The fluorescent protein iLOV as a reporter for screening of high‐yield production of antimicrobial peptides in Pichia pastoris

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), pp. 2126-2139. (doi: 10.1111/1751-7915.14034) (PMID:35312165) (PMCID:PMC9249318)

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Abstract

The methylotrophic yeast Pichia pastoris is commonly used for the production of recombinant proteins at scale. The identification of an optimally overexpressing strain following transformation can be time and reagent consuming. Fluorescent reporters like GFP have been used to assist identification of superior producers, but their relatively big size, maturation requirements and narrow temperature range restrict their applications. Here, we introduce the use of iLOV, a flavin-based fluorescent protein, as a fluorescent marker to identify P. pastoris high-yielding strains easily and rapidly. The use of this fluorescent protein as a fusion partner is exemplified by the production of the antimicrobial peptide NI01, a difficult target to overexpress in its native form. iLOV fluorescence correlated well with protein expression level and copy number of the chromosomally integrated gene. An easy and simple medium-throughput plate-based screen directly following transformation is demonstrated for low complexity screening, while a high-throughput method using fluorescence-activated cell sorting (FACS) allowed for comprehensive library screening. Both codon optimization of the iLOV_NI01 fusion cassettes and different integration strategies into the P. pastoris genome were tested to produce and isolate a high-yielding strain. Checking the genetic stability, process reproducibility and following the purification of the active native peptide are eased by visualization of and efficient cleavage from the iLOV reporter. We show that this system can be used for expression and screening of several different antimicrobial peptides recombinantly produced in P. pastoris.

Item Type:Articles
Additional Information:This work was supported by funding from the UK Biotechnology and Biological Sciences Research Council (BB/R001499/1 and BB/V00056X/1 to J.M.C) and the Industrial Biotechnology Innovation Centre (IBioIC) to A.M.K. This work was funded in part by InnovateUK grant 103358.
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Christie, Professor John and Kjeldsen, Miss Annemette and Smith, Dr Brian
Authors: Kjeldsen, A., Kay, J. E., Baxter, S., McColm, S., Serrano-Amatriain, C., Parker, S., Robb, E., Arnold, S. A., Gilmour, C., Raper, A., Robertson, G., Fleming, R., Smith, B. O., Fotheringham, I. G., Christie, J. M., and Magneschi, L.
College/School:College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Journal Name:Microbial Biotechnology
Publisher:Wiley
ISSN:1751-7915
ISSN (Online):1751-7915
Published Online:21 March 2022
Copyright Holders:Copyright © 2022 The Authors
First Published:First published in Microbial Biotechnology 15(7): 2126-2139
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
301413How do Phototropin Receptor Kinases Initiate Signalling from the PlasmaJohn ChristieBiotechnology and Biological Sciences Research Council (BBSRC)BB/R001499/1Institute of Molecular, Cell & Systems Biology
310016Co-ordinated Photoreceptor Engineering for Improved Biomass ProductionJohn ChristieBiotechnology and Biological Sciences Research Council (BBSRC)BB/V00056X/1Institute of Molecular, Cell & Systems Biology