Pseudomonas putida JM 37 metabolized glyoxylate at a specific rate of 55 g/g dry biomass/day. In order to investigate their role, three genes encoding enzymes that potentially convert glyoxylate were disrupted, namely tartronate semialdehyde synthase (gcl), malate synthase (glcB) and isocitrate lyase (aceA). Strains with transposon insertion in either of these genes were isolated from a 50,000 clone library employing a PCR-guided enrichment strategy. Further, all three respective double mutants were constructed via site-directed insertion of a knock-out plasmid. Neither mutation of gcl, glcB, aceA nor any of the respective double mutation influenced glyoxylic acid conversion, indicating that P. putida JM37 may possess other enzymes and pathways for glyoxylate metabolism.