Dwindling fossil fuel resources and food security concerns motivate production of a wide range of chemicals and materials from renewable, abundant sources such as lignocellulosic biomass. Treatment of lignocellulosic biomass releases an array of sugars that are used by microbial cell factories (e.g. Saccharomyces cerevisiae) for biofuel-, biomolecule-, and biomaterials production. Economic productivity on lignocellulosic hydrolysates using S. cerevisiae requires co-utilization of abundant hexose and pentose sugars during fermentation. However, metabolically engineered strains will preferentially utilize D-glucose resulting from poor transport of other relatively abundant sugars (e.g. D-xylose) arising from competitive inhibition. Realizing sugar co-utilization for bioconversion purposes requires additional novel carbohydrate transporter discovery and/or protein engineering efforts. Anaerobic gut fungi produce and utilize an array of sugars from raw lignocellulosic biomass in a highly competitive environment, and therefore they are an attractive platform for transporter discovery. We have identified more than 100 carbohydrate transporter sequences from the assembled transcriptomes of three gut fungal isolates, including families novel to fungal organisms (i.e. ABC and SWEET systems). In this study, putative gut fungal carbohydrate transporters were functionally characterized in S. cerevisiae for the first time. Membrane proteomics were used to validate in silico identification and functional annotation of these transporters. In parallel, recombinant expression in the transporter-deficient strain EBY.VW4000 assayed production compatibility and carbohydrate transport function, and showed that gut fungal transporters can recover transport of select carbohydrates. Novel transporter characteristics were characterized using FRET methods and liquid scintillation counting. Furthermore, proof-of-principle evolutionary protein engineering sought to eliminate glucose transport in these transporters in favor of other hexose and pentose sugars. Future work aims to expand discovery using native consortia metatranscriptomes and to realize selective transport through additional engineering efforts.

Engineering gut fungal membrane-embedded transporters for metabolic engineering in Saccharomyces cerevisiae

255th National Meeting (2018)

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