resumo
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 Cmolg(P(3HB))/Cmol(lac) vs 0.10 Cmolg(P(3HB))/Cmol(lac) of the original strain). (C) 2013 Elsevier Inc. All rights reserved.
palavras-chave
FED-BATCH CULTURE; POLY(3-HYDROXYBUTYRATE) PRODUCTION; DIRECT SELECTION; X-GAL; MUTANTS; RESISTANCE; PATHWAYS; SOLVENT; BIOMASS; ACIDS
categoria
Biotechnology & Applied Microbiology
autores
Pais, J; Farinha, I; Freitas, F; Serafim, LS; Martinez, V; Martinez, JC; Arevalo-Rodriguez, M; Prieto, MA; Reis, MAM
nossos autores
Luísa Seuanes Serafim
Professor Auxiliaragradecimentos
Filomena Freitas acknowledges Fundacao para a Ciencia e a Tecnologia (FC&T, Portugal) for PostDoctoral fellowship SFRH/BPD/72280. Joana Pais and Ines Farinha acknowledge FC&T for PhD fellowships SFFRH/BD/44695/ and SFRH/BDE/52089/2013, respectively. This work was supported by FC&T through projects PEst-C/EQB/LA0006/2011 and PEST-C/CTM/LA/0011/2011. M. Auxiliadora Prieto and Virginia Martinez acknowledge funding grants NMP2-CT-2007-026515 and BIO2010-21049. The authors acknowledge funding from project BIOPRODUCTION FP6-NMP 26515. Cristina Perez, Marta Bonilla and Leonor Matas are gratefully acknowledged for excellent technical support.