1. Transportome-wide engineering of Saccharomyces cerevisiae
- Author
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Irina Borodina, Behrooz Darbani, Vasil D'ambrosio, Guokun Wang, Iben Møller-Hansen, Michael Krogh Jensen, Hanne Bjerre Christensen, and Mahsa Babaei
- Subjects
0106 biological sciences ,Muconic acid ,Saccharomyces cerevisiae Proteins ,Cell factory ,Antiporter ,Metabolite ,Saccharomyces cerevisiae ,Protocatechuic acid ,Bioengineering ,Betaxanthins ,01 natural sciences ,Applied Microbiology and Biotechnology ,Antiporters ,Article ,Metabolic engineering ,03 medical and health sciences ,chemistry.chemical_compound ,010608 biotechnology ,030304 developmental biology ,0303 health sciences ,biology ,Transporter ,biology.organism_classification ,Transporter protein ,Sorbic Acid ,Transport protein ,Metabolic Engineering ,Biochemistry ,chemistry ,Synthetic Biology ,Biotechnology - Abstract
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., Highlights • Transporter engineering is important in strain development. • 361 non-essential native transporters can be disrupted via CRISPR-Cas9 in yeast S. cerevisiae. • Biosensors enable high-throughput screening of transporter mutants. • Transport of cis,cis-muconic (CCM), protocatechuic (PCA) acids, and betaxanthins was engineered. • Tpo2p was validated as an importer of CCM and PCA in Xenopus expression assays.
- Published
- 2021