Microbial production of xylonic acid can be achieved via xylose oxidation catalyzed by the xylose dehydrogenase enzyme. In the present study, 11 putative xylose dehydrogenases (XDH) from bacterial and fungi were identified through in silico analysis using the sequences of the two XDH previously described. Among them, 6 genes were successfully cloned and expressed in Komagataella phaffii (Pichia pastoris) as demonstrated by the ability of the strains to produce xylonic acid. The best strain was capable of producing up to 37.1 ± 1.9 and 11.7 ± 1.6 g/L of xylonic acid with yields of 0.96 ± 0.02 and 0.40 ± 0.06 g/g in mineral medium and sugarcane bagasse hydrolysate, respectively. The results presented here demonstrated the functionality of 6 newly identified xylose dehydrogenases, increasing the number of XDH described. In addition, the efficiency of K. phaffii as a xylonic acid producer is showed for the first time. Display omitted •Six new XDHs were identified and functionally expressed in the yeast K. phaffii.•Recombinant strains were able to produce xylonic acid from renewable sugar.•0.96 g xylonic acid/g xylose was obtained in the bioreactor with the best strain.•Sugarcane bagasse hydrolysate were used as a substrate for xylonic acid production.•K. phaffii can be used as an efficient microbial platform to produce organic acids.