Abstract

D-xylose is the major component of hemicellulose and is the second most abundant sugar in nature. However, few microorganisms are able to consume it in an efficient manner. Four different pathways have been identified in diverse microorganisms. Weimberg pathway has as first step the oxidation of xylose into xylono-lactone by a xylose dehydrogenase, and produces α-ketoglutarate as final product. Dahms pathway has some steps in common with Weimberg route, but produces pyruvate and glycoladehyde as final metabolites. Xylose isomerase pathway converts xylose into xylulose-5-phosphate, a pentose phosphate pathway intermediate. Finally, the oxo-reductive pathway, originally described in fungi, involves conversion of xylose into xylitol and then into xylulose-5-phosphate. Herbaspirillum seropedicae are β-proteobacteria isolated as endophytes from sugarcane, rice and other plants. When xylose is used as sole carbon source, strain Z69 of this species can grow and accumulate up to 50% of its dry weight as poly-3-hydroxybutyrate (PHB). The catabolism of D-xylose and the metabolic pathways involved have not been yet described in H. seropedicae. The objective of this work is to identify the metabolic network for catabolism of xylose in H. seropedicae Z69. This includes the identification of relevant genes and their expression. This will help us to optimize the production of PHB from raw materials rich in xylose, like lignocellulosic biomass. Only the genome sequence of H. seropedicae Smr1 is currently available. Considering that strains Z69 and Smr1 are highly similar, genome sequences of strain Smr1 were used to identify ORFs potentially able to encode for enzymes expressed for xylose metabolism. Based on these DNA sequences, a collection of knock-out mutants derived from Z69 were constructed, including simple and double mutants. One of these mutants has a deletion in ORF Hsero_4497 (Z69ΔfabG). This gene, annotated as fabG, was selected as a candidate to encode a xylose dehydrogenase. Compared with wild-type, the specific growth rate of Z69ΔfabG grown on xylose was reduced by 50%. This mutant also showed a drastic reduction in xylose dehydrogenase activity compared with the wild-type strain. Cell extracts of the wild-type strain grown on xylitol as sole carbon source had an appreciable activity of xylitol dehydrogenase (XDH), absent in extracts prepared from cells grown on xylose or glucose. However, the XDH activity in mutant Z69ΔfabG grown on xylose was significantly higher than the wild-type grown under the same conditions. This suggests an alternative oxo-reductive pathway is expressed in the absence of a functional Weimberg pathway. The identification of gene(s) and genetic mechanisms responsible for oxo-reductive pathway is in progress: mutants in ORF Hsero_3396 (candidate to encode a XDH) derived from strain Z69 and Z69ΔfabG are being constructed to test this hypothesis.