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

J. Philipp Benz; Ryan J. Protzko; Jonas M.S. Andrich; Stefan Bauer; John E. Dueber; Chris R. Somerville

doi:10.1186/1754-6834-7-20

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

J. Philipp Benz, Ryan J. Protzko, Jonas M.S. Andrich, Stefan Bauer, John E. Dueber, Chris R. Somerville

Research output: Contribution to journal › Article › peer-review

55 Scopus citations

Abstract

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.

Original language	English
Article number	20
Journal	Biotechnology for Biofuels
Volume	7
Issue number	1
DOIs	https://doi.org/10.1186/1754-6834-7-20
State	Published - 6 Feb 2014
Externally published	Yes

Keywords

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

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1186/1754-6834-7-20

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@article{aa74b34c2a034b6a8e02ae7b22d4a67b,

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

abstract = "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.",

keywords = "Bioconversion, D-galacturonic acid, L-galactonic acid, Meso-galactaric acid, Metabolic engineering, Neurospora crassa, Pectin, Saccharomyces cerevisiae, Sugar transport",

author = "Benz, {J. Philipp} and Protzko, {Ryan J.} and Andrich, {Jonas M.S.} and Stefan Bauer and Dueber, {John E.} and Somerville, {Chris R.}",

note = "Funding Information: We want to thank Ana B Ib{\'a}{\~n}ez for excellent technical assistance, Jon Galazka, Yuping Lin and Ligia Acosta-Sampson for help with the set-up of the tritiated kinetics experiments, and Shu-Lun Tang for help with the ChemDraw structures. We thank Luke Latimer for helpful advice and for his comments on the manuscript. We furthermore acknowledge the FGSC and the Neurospora Genome Project for continuous support. This work was supported by grants from the Energy Biosciences Institute to CRS and JED. JPB was supported by a Feodor-Lynen fellowship from the Humboldt-Foundation (GER).",

year = "2014",

month = feb,

day = "6",

doi = "10.1186/1754-6834-7-20",

language = "English",

volume = "7",

journal = "Biotechnology for Biofuels",

issn = "1754-6834",

publisher = "BioMed Central Ltd.",

number = "1",

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TY - JOUR

T1 - Identification and characterization of a galacturonic acid transporter from Neurospora crassa and its application for Saccharomyces cerevisiae fermentation processes

AU - Benz, J. Philipp

AU - Protzko, Ryan J.

AU - Andrich, Jonas M.S.

AU - Bauer, Stefan

AU - Dueber, John E.

AU - Somerville, Chris R.

N1 - Funding Information: We want to thank Ana B Ibáñez for excellent technical assistance, Jon Galazka, Yuping Lin and Ligia Acosta-Sampson for help with the set-up of the tritiated kinetics experiments, and Shu-Lun Tang for help with the ChemDraw structures. We thank Luke Latimer for helpful advice and for his comments on the manuscript. We furthermore acknowledge the FGSC and the Neurospora Genome Project for continuous support. This work was supported by grants from the Energy Biosciences Institute to CRS and JED. JPB was supported by a Feodor-Lynen fellowship from the Humboldt-Foundation (GER).

PY - 2014/2/6

Y1 - 2014/2/6

N2 - 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.

AB - 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.

KW - Bioconversion

KW - D-galacturonic acid

KW - L-galactonic acid

KW - Meso-galactaric acid

KW - Metabolic engineering

KW - Neurospora crassa

KW - Pectin

KW - Saccharomyces cerevisiae

KW - Sugar transport

UR - http://www.scopus.com/inward/record.url?scp=84897671128&partnerID=8YFLogxK

U2 - 10.1186/1754-6834-7-20

DO - 10.1186/1754-6834-7-20

M3 - Article

AN - SCOPUS:84897671128

SN - 1754-6834

VL - 7

JO - Biotechnology for Biofuels

JF - Biotechnology for Biofuels

IS - 1

M1 - 20

ER -

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

Abstract

Keywords

UN SDGs

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