Identification of the 2-Methylcitrate Pathway Involved in the Catabolism of Propionate in the Polyhydroxyalkanoate-Producing Strain Burkholderia sacchari IPT101 T and Analysis of a Mutant Accumulating a Copolyester with Higher 3-Hydroxyvalerate Content

Abstract

ABSTRACT Burkholderia sacchari IPT101 T induced the formation of 2-methylcitrate synthase and 2-methylisocitrate lyase when it was cultivated in the presence of propionic acid. The prp locus of B. sacchari IPT101 T is required for utilization of propionic acid as a sole carbon source and is relevant for incorporation of 3-hydroxyvalerate (3HV) into copolyesters, and it was cloned and sequenced. Five genes ( prpR , prpB , prpC , acnM , and ORF5) exhibited identity to genes located in the prp loci of other gram-negative bacteria. prpC encodes a 2-methylcitrate synthase with a calculated molecular mass of 42,691 Da. prpB encodes a 2-methylisocitrate lyase. The levels of PrpC and PrpB activity were much lower in propionate-negative mutant IPT189 obtained from IPT101 T and were heterologously expressed in Escherichia coli. The acnM gene (ORF4) and ORF5, which are required for conversion of 2-methylcitric acid to 2-methylisocitric acid in Ralstonia eutropha HF39, are also located in the prp locus. The translational product of ORF1 ( prpR ) had a calculated molecular mass of 70,598 Da and is a putative regulator of the prp cluster. Three additional open reading frames (ORF6, ORF7, and ORF8) whose functions are not known were located adjacent to ORF5 in the prp locus of B. sacchari , and these open reading frames have not been found in any other prp operon yet. In summary, the organization of the prp genes of B. sacchari is similar but not identical to the organization of these genes in other bacteria investigated recently. In addition, this study provided a rationale for the previously shown increased molar contents of 3HV in copolyesters accumulated by a B. sacchari mutant since it was revealed in this study that the mutant is defective in prpC .