Identification and characterization of a galacturonic acid transporter from Neurospora crassa and its application for Saccharomyces cerevisiae fermentation processes

Biotechnology for Biofuels

Chris R Somerville²  John E Dueber⁴  Stefan Bauer⁵  Jonas MS Andrich³  Ryan J Protzko¹  J Philipp Benz⁵ 

[1]Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA

[2]Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA, USA

[3]present address: Institute of Environmental and Sustainable Chemistry, Technische Universität Braunschweig, Braunschweig, Germany

[4]Department of Bioengineering, University of California Berkeley, Berkeley, CA, USA

[5]Energy Biosciences Institute, University of California Berkeley, Berkeley, CA, USA

关键词: L-galactonic acid;    Meso-galactaric acid;    Bioconversion;    Metabolic engineering;    Saccharomyces cerevisiae;    Sugar transport;    Neurospora crassa;    D-galacturonic acid;    Pectin;   

Others  :  793721
DOI  :  10.1186/1754-6834-7-20

 received in 2013-11-05, accepted in 2014-01-15,  发布年份 2014

【 摘 要 】

Background

Pectin-rich agricultural wastes potentially represent favorable feedstocks for the sustainable production of alternative energy and bio-products. Their efficient utilization requires the conversion of all major constituent sugars. The current inability of the popular fermentation host Saccharomyces cerevisiae to metabolize the major pectic monosaccharide D-galacturonic acid (D-GalA) significantly hampers these efforts. While it has been reasoned that the optimization of cellular D-GalA uptake will be critical for the engineering of D-GalA utilization in yeast, no dedicated eukaryotic transport protein has been biochemically described. Here we report for the first time such a eukaryotic D-GalA transporter and characterize its functionality in S. cerevisiae.

Results

We identified and characterized the D-GalA transporter GAT-1 out of a group of candidate genes obtained from co-expression analysis in N. crassa. The N. crassa Δgat-1 deletion strain is substantially affected in growth on pectic substrates, unable to take up D-GalA, and impaired in D-GalA-mediated signaling events. Moreover, expression of a gat-1 construct in yeast conferred the ability for strong high-affinity D-GalA accumulation rates, providing evidence for GAT-1 being a bona fide D-GalA transport protein. By recombinantly co-expressing D-galacturonate reductase or uronate dehydrogenase in yeast we furthermore demonstrated a transporter-dependent conversion of D-GalA towards more reduced (L-galactonate) or oxidized (meso-galactaric acid) downstream products, respectively, over a broad concentration range.

Conclusions

By utilizing the novel D-GalA transporter GAT-1 in S. cerevisiae we successfully generated a transporter-dependent uptake and catalysis system for D-GalA into two products with high potential for utilization as platform chemicals. Our data thereby provide a considerable first step towards a more complete utilization of biomass for biofuel and value-added chemicals production.

【 授权许可】

   
2014 Benz et al.; licensee BioMed Central Ltd.

【 图 表 】

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