PXD042326

PXD042326 is an original dataset announced via ProteomeXchange.

Title	Engineering stringent genetic biocontainment of yeast with a protein stability switch
Description	Synthetic biology holds immense promise to tackle key problems we are facing, for instance in resource use, environmental health, and human health care. However, comprehensive safety measures are needed to deploy genetically engineered microorganisms in open-environment applications. Intrinsic, genetically encoded biocontainment systems, which control cell survival based on environmental cues, can solve this issue. Here, we describe a genetic biocontainment system based on conditional stability of essential proteins. We used a yeast-adapted destabilizing domain degron, which can be stabilized by estradiol addition (ERdd). Leveraging the yeast GFP collection and lab automation platforms, we ERdd-tagged 775 essential genes and screened for strains with estradiol dependent growth. Three genes, SPC110, DIS3 and RRP46, were found to be particularly suitable targets. Respective strains showed no growth defect in the presence of estradiol and strong growth inhibition in its absence. SPC110-ERdd offered the most stringent containment, with an escape frequency of 7.0x10-8, and full growth restoration at 100 nM estradiol. By systematically analyzing the containment escapees, we identified the non-essential C-terminal region of SPC110 as target for escape mutations. Its removal decreased the escape frequency with a single ERdd tag further to 4.9x10-9. Combining SPC110-ERdd with a second ERdd tag on either DIS3 or RRP46 resulted in escape frequencies below the detection limit of the used assay (<2x10-10). Being based on conditional protein stability, this approach is mechanistically orthogonal to previously reported intrinsic biocontainment systems. It thus can be readily combined with other systems, for instance ones based on transcriptional or translational control of essential gene expression, to achieve multiplexed, extremely stringent control over the survival of engineered organisms.
HostingRepository	PRIDE
AnnounceDate	2024-02-05
AnnouncementXML	Submission_2024-02-05_08:32:10.652.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD042326
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Stefan Hoffmann
SpeciesList	scientific name: Saccharomyces cerevisiae (Baker's yeast); NCBI TaxID: 4932;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Exploris 480

Revision	Datetime	Status	ChangeLog Entry
0	2023-05-18 07:17:07	ID requested
⏵ 1	2024-02-05 08:32:11	announced

10.6019/PXD042326;

10.1038/S41467-024-44988-8;

submitter keyword: genomic safeguards,biocontainment, biosafety, high-throughput genome engineering

Yizhi Cai
contact affiliation	Manchester Institute of Biotechnology, The University of Manchester, UK
contact email	yizhi.cai@manchester.ac.uk
lab head
Stefan Hoffmann
contact affiliation	University of Manchester, Manchester Institute of Biotechnology
contact email	stefan.hoffmann@manchester.ac.uk
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD042326
     1. Label: PRIDE project
     2. Name: Engineering stringent genetic biocontainment of yeast with a protein stability switch