Disruption of the 2-methylcitric acid cycle and evaluation of poly-3-hydroxybutyrate-co-3- hydroxyvalerate biosynthesis suggest alternate catabolic pathways of propionate in Burkholderia sacchari Erica Mendes Pereira, Sonia Regina Silva-Queiroz, Jose ´ Grego ´ rio Cabrera Gomez, and Luiziana Ferreira Silva Abstract: The objective of the present work was to evaluate the relevance of the 2-methylcitric acid cycle (2MCC) to the catabolism of propionate in Burkholderia sacchari. Two B. sacchari mutants unable to grow on propionate were obtained: one disrupted in acnM, and the other in acnM and prpC deleted. An operative 2MCC significantly reduces the bacterial ability to incorporate 3-hydroxyvalerate (3HV) into a biodegradable copolyester accumulated from carbohydrates plus pro- pionate. The efficiency of the mutants in converting propionate to 3HV units (Y3HV/prp) increased from 0.09 gÁg –1 to 0.81– 0.96 gÁg –1 , indicating that acnM and prpC are both essential for growth on propionate. None of the mutations resulted in achievement of the maximum theoretical Y3HV/prp (1.35 gÁg –1 ). When increasing concentrations of propionate were supplied, decreasing values of Y3HV/prp were observed. The results obtained corroborate the hypothesis of the presence of other propi- onate catabolic pathways in B. sacchari. The 2MCC would be the more operative pathway, but a second pathway, which remains to be elucidated, would assume more importance under propionate concentrations of 1 gÁL –1 or higher. The effi- ciency in converting propionate to 3HV units can be improved by decreasing the propionate concentrations, owing to the role of the 2MCC. Key words: Burkholderia sacchari, 2-methylcitric acid cycle, polyhydroxyalkanoate, 3-hydroxyvalerate, propionate catabo- lism. Re ´sume ´: L’objectif de ce travail e ´tait d’e ´valuer l’implication du cycle du 2-me ´thyl citrate (C2MC) sur le catabolisme du propionate chez Burkholderia sacchari. Deux mutants de B. sacchari incapables de croı ˆtre sur le propionate ont e ´te ´ obte- nus : un mutant dont le ge `ne acnM e ´tait interrompu et un mutant dont les ge `nes acnM et prpC e ´taient supprime ´s. Un C2MC fonctionnel re ´duisait significativement la capacite ´ bacte ´rienne a ` incorporer le 3-hydroxyvale ´rate (3HV) dans le co- polyester biode ´gradable accumule ´a ` partir des hydrates de carbone et du propionate. L’efficacite ´a ` convertir le propionate en unite ´s de 3HV (Y3HV/prp) de ´montre ´e par les mutants augmentait de 0,09 gÁg –1 a ` 0,81–0,96 gÁg –1 , indiquant que les ge `nes acnM et prpC sont tous deux essentiels a ` la croissance sur propionate. Aucune des mutations ne conduisait a ` l’atteinte du Y3HV/prp maximal the ´orique (1,35 gÁg –1 ). Lorsque des concentrations croissantes de propionate e ´taient ajoute ´es, des valeurs de Y3HV/prp de ´croissantes e ´taient observe ´es. Les re ´sultats obtenus corroborent l’hypothe `se de la pre ´sence d’autres sentiers cataboliques du propionate chez B. sacchari. Le C2MC serait plus ope ´rant, mais un deuxie `me sentier qui reste a `e ´lucider serait plus important a ` des concentrations de propionate de 1 gÁL –1 ou plus. L’efficacite ´ de conversion du propionate en unite ´s de 3HV peut e ˆtre ame ´liore ´e en diminuant les concentrations de propionate, a ` cause du ro ˆle joue ´ par le C2MC. Mots-cle ´s : Burkholderia sacchari, cycle du 2-me ´thylcitrate, polyhydroxyalcanoate, 3-hydroxyvale ´rate, catabolisme du pro- pionate. [Traduit par la Re ´daction] Introduction Poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P3HB-co- 3HV) is a biodegradable polyester of industrial interest that is accumulated, as intracellular granules, by bacteria mainly under unbalanced growth conditions (Page 1995; Steinbu ¨- chel and Valentin 1995). Studies to produce P3HB-co-3HV using sugarcane derivatives and propionate selected Burkholderia sacchari as a promising bacterial strain (Gomez et al. 1996; Gomez and Bueno Netto 1997; Bra ¨mer and Steinbu ¨chel 2001; Nonato et al. 2001; Silva et al. 2004). However, as in other bacteria, propionate is devi- Received 7 January 2009. Accepted 27 January 2009. Published on the NRC Research Press Web site at cjm.nrc.ca on 23 June 2009. E.M. Pereira, S.R. Silva-Queiroz, J.G.C. Gomez, and L.F. Silva. 1 Departamento de Microbiologia, Instituto de Cie ˆncias Biome ´dicas, Universidade de Sa ˜o Paulo, Av. Prof. Lineu Prestes 1374, CEP 05508-000, Sa ˜o Paulo SP, Brazil; Programa de Po ´s-Graduac ¸a ˜o Interunidades em Biotecnologia Universidade de Sa ˜o Paulo, Av. Prof. Lineu Prestes 1730, CEP 05508-900, Brazil. 1 Corresponding author (e-mail: lukneif@usp.br). 688 Can. J. Microbiol. 55: 688–697 (2009) doi:10.1139/W09-018 Published by NRC Research Press Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by USP-SIBI/Sistema Integrado on 10/08/12 For personal use only.