Tartronate semialdehyde reductase defines a novel rate-limiting step in assimilation and bioconversion of glycerol in Ustilago maydis

Abstract

Background: Glycerol is a by-product of biodiesel production. Currently, it has limited applications with low bioconversion efficiency to most metabolites reported. This is partly attributed to the poor knowledge on the glycerol metabolic pathway in bacteria and fungi.

Methodology/principal findings: We have established a fast screening method for identification of genes that improve glycerol utilization in Ustilago maydis. This was done by comparing the growth rates of T-DNA tagged mutant colonies on solid medium using glycerol as the sole carbon source. We present a detailed characterization of one of the mutants, GUM1, which contains a T-DNA element inserted into the promoter region of UM02592 locus (MIPS Ustilago maydis database, MUMDB), leading to enhanced and constitutive expression of its mRNA. We have demonstrated that um02592 encodes a functional tartronate semialdehyde reductase (Tsr1), which showed dual specificity to cofactors NAD(+) and NADP(+) and strong substrate specificity and enantioselectivity for D-glycerate. Improved glycerol assimilation in GUM1 was associated with elevated expression of tsr1 mRNA and this could be phenocopied by over-expression of the gene. Glycolipid accumulation was reduced by 45.2% in the knockout mutant whereas introduction of an extra copy of tsr1 driven by the glyceraldehyde phosphate dehydrogenase promoter increased it by 40.4%.

Conclusions/significance: Our results demonstrate that tartronate semialdehyde reductase (TSR) plays an important role in glycerol assimilation in U. maydis and defines a novel target in genetic engineering for improved conversion of glycerol to higher value products. Our results add significant depth to the understanding of the glycerol metabolic pathway in fungi. We have demonstrated, for the first time, a biological role of a eukaryotic TSR.

Figure 1. Tsr1-catalyzed reactions.

Figure 2. Phenotypes of GUM1 and GUM2.

A. Screening for glycerol utilization mutants. Cultures of selected mutants were normalized to 1.0 OD₆₀₀ and spotted on MinG agar plate in 10-fold serial dilutions. The photo was taken after incubation at 28°C for 2 days. B. Glycolipid profiles of WT, GUM1 and GUM2 in glucose or glycerol-containing medium. The MEL and UA species are indicated on the left. WT: wild-type strain (U. maydis L8 strain); D: dextrose as carbon source; G: glycerol as carbon source.

Figure 3. Characterization of GUM1.

A. Southern blot analysis of GUM1 and GUM2. Genomic DNA (10 µg) of WT, GUM1 and GUM2 were digested with BamHI and probed with a digoxigenin-labeled hpt DNA fragment. B. Schematic illustration of T-DNA insertion site in GUM1. LB: left border of T-DNA; Pgpd: gpd promoter; hpt: coding sequence of Hygromycin resistance gene; tsr1L and tsr1R: homologous regions used for knockout of tsr1. C. Expression of um10280 and um02592. WT and GUM1 cells were shifted to YPG medium and harvested after the time indicated (hours), and mRNAs were detected using the probes as illustrated in Figure 2B. Hybridization with ppi probe was used as the control for constitutive expression.

Figure 4. Sequence analysis of β-hydroxyacid dehydrogenases.

A. Phylogenic tree of selected β-hydroxyacid dehydrogenases. Sequence alignment and phylogenic tree was created with Jalview 2.5 program using the average distance method with BLOSUM62 matrix. The sequences and origins can be found in the following Protein Identifiers from GenBank: 145698315 (EcTSR, Escherichia coli str. K-12 substr. MG1655); 16766546 (SeTSR, Salmonella enterica subsp. enterica serovar Typhimurium str. LT2); 152972053 (KpTSR, Klebsiella pneumoniae subsp. pneumoniae MGH 78578); 57226089 (CnTsr, putative TSR of Cryptococcus neoformans var. neoformans JEC21); 46098529 (Um02592, Ustilago maydis 521); 242207395 (PpTsr, putative Tsr of Postia placenta Mad-698-R); 16272945 (HiHIBD, Haemophilus influenzae Rd KW20); 23308751 (HsHIBD, Homo sapiens); 83977457 (RnHIBD, Rattus norvegicus); 39952089 (MgHIBD, Magnaporthe grisea 70-15); 32411867 (NcHIBD, Neurospora crassa OR74A); 18415593 (AtHIBD, Arabidopsis thaliana); 19111887 (SpPGD, Schizosaccharomyces pombe); 40068518 (HsPGD, Homo sapiens); 145611724 (MgPGD, Magnaporthe grisea 70-15); 458910 (ScPGD, Saccharomyces cerevisiae); 15232888 (AtPGD, Arabidopsis thaliana); 46098078 (Um02189, Ustilago maydis 521) and 71014537 (Um02577, Ustilago maydis 521). B. Conserved motifs. *, ∧ and > indicate functionally important residues .

Figure 5. Tsr1-catalyzed reactions.

A. Oxidation of various substrates. β-HBA: β-hydroxybutyric acid; 6-PGA: 6-phosphogluconate acid; D-thr: _D-threonine. B. Effects of cofactors on reduction and oxidation reactions. Standard deviation (SD) derived from triplicates. * and ** indicate significant differences in the group (p<0.05 and 0.001 respectively) based on one-way ANOVA test.

Figure 6. Effects of tsr1 knockout and over-expression.

A. Relative TSR activity in various strains. Tsr1 activity was assayed using _DL-glycerate as substrate and NADP⁺ as cofactor according to the standard enzyme assay method. The activity of WT is set at 100%. tsr1Δ and tsr1^gpd are the knockout and over-expression strains in L8 background, respectively. Tsr1 activity was assayed in triplicates. B. Expression of tsr1 mRNA in various strains. Strains were cultured in YPD medium to late exponential phase (time zero) before shifted to MinG medium for the duration indicated. The blot was probed with digoxigenin-labeled tsr1 cDNA. In tsr1^gpd, the upper band is the overexpressed tsr1-egfp mRNA. The rRNA bands were stained with Methylene Blue. C. Cell morphology and glycolipids accumulation. Cells of WT, tsr1^gpd and tsr1Δ strains that were cultured for 14 days in MinG medium. The needle-like or fibrous material around the cells is UA. The scale bar represents 10 µm.

Figure 7. Effects of glycerate supplementation.

A. Glycolipid profiles of WT and tsr1Δ. Single colonies of WT and tsr1Δ were cultured in MinG (10 g/l glycerol) with (+) or without (-) _DL-glycerate (1%, v/v) at 30°C, 250 rpm for 5 days. B. Glycerol utilization in WT and tsr1Δ. Residual glycerol was quantified by HPLC after a 5-day culture in MinG medium and the relative glycerol utilization was calculated from three independent repeats. ** indicates statistical significance (p<0.001) by one-way ANOVA test.

Figure 8. Deletion analysis of tsr1 promoter.

A. Schematic illustration of reporter constructs. The location of T-DNA insertion site in GUM1 is indicated. Tnos: transcriptional terminator of A. tumefaciens nopaline synthase gene. B. Relative fluorescence intensity of reporter constructs. The normalized fluorescence intensity of the -926 promoter was set at 100% and results were average of triplicates.

Figure 9. Model of glycerol metabilosm in U. maydis.

The pathway is based on Tom et al. and the enzymes for various steps are based on the current information in the KEGG DataBase (http://www.genome.jp/kegg/) and the MIPS Ustilago maydis Genome Database (http://mips.helmholtz-muenchen.de/genre/proj/ustilago/).