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.

Figure 1

Identification of NCU00988 as a candidate D-galacturonic acid (D-GalA) transporter. (A) Induction of selected transporter genes after transfer for 4 h to pectin versus sucrose. The log₂-fold induction of seven major facilitator superfamily (MFS)-type transporters from the most pectin-specific co-expression cluster as determined by Cuffdiff [37] is shown. Data were calculated from independent triplicate cultures. (B) Relative expression levels of the top three pectin-induced transporter genes in N. crassa as determined by quantitative PCR. Sucrose pre-grown mycelia were transferred to Vogel’s medium with 2 μM D-GalA or w/o carbon source (no carbon). Samples were taken 4 h after transfer. Relative transcript quantities (RQ) are depicted where 1 represents the transcription level on medium w/o carbon source. Only NCU00988 showed a response to the presence of D-GalA. Bars represent standard deviations (n = 3). (C) Pectin consumption phenotype. The deletion strains of the top three pectin-responsive transporters were grown for 4 days on 1% pectin and the consumption of pectin followed over time by measuring the remaining substrate concentration in the culture supernatants by the phenol-sulfuric acid method. Data represent the mean of triplicates and are plotted relative to the values at day 1 (as 100%). Only the deletion strain for NCU00988 (black triangle) displayed a strong delay in pectin consumption. WT, wild-type.

Figure 2

The NCU00988 deletion strain Δgat-1 is unable to take up D-galacturonic acid (D-GalA). (A) Carbon source-specific growth phenotype. Conidia of N. crassa wild-type (WT) and the ∆gat-1 deletion strain were inoculated into 3-mL cultures containing various 2% carbon sources and incubated at 25°C, 250 rpm in the light. At the indicated times (1.5 to 4.0 days) the mycelia were harvested and the dry weight determined. Bars represent standard deviations (n = 5). The ∆gat-1 strain displayed a specific growth reduction on pectic substrates (polygalacturonic acid (PGA) and pectin from citrus peel). The extent of the biomass reduction is indicated (in % of WT). (B,C) Monosaccharide transport assays. Sucrose pre-grown N. crassa WT and ∆gat-1 mycelia were transferred for 4 h to 0.5% pectin to induce the pectinolytic response and subsequently to the reaction solution containing 90 μM each of the indicated monosaccharides and Vogel’s salts (at pH 5.8). The cultures were incubated in the reaction solution for 30 minutes at 25°C, 250 rpm in the light. Aliquots of the supernatant were taken at regular intervals and the remaining sugar concentrations analyzed by HPAEC-PAD. Bars represent standard deviations (n = 3). ∆gat-1 was found to be unable to transport D-GalA as well as D-glucuronic acid (D-GlcA). Suc, sucrose.

Figure 3

GAT-1 is required for D-galacturonic acid (D-GalA)-mediated induction of pectinases. (A,B) Relative expression levels of the exo-polygalacturonase gene gh28-2 (NCU06961) in N. crassa as determined by quantitative PCR. Sucrose pre-grown WT and ∆gat-1 mycelia were transferred to medium with 0.5% citrus peel pectin, 2 μM D-GalA or w/o carbon source (NoC). Samples were taken 4 h after transfer. Relative transcript quantities (RQ) are depicted, where 1 represents the transcription level on NoC. The ∆gat-1 deletion strain displayed a strong reduction of pectinase induction in both conditions. WT, wild-type.

Figure 4

Heterologous expression of gat-1 in yeast confers the ability to uptake D-galacturonic acid (D-GalA) with high affinity. The cDNA of gat-1 was fused to the super folder-green fluorescent protein (sfGFP) under the control of the phosphoglycerate kinase 1 (PGK1) promoter and transformed into yeast (S. cerevisiae strain BY4742). (A) The incorporation of the construct into the plasma membrane was followed by confocal microscopy (100 × oil; upper panels), whereas no GFP fluorescence could be observed in the vector-only transformed control cells (lower panels). (B) D-GalA transport by the S. cerevisiae strains as described above. Shown is D-GalA transport by yeast with (closed circles) or without (open circles) GAT-1. The initial concentration of D-GalA was 50 μM. All values are the mean of two measurements. (C) Kinetics of D-GalA transport by GAT-1. The transport rate was determined as a function of D-GalA concentration (ranging from 0.1 μM to 10 μM) by yeast strains expressing gat-1-sfGFP and was normalized by total protein concentration.

Figure 5

Bioconversion of D-galacturonic acid (D-GalA) to downstream products by genetically engineered yeast strains. (A) D-GalA can be converted to meso-galactaric acid (GalAA) and L-galactonate (L-GalOA) in S. cerevisiae strains heterologously expressing uronate dehydrogenase (UDH) or D-galacturonic acid reductase (GAAA), respectively, using endogenous cofactors. (B) Bioconversion yeast strains expressing GAT-1 exhibit rapid, high-affinity uptake of D-GalA (at pH 5.8 and an initial D-GalA concentration of 90 μM). (C) Intracellular products were detected by liquid chromatography coupled to tandem mass-spectrometry of chloroform:methanol:water-extracted yeast cells from the 1-h time point samples and their accumulation was found to be transporter-dependent. (D) Even at high D-GalA conditions (100 mM, pH 6.0) co-expression of GAT-1 in GAAA- or UDH-expressing yeast strains increases bioconversion product accumulation by an average 1.8- and 2.1-fold, respectively. NAD⁺, Nicotinamide adenine dinucleotide; NADPH, Nicotinamide adenine dinucleotide phosphate (reduced); GFP, green fluorescent protein.