Johari, Y.B., Brown, A.J., Alves, C.S. et al. (5 more authors) (2019) CHO genome mining for synthetic promoter design. Journal of Biotechnology, 294. pp. 1-13. ISSN 0168-1656
Abstract
Synthetic promoters are an attractive alternative for use in mammalian hosts such as CHO cells as they can be designed de novo with user-defined functionalities. In this study, we describe and validate a method for bioprocess-directed design of synthetic promoters utilizing CHO genomic sequence information. We designed promoters with two objective features, (i) constitutive high-level recombinant gene transcription, and (ii) upregulated transcription under mild hypothermia or late-stage culture. CHO genes varying in transcriptional activity were selected based on a comparative analysis of RNA-Seq transcript levels in normal and biphasic cultures in combination with estimates of mRNA half-life from published genome scale datasets. Discrete transcription factor regulatory elements (TFREs) upstream of these genes were informatically identified and functionally screened in vitro to identify a subset of TFREs with the potential to support high activity recombinant gene transcription during biphasic cell culture processes. Two libraries of heterotypic synthetic promoters with varying TFRE combinations were then designed in silico that exhibited a maximal 2.5-fold increase in transcriptional strength over the CMV-IE promoter after transient transfection into host CHO-K1 cells. A subset of synthetic promoters was then used to create stable transfectant pools using CHO-K1 cells under glutamine synthetase selection. Whilst not achieving the maximal 2.5-fold increase in productivity over stable pools harboring the CMV promoter, all stably transfected cells utilizing synthetic promoters exhibited increased reporter production - up to 1.6-fold that of cells employing CMV, both in the presence or absence of intron A immediately downstream of the promoter. The increased productivity of stably transfected cells harboring synthetic promoters was maintained during fed-batch culture, with or without a transition to mild hypothermia at the onset of stationary phase. Our data exemplify that it is important to consider both host cell and intended bioprocess contexts as design criteria in the de novo construction of synthetic genetic parts for mammalian cell engineering.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2019 Elsevier. This is an author produced version of a paper subsequently published in Journal of Biotechnology. Uploaded in accordance with the publisher's self-archiving policy. Article available under the terms of the CC-BY-NC-ND licence (https://creativecommons.org/licenses/by-nc-nd/4.0/). |
Keywords: | CHO genome; Chinese hamster ovary cells; recombinant protein expression; synthetic biology; synthetic promoter; transcriptomics |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Engineering (Sheffield) > Department of Chemical and Biological Engineering (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 04 Feb 2019 16:18 |
Last Modified: | 17 Nov 2021 09:24 |
Status: | Published |
Publisher: | Elsevier |
Refereed: | Yes |
Identification Number: | 10.1016/j.jbiotec.2019.01.015 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:142042 |
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