title: Enhanced 3-methylcatechol production by Pseudomonas putida TODE1 in a two-phase biotransformation system.
abstract: In this study, genetically engineered Pseudomonas putida TODE1 served as a biocatalyst for the bioproduction of valuable 3-methylcatechol (3MC) from toluene in an aqueous-organic two-phase system. The two-phase system was used as an approach to increase the biocatalyst efficiency. Among the organic solvent tested, n-decanol offered several benefits including having the highest partitioning of 3MC, with a high 3MC yield and low cell toxicity. The effect of media supplementation with carbon/energy sources (glucose, glycerol, acetate and succinate), divalent metal cations (Mg(2+), Ca(2+), Mn(2+) and Fe(2+)), and short-chain alcohols (ethanol, n-propanol and n-butanol) as a cofactor regeneration system on the toluene dioxygenase (TDO) activity, cell viability, and overall 3MC yield were evaluated. Along with the two-step cell preparation protocol, supplementation of the medium with 4 mM glycerol as a carbon/energy source, and 0.4 mM Fe(2+) as a cofactor for TDO significantly enhanced the 3MC production level. When in combination with the use of n-decanol and n-butanol as the organic phase, a maximum overall 3MC concentration of 31.8 mM (166 mM in the organic phase) was obtained in a small-scale production, while it was at 160.5 mM (333.2 mM in the organic phase) in a 2-L scale. To our knowledge, this is the highest 3MC yield obtained from a TDO-based system so far.
authors.affiliation: Graduate Program in Biotechnology, Faculty of Science, Chulalongkorn University.
authors.forename: Ajiraporn
authors.initials: A
authors.lastname: Kongpol
authors.forename: Junichi
authors.initials: J
authors.lastname: Kato
authors.forename: Takahisa
authors.initials: T
authors.lastname: Tajima
authors.forename: Thunyarat
authors.initials: T
authors.lastname: Pongtharangkul
authors.forename: Alisa S
authors.initials: AS
authors.lastname: Vangnai
mesh_terms: D001711:Biotransformation; D002396:Catechols / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry; D060847:Metabolic Engineering; D050296:Microbial Viability; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism*; D014050:Toluene / Q000378:metabolism*
chemical_list: D002396:Catechols; D003470:Culture Media; C031136:3-methylcatechol; D014050:Toluene