Biosensor‐assisted engineering of a high‐yield Pichia pastoris cell‐free protein synthesis platform
File(s)
Accepted version
Accepted version
Author(s)
Aw, Rochelle
Polizzi, Karen M
Type
Journal Article
Abstract
Cell‐free protein synthesis (CFPS) has recently undergone a resurgence partly due to the proliferation of synthetic biology. The variety of hosts used for cell‐free extract production has increased, which harnesses the diversity of cellular biosynthetic, protein folding, and posttranslational modification capabilities available. Here we describe a CFPS platform derived from Pichia pastoris, a popular recombinant protein expression host both in academia and the biopharmaceutical industry. A novel ribosome biosensor was developed to optimize the cell extract harvest time. Using this biosensor we identified a potential bottleneck in ribosome content. Therefore, we undertook strain engineering to overexpress global regulators of ribosome biogenesis to increase in vitro protein production. CFPS extracts from the strain overexpressing FHL1 had a 3‐fold increase in recombinant protein yield compared to those from the wild‐type X33 strain. Furthermore, our novel CFPS platform can produce complex therapeutic proteins, as exemplified by the production of human serum albumin to a final yield of 48.1 μg mL‐1. Therefore, this work not only adds to the growing number of CFPS systems from diverse organisms, but also provides a blueprint for rapidly engineering new strains with increased productivity in vitro that could be applied to other organisms.
Date Issued
2019-03-01
Date Acceptance
2018-12-14
Citation
Biotechnology and Bioengineering, 2019, 116 (3), pp.656-666
ISSN
0006-3592
Publisher
Wiley
Start Page
656
End Page
666
Journal / Book Title
Biotechnology and Bioengineering
Volume
116
Issue
3
Copyright Statement
© 2018 Wiley. This is the accepted version of the following article: Aw, R. and Polizzi, K. M. (2018), Biosensor‐Assisted Engineering of a High‐Yield Pichia pastoris Cell‐Free Protein Synthesis Platform. Biotechnology and Bioengineering. Accepted Author Manuscript., which has been published in final form at https://dx.doi.org/10.1002/bit.26901
Sponsor
Engineering & Physical Science Research Council (EPSRC)
Identifier
https://onlinelibrary.wiley.com/doi/full/10.1002/bit.26901
Grant Number
EP/K038648/1
Subjects
Science & Technology
Life Sciences & Biomedicine
Biotechnology & Applied Microbiology
cell-free protein synthesis (CFPS)
in vitro transcription translation
Pichia pastoris/Komagataella phaffi
synthetic biology
yeast
TRANSCRIPTION-TRANSLATION SYSTEM
SERUM-ALBUMIN PRODUCTION
SACCHAROMYCES-CEREVISIAE
GROWTH-RATE
EXPRESSION
YEAST
OPTIMIZATION
DISRUPTION
EFFICIENT
LYMPHOMA
Pichia pastoris/Komagataella phaffi
cell-free protein synthesis (CFPS)
in vitro transcription translation
synthetic biology
yeast
Biosensing Techniques
Cell-Free System
Genetic Engineering
Pichia
Protein Biosynthesis
Recombinant Proteins
Synthetic Biology
Cell-Free System
Pichia
Recombinant Proteins
Genetic Engineering
Biosensing Techniques
Protein Biosynthesis
Synthetic Biology
Biotechnology
Publication Status
Published
Date Publish Online
2018-12-14