Synthetic biology enables the production of small molecules by recombinant microbes for pharma, food, and materials applications. The secretion of products reduces the cost of separation and purification, but it is challenging to engineer due to the limited understanding of the transporter proteins' functions. Here we describe a method for genome-wide transporter disruption that, in combination with a metabolite biosensor, enables the identification of transporters impacting the production of a given target metabolite in yeast Saccharomyces cerevisiae. We applied the method to study the transport of xenobiotic compounds, cis,cis-muconic acid (CCM), protocatechuic acid (PCA), and betaxanthins. We found 22 transporters that influenced the production of CCM or PCA. The transporter of the 12-spanner drug:H(+) antiporter (DHA1) family Tpo2p was further confirmed to import CCM and PCA in Xenopus expression assays. We also identified three transporter proteins (Qdr1p, Qdr2p, and Apl1p) involved in betaxanthins transport. In summary, the described method enables high-throughput transporter identification for small molecules in cell factories.
Bibliographical noteFunding Information:
This project has received funding from the European Research Council under the European Union's Horizon 2020 research and innovation programme (YEAST-TRANS project, Grant Agreement No. 757384 ) and the European Union's Horizon 2020 research and innovation programme under grant agreement No. 720770 (DAFIA project). IB also acknowledges the financial support from the Novo Nordisk Foundation (Grant agreement No. NNF20CC0035580 and No. NNF20OC0060809 >).
© 2021 The Authors
- Cell factory
- Metabolic engineering
- Muconic acid
- Protocatechuic acid
- Transporter protein