Hoffmann, S. A. | bioRxiv Science

bioRxiv2025

Synthetic yeast genome SCRaMbLEing uncovers a new role for ribosomal proteins in genetic code expansion

The translation of proteins with non-canonical amino acids (ncAAs) has emerged as a powerful technology for embedding new functional elements into proteins, enabling the development of novel enzymes, materials, and biopharmaceuticals. However, the utility of this approach has been hindered by weak translation efficiencies. To address this challenge, we sought to substantially improve orthogonal translation in Saccharomyces cerevisiae. We first evaluated recently described {Delta}NPylRS-class pyrrolysyl-tRNA synthetase systems and identified a homolog with [~]5.4-fold higher activity than the best previously reported pyrrolysyl system. Building on this advance, we leveraged the SCRaMbLE system in the semi-synthetic yeast strain Syn6.5 to generate structural genomic variation, and identified strains with enhanced ncAA incorporation. Pooling genomic alterations across enhanced strains, we identified an association for the deletion of several ribosomal protein genes with the increased production of ncAA-containing protein. These findings demonstrate a previously unrecognized role for ribosomal proteins in enabling alternate genetic codes.

synthetic biology↗

bioRxiv2022

Engineering stringent genetic biocontainment of yeast with a protein stability switch

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. 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 4.3x10-9. 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. SignificanceSynthetic biology holds enormous potential to tackle key issues humanity is facing and can for instance revolutionize agriculture, bioremediation or health care. In each case, the unchecked spread of engineered organisms in natural environments must be prevented. This is particularly problematic with use cases of engineered microbes in open environments. Intrinsic, genetically encoded biocontainment systems, which control cell survival based on environmental cues, can solve this issue. We have developed such a genetic biocontainment system acting on the stability of essential proteins, leveraging a switchable degron. Through a large-scale screening for suitable essential target genes, we were able to create yeast strains that are strictly dependent on estradiol. Supplied with this small molecule, the engineered cells maintain high fitness and grow as robustly as the unmodified strains.

synthetic biology↗