Bioconversion of D-galacturonate to keto-deoxy-L-galactonate (3-deoxy-L-threo-hex-2-ulosonate) using filamentous fungi

<p>Abstract</p> <p>Background</p> <p>The D-galacturonic acid derived from plant pectin can be converted into a variety of other chemicals which have potential use as chelators, clarifiers, preservatives and plastic precursors. Among these is the deoxy-keto acid derived from L-galactonic acid, keto-deoxy-L-galactonic acid or 3-deoxy-L-<it>threo</it>-hex-2-ulosonic acid. The keto-deoxy sugars have been found to be useful precursors for producing further derivatives. Keto-deoxy-L-galactonate is a natural intermediate in the fungal D-galacturonate metabolic pathway, and thus keto-deoxy-L-galactonate can be produced in a simple biological conversion.</p> <p>Results</p> <p>Keto-deoxy-L-galactonate (3-deoxy-L-<it>threo</it>-hex-2-ulosonate) accumulated in the culture supernatant when <it>Trichoderma reesei </it>Δ<it>lga1 </it>and <it>Aspergillus niger </it>Δ<it>gaaC </it>were grown in the presence of D-galacturonate. Keto-deoxy-L-galactonate accumulated even if no metabolisable carbon source was present in the culture supernatant, but was enhanced when D-xylose was provided as a carbon and energy source. Up to 10.5 g keto-deoxy-L-galactonate l^-1was produced from 20 g D-galacturonate l^-1and <it>A. niger </it>Δ<it>gaaC </it>produced 15.0 g keto-deoxy-L-galactonate l^-1from 20 g polygalacturonate l^-1, at yields of 0.4 to 1.0 g keto-deoxy-L-galactonate [g D-galacturonate consumed]^-1. Keto-deoxy-L-galactonate accumulated to concentrations of 12 to 16 g l^-1intracellularly in both producing organisms. This intracellular concentration was sustained throughout production in <it>A. niger </it>Δ<it>gaaC</it>, but decreased in <it>T. reesei</it>.</p> <p>Conclusions</p> <p>Bioconversion of D-galacturonate to keto-deoxy-L-galactonate was achieved with both <it>A. niger </it>Δ<it>gaaC </it>and <it>T. reesei </it>Δ<it>lga1</it>, although production (titre, volumetric and specific rates) was better with <it>A. niger </it>than <it>T. reesei</it>. <it>A. niger </it>was also able to produce keto-deoxy-L-galactonate directly from pectin or polygalacturonate demonstrating the feasibility of simultaneous hydrolysis and bioconversion. Although keto-deoxy-L-galactonate accumulated intracellularly, concentrations above ~12 g l^-1were exported to the culture supernatant. Lysis may have contributed to the release of keto-deoxy-L-galactonate from <it>T. reesei </it>mycelia.</p

Raspberry Ketone Accumulation in Nicotiana benthamiana and Saccharomyces cerevisiae by Expression of Fused Pathway Genes

Raspberry ketone has generated interest in recent years both as a flavor agent and as a health promoting supplement. Raspberry ketone can be synthesized chemically, but the value of a natural nonsynthetic product is among the most valuable flavor compounds on the market. Coumaroyl-coenzyme A (CoA) is the direct precursor for raspberry ketone but also an essential precursor for flavonoid and lignin biosynthesis in plants and therefore highly regulated. The synthetic fusion of 4-coumaric acid ligase (4CL) and benzalacetone synthase (BAS) enables the channeling of coumaroyl-CoA from the ligase to the synthase, proving to be a powerful tool in the production of raspberry ketone in both N. benthamiana and S. cerevisiae. To the best of our knowledge, the key pathway genes for raspberry ketone formation are transiently expressed in N. benthamiana for the first time in this study, producing over 30 μg/g of the compound. Our raspberry ketone producing yeast strains yielded up to 60 mg/L, which is the highest ever reported in yeast

Performance of Leuconostoc citreum FDR241 during wheat flour sourdough type I propagation and transcriptional analysis of exopolysaccharides biosynthesis genes

This study focused on the performance of the dextran producer Leuconostoc citreum as starter culture during 30 days of wheat flour type I sourdough propagation (back-slopping). As confirmed by RAPD-PCR analysis, the strain dominated throughout the propagation procedure, consisting of daily fermentations at 20 °C. The sourdoughs were characterized by consistent lactic acid bacteria cell density and acidification parameters, reaching pH values of 4.0 and mild titratable acidity. Carbohydrates consumption remained consistent during the propagation procedure, leading to formation of mannitol and almost equimolar amount of lactic and acetic acid. The addition of sucrose enabled the formation of dextran, inducing an increase in viscosity of the sourdough of 2–2.6 fold, as well as oligosaccharides. The transcriptional analysis based on glucosyltransferases genes (GH70) showed the existence in L. citreum FDR241 of at least five different dextransucrases. Among these, only one gene, previously identified as forming only α-(1–6) glycosidic bonds, was significantly upregulated in sourdough fermentation conditions, and the main responsible of dextran formation. A successful application of a starter culture during long sourdough back-slopping procedure will depend on the strain robustness and fermentation conditions. Transcriptional regulation of EPS-synthetizing genes might contribute to increase the efficiency of industrial processes.Peer reviewe

Transcriptome of Saccharomyces cerevisiae during production of D-xylonate

BACKGROUND: Production of D-xylonate by the yeast S. cerevisiae provides an example of bioprocess development for sustainable production of value-added chemicals from cheap raw materials or side streams. Production of D-xylonate may lead to considerable intracellular accumulation of D-xylonate and to loss of viability during the production process. In order to understand the physiological responses associated with D-xylonate production, we performed transcriptome analyses during D-xylonate production by a robust recombinant strain of S. cerevisiae which produces up to 50 g/L D-xylonate. RESULTS: Comparison of the transcriptomes of the D-xylonate producing and the control strain showed considerably higher expression of the genes controlled by the cell wall integrity (CWI) pathway and of some genes previously identified as up-regulated in response to other organic acids in the D-xylonate producing strain. Increased phosphorylation of Slt2 kinase in the D-xylonate producing strain also indicated that D-xylonate production caused stress to the cell wall. Surprisingly, genes encoding proteins involved in translation, ribosome structure and RNA metabolism, processes which are commonly down-regulated under conditions causing cellular stress, were up-regulated during D-xylonate production, compared to the control. The overall transcriptional responses were, therefore, very dissimilar to those previously reported as being associated with stress, including stress induced by organic acid treatment or production. Quantitative PCR analyses of selected genes supported the observations made in the transcriptomic analysis. In addition, consumption of ethanol was slower and the level of trehalose was lower in the D-xylonate producing strain, compared to the control. CONCLUSIONS: The production of organic acids has a major impact on the physiology of yeast cells, but the transcriptional responses to presence or production of different acids differs considerably, being much more diverse than responses to other stresses. D-Xylonate production apparently imposed considerable stress on the cell wall. Transcriptional data also indicated that activation of the PKA pathway occurred during D-xylonate production, leaving cells unable to adapt normally to stationary phase. This, together with intracellular acidification, probably contributes to cell death. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-763) contains supplementary material, which is available to authorized users

Pdc2 coordinates expression of the THI regulon in the yeast Saccharomyces cerevisiae

Coordination of gene expression in response to different metabolic signals is crucial for cellular homeostasis. In this work, we addressed the role of Pdc2 in the coordinated control of biosynthesis and demand of an essential metabolic cofactor, thiaminediphosphate (ThDP). The DNA binding protein Pdc2 was initially identified as a regulator of the genes PDC1 and PDC5, which encode isoforms of the glycolytic enzyme pyruvate decarboxylase (Pdc). The Pdc2 has also been implicated as a regulator of genes encoding enzymes in ThDP metabolism. The ThDP is the cofactor of Pdc. Using global and gene-specific expression analysis, we show that Pdc2 is required for the upregulation of all genes controlled by thiamine availability. The Pdc2 seems to act together with Thi2, a known transcriptional regulator of THI genes. The requirement for these two factors differs in a gene-specific manner. While the Thi2, in conjunction with Thi3, seems to control expression of THI genes with respect to thiamine availability, the Pdc2 may link the ThDP demand to carbon source availability. Interestingly, the enzymes Pdc1 and Pdc5 are enriched in the nucleus. Both are known to affect gene expression in an autoregulatory mechanism and expression of both is regulated by glucose and Pdc2, further pointing to a role of Pdc2 in coordinating different metabolic signals. Our analysis helps to further define the THI regulon and hence the spectrum of genes/proteins involved in the ThDP homeostasis. In particular, we identify novel proteins putatively involved in thiamine and/or ThDP transport across the plasma and the mitochondrial membrane. In conclusion, the THI regulon is the most interesting system to study principles of genes expression and metabolic coordination and deserves further attention