title: Improvement on the yield of polyhydroxyalkanotes production from cheese whey by a recombinant Escherichia coli strain using the proton suicide methodology.
abstract: In this work Escherichia coli strain CML3-1 was engineered through the insertion of Cupriavidus necator P(3HB)-synthesis genes, fused to a lactose-inducible promoter, into the chromosome, via transposition-mediated mechanism. It was shown that polyhydroxyalkanotes (PHAs) production by this strain, using cheese whey, was low due to a significant organic acids (OA) synthesis. The proton suicide method was used as a strategy to obtain an E. coli mutant strain with a reduced OA-producing capacity, aiming at driving bacterial metabolism toward PHAs synthesis. Thirteen E. coli mutant strains were obtained and tested in shake flask assays, using either rich or defined media supplemented with lactose. P8-X8 was selected as the best candidate strain for bioreactor fed-batch tests using cheese whey as the sole carbon source. Although cell growth was considerably slower for this mutant strain, a lower yield of OA on substrate (0.04 Cmol(OA)/Cmol(lac)) and a higher P(3HB) production (18.88 g(P(3HB))/L) were achieved, comparing to the original recombinant strain (0.11 Cmol(OA)/Cmol(lac) and 7.8 g(P(3HB))/L, respectively). This methodology showed to be effective on the reduction of OA yield by consequently improving the P(3HB) yield on lactose (0.28 Cmol (P(3HB))/Cmol(lac) vs 0.10 Cmol(P(3HB))/Cmol(lac) of the original strain).
authors.affiliation: REQUIMTE/CQFB, Chemistry Department, FCT/Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
authors.forename: Joana
authors.initials: J
authors.lastname: Pais
authors.affiliation: REQUIMTE/CQFB, Chemistry Department, FCT/Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
authors.forename: Inês
authors.initials: I
authors.lastname: Farinha
authors.affiliation: REQUIMTE/CQFB, Chemistry Department, FCT/Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
authors.forename: Filomena
authors.initials: F
authors.lastname: Freitas
authors.affiliation: CICECO, Departamento de Química, Universidade de Aveiro, Campus Universitário de Santiago, P-3810-193 Aveiro, Portugal.
authors.forename: Luísa S
authors.initials: LS
authors.lastname: Serafim
authors.affiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, C/Ramiro de Maeztu, 9, 28040 Madrid, Spain.
authors.forename: Virginia
authors.initials: V
authors.lastname: Martínez
authors.affiliation: Biomedal S.L., Avda. Américo Vespucio 5E. 1ª M12, 41092 Seville, Spain.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Martínez
authors.affiliation: Biomedal S.L., Avda. Américo Vespucio 5E. 1ª M12, 41092 Seville, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Arévalo-Rodríguez
authors.affiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, C/Ramiro de Maeztu, 9, 28040 Madrid, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Auxiliadora Prieto
authors.affiliation: REQUIMTE/CQFB, Chemistry Department, FCT/Universidade Nova de Lisboa, 2829-516 Caparica, Portugal. Electronic address: amr@fct.unl.pt.
authors.forename: Maria A M
authors.initials: MA
authors.lastname: Reis
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001431:Bacteriological Techniques; D019149:Bioreactors; D001959:Bromates / Q000494:pharmacology; D001965:Bromides / Q000494:pharmacology; D002611:Cheese; D003470:Culture Media / Q000494:pharmacology; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005813:Genes, Synthetic; D006885:Hydroxybutyrates / Q000378:metabolism*; D007220:Industrial Waste; D007785:Lactose / Q000378:metabolism; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D054813:Polyhydroxyalkanoates / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012641:Selection, Genetic
chemical_list: D001426:Bacterial Proteins; D001959:Bromates; D001965:Bromides; D003470:Culture Media; D006885:Hydroxybutyrates; D007220:Industrial Waste; D009097:Multienzyme Complexes; D054813:Polyhydroxyalkanoates; D011993:Recombinant Fusion Proteins; D007785:Lactose
keywords: CFU; CW; DCW; LB; Luria Bertani; OA; OA production yield on lactose; OA volumetric production rate; Organic acids production inhibition; P(3HB); P(3HB) volumetric production rate; PHA; Polyhydroxyalkanoates (PHAs); Proton suicide method; Recombinant E. coli; X; Y(OA/X); Y(OA/lac); Y(P(3HB)/lac); Y(X/lac); active biomass; cheese whey; colony forming units; dry cell weight; growth yield on lactose; lactose volumetric consumption rate; organic acids; polyhydroxyalkanoates; polyhydroxybutyrate; q(lac); r(Lac); r(OA); r(P(3HB)); specific OA yield; specific lactose consumption rate; storage yield on lactose