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
Transport of substances across biological cell membranes is essential for nutrient uptake (Davidson and Chen, 2004; McCracken and Edinger, 2013), maintenance of cellular homeostasis (Park et al, 2004; Rink and Haase, 2007), and intercellular communication (Record et al, 2014)
We demonstrate a method for efficient CRISPR-Cas9 mediated transporter disruption and apply this to rapidly characterize trans porters with activity towards the endogenously-produced xenobiotic compounds CCM, protocatechuic acid (PCA), and betaxanthins
The use of a simple plasmid transformation for CRISPR-Cas9 medi ated gene disruption was adapted to generate a library of engineered S. cerevisiae strains (Roy et al, 2018)
Summary
Transport of substances across biological cell membranes is essential for nutrient uptake (Davidson and Chen, 2004; McCracken and Edinger, 2013), maintenance of cellular homeostasis (Park et al, 2004; Rink and Haase, 2007), and intercellular communication (Record et al, 2014). Transporter engi neering can result in more efficient cell factories for the production of chemicals by fermentation of renewable feedstocks (Park et al, 2014; Li et al, 2019). Transporter engineering strategies altering the intracellular transport of compounds (Cardenas and Da Silva, 2016), blocking the export of the intermediate metabolites (Park et al, 2014; Li et al, 2019), or enhancing the export of the final products (Doshi et al, 2013; Hara et al, 2017; Hu et al, 2018) have been shown to improve cell factories. The engineering strategies were based on the knowledge about transporter function and specificity, which is only available for a limited number of transporters. There is a need for new high-throughput methods for in vivo transporter characterization
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