XylR regulates genes at xyl cluster, involved in D-xylose catabolism in Herbaspirillum seropedicae Z69.

D-xylose, one of the most abundant sugars in lignocellulosic biomass, is not widely used to produce bioproducts with added value, in part due to the absence of industrial microorganisms able to metabolize it efficiently. Herbaspirillum seropedicae Z69 is a ß-proteobacterium able to accumulate poly-3-hydroxybutyrate, a biodegradable thermoplastic biopolymer, with contents higher than 50%. It metabolizes D-xylose by non-phosphorylative pathways. In the genome of Z69, we found the genes xylFGH (ABC D-xylose transporter), xylB, xylD, and xylC (superior non-phosphorylative pathway), and the transcriptional regulator xylR, forming the xyl cluster. We constructed the knock-out mutant Z69ΔxylR that has a reduced growth in D-xylose and in D-glucose, compared with Z69. In addition, we analyzed the expression of xyl genes by RT-qPCR and promoter fusion. These results suggest that XylR activates the expression of genes at the xyl cluster in the presence of D-xylose. On the other hand, XylR does not regulate the expression of xylA, mhpD (lower non-phosphorylative pathways) and araB (L-arabinose dehydrogenase) genes. The participation of D-glucose in the regulation mechanism of these genes must still be elucidated. These results contribute to the development of new strains adapted to consume lignocellulosic sugars for the production of value-added bioproducts.

Herbaspirillum seropedicae expresses non-phosphorylative pathways for D-xylose catabolism.

Herbaspirillum seropedicae is a ß-proteobacterium that establishes as an endophyte in various plants. These bacteria can consume diverse carbon sources, including hexoses and pentoses like D-xylose. D-xylose catabolic pathways have been described in some microorganisms, but databases of genes involved in these routes are limited. This is of special interest in biotechnology, considering that D-xylose is the second most abundant sugar in nature and some microorganisms, including H. seropedicae, are able to accumulate poly-3-hydroxybutyrate when consuming this pentose as a carbon source. In this work, we present a study of D-xylose catabolic pathways in H. seropedicae strain Z69 using RNA-seq analysis and subsequent analysis of phenotypes determined in targeted mutants in corresponding identified genes. G5B88_22805 gene, designated xylB, encodes a NAD+-dependent D-xylose dehydrogenase. Mutant Z69∆xylB was still able to grow on D-xylose, although at a reduced rate. This appears to be due to the expression of an L-arabinose dehydrogenase, encoded by the araB gene (G5B88_05250), that can use D-xylose as a substrate. According to our results, H. seropedicae Z69 uses non-phosphorylative pathways to catabolize D-xylose. The lower portion of metabolism involves co-expression of two routes: the Weimberg pathway that produces α-ketoglutarate and a novel pathway recently described that synthesizes pyruvate and glycolate. This novel pathway appears to contribute to D-xylose metabolism, since a mutant in the last step, Z69∆mhpD, was able to grow on this pentose only after an extended lag phase (40-50 h). KEY POINTS: • xylB gene (G5B88_22805) encodes a NAD+-dependent D-xylose dehydrogenase. • araB gene (G5B88_05250) encodes a L-arabinose dehydrogenase able to recognize D-xylose. • A novel route involving mhpD gene is preferred for D-xylose catabolism.

Propionic acid metabolism and poly-3-hydroxybutyrate-co-3-hydroxyvalerate production by a prpC mutant of Herbaspirillum seropedicae Z69.

Polyhydroxyalkanoates (PHAs) are thermoplastic polyesters produced by a wide range of bacteria as carbon and energy reserves. PHA accumulation is typically increased under unbalanced growth conditions and with carbon source in excess. Although polyhydroxybutyrate (PHB) could be used for specific applications, it is brittle and not a useful alternative for plastics like polypropylene. Far more useful polypropylene-like PHAs, are copolymers composed of 3-hydroxybutyrate and 3-hydroxyvalerate, P(3HB-co-3HV). Propionic acid is one of the carbon sources that can be used to generate 3HV. A mutant derived from Herbaspirillum seropedicae Z69, a strain previously described as capable of producing P(3HB-co-3HV) from propionic acid, was constructed to increase 3HV biosynthetic efficiency. The strategy involved elimination of a catabolic route for propionyl-CoA by deficiency marker exchange of a selected gene. The mutant (Z69Prp) was constructed by elimination of the 2-methylcitrate synthase (PrpC) gene of the 2-methylcitrate cycle for propionate catabolism. Strain Z69Prp was unable to grow on sodium propionate, but in cultures with glucose-propionate accumulated 50% of its dry weight as copolymer. Z69Prp had 14.1 mol% 3HV; greater than that of strain Z69 (2.89 mol%). The 3HV yield from propionic acid (Y3HV/prop) was 0.80 g g-1, and below the maximum theoretical value (1.35 g g-1).