Deciphering the transport mechanisms of small xenobiotic molecules in synthetic yeast cell factories

Project Information

YEAST-TRANS

Grant agreement ID: 757384

DOI

10.3030/757384

Project closed

EC signature date 27 October 2017

Start date 1 December 2017

End date 30 November 2022

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 423 357,50

EU contribution

€ 1 423 357,50

Coordinated by

DANMARKS TEKNISKE UNIVERSITET
Denmark

Periodic Reporting for period 4 - YEAST-TRANS (Deciphering the transport mechanisms of small xenobiotic molecules in synthetic yeast cell factories)

Reporting period: 2022-06-01 to 2022-11-30

Summary of the context and overall objectives of the project

Industrial biotechnology employs synthetic cell factories to create bulk and fine chemicals and fuels from renewable resources. The major part of the wanted bio-based chemicals are not native to the host cell, such as yeast, i.e. they are xenobiotic. Some xenobiotic compounds are readily secreted by synthetic cells, some are poorly secreted, and some are not secreted at all, but how does this transport occur? YEAST-TRANS addresses these fundamental questions. The overall objective is to uncover the transport mechanisms of small xenobiotic molecules by synthetic yeast cells and to apply this knowledge to engineer more efficient cell factories for the bio-based production of fuels and chemicals. If we can understand and improve the section of small molecules, we can bring down the cost of biotechnological processes and enable the emergence of novel bio-based products.
The project has achieved its major objectives. We have investigated the plasticity of yeast transporters towards small metabolites, developed novel workflows for faster and easier transporter characterization and computational workflows for transporter prediction, and engineered the transport of various small metabolites in yeast cell factories. The results were published in 28 peer-reviewed papers and presented at over 25 conferences. A technology for the fermentative production of ergothioneine has been developed and licensed to the industry.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

During the 5-year project, we worked on developing experimental methods for the characterization of transporters, computational methods for transporter discovery, and engineering the transport of small molecules in yeast cell factories. Below, several achievements are highlighted.
1. Experimental proof of high transportome plasticity.
We performed the first systematic study of transporter substrate specificity, where we assayed 30 transporters of different families from yeast Saccharomyces cerevisiae against six diverse small metabolites, both native and xenobiotic. We discovered that transporters were greatly promiscuous in equilibrative transport, where 20 transporters facilitated the uptake of one or several tested compounds. When it came to concentrative transport, transporters were more substrate-specific. High substrate promiscuity of transporters has been suggested and experimentally shown for some multidrug transporters in bacteria (Cudkowicz &Schuldiner, 2019, PLoS One; Kermani et al, 2020, Nat Commun), but our study shows that it is a very general phenomenon, not limited to specific transporter family and antibiotics.
2. Novel, faster, and easier methods for functional characterization of transporters.
We addressed one of the most critical problems in the field - the lack of assays for functional determination. We have established a new workflow that combines cell-free transporter protein expression and solid supported membrane electrophysiology and allows functional characterization of transporters within ca. five days. Further, we have developed a promising scalable method based on Xenopus expression and C13-labelled substrate blends that allows characterizing a given transporter against multiple compounds in a single assay.
3. Web application for predicting transporter candidates for a given molecule at www.transporterpal.com
TransporterPAL predicts suitable transporters for compounds by interconnecting data for biosynthetic genes and their interactions with transporters. The web application queries the STITCH, STRING, and UniProtKB databases via their respective APIs and returns a set of potential transporters based on a compound and, optionally, the organism as input. This is the first bioinformatics tool that can predict transporter candidates.
4. Methods for transporter engineering in cell factories.
We have developed a method for transportome-wide engineering and made the plasmids library available to the scientific community through AddGene material repository (Wang et al, 2021, Metab Eng). We have shown on various products how transporter engineering can be applied in the context of cell factory engineering and disseminated these results through papers and conference presentations. We also created a technology for producing the anti-aging compound ergothioneine that was licensed to the industry.
The project results were published in 28 peer-reviewed papers and presented as talks and/or posters at over 25 international conferences. The project also resulted in a patent on ergothioneine that was licensed to the industry, four press releases, and a popular science article.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

• One of the project's key objectives was to investigate the plasticity of the transportome in a model eukaryotic organism. We performed the first systematic study of transporter substrate specificity, where we assayed 30 transporters of different families from yeast Saccharomyces cerevisiae against six diverse small metabolites, both native and xenobiotic. We found that transporters were more promiscuous for equilibrative transport but less for concentrative. This finding dramatically changes our understanding of transporter specificities and functions.
• The development of a methodology for transporter characterization by cell-free synthesis and SSME also represents a significant advance in the field. There are only a few reports on the cell-free synthesis of membrane proteins, mainly GPCRs and a few ion channels. At the same time, SSME experiments are limited by the tedious process of purifying transmembrane transporter proteins. By combining cell-free expression and SSME technologies, we enable a convenient 5-day method for transporter characterization. We demonstrate the utility of this workflow by assaying five diverse transporters, where we determined substrate specificity, kinetic parameters, pH dependency and obtained mechanistic insights into transporter functionalities. (Dong et al, in preparation)
• We have created a library for genome-wide disruption of transporter genes in S. cerevisiae (Wang et al., 2021, Metab Eng). The transporter disruption library allows scientists to disrupt all transporters individually in any production strain. This makes transporter engineering an accessible tool for strain optimization. All plasmids carrying transporter genes for expression in Xenopus oocytes are publicly available through Addgene (ID 79999).
• We have made a computational algorithm TransporterPAL for predicting transporter candidates for a metabolite of choice, and we built it into a web application to facilitate its use (www.transporterpal.com). TransporterPAL queries the STITCH, STRING, and UniProtKB databases via their respective APIs and returns a set of potential transporters based on a compound and, optionally, the organism as input (Dyekjær et al., www.biorxiv.org/content/10.1101/2022.09.05.506577v1). It is the first transporter-by-substrate prediction tool.
• We have developed and patented a technology for the production of a longevity compound ergothioneine by yeast fermentation (Borodina et al, 2020, Nutr Res Rev). The technology and IP have been licensed to a company that will bring affordable and sustainable ergothioneine to the consumer. Transporter engineering was a part of the ergothioneine strain optimization. (van der Hoek et al, 2019, Front Bioeng Biotechnol; van der Hoek et al, 2022, Metab Eng; van der Hoek et al, 2022, FEBS Lett)
These discoveries and methods will facilitate future research in the field.

Understanding the transport of metabolites helps to improve cell factories