development.”

Congratulations to the team for this remarkable scientific contribution!

  1. Kjeldsen, A. et al. 2022. The fluorescent protein iLOV as a reporter for screening of high‐yield production of antimicrobial peptides in Pichia pastoris. Microbial Biotechnology, 15(7):2126-2139. doi:10.1111/1751-7915.14034. ↩︎
  2. Chapman, S. et al. 2008. The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection. Proceedings of the National Academy of Sciences, 105(50):20038-20043. doi:10.1073/pnas.0807551105. ↩︎