title: Plasmid-encoded biosynthetic genes alleviate metabolic disadvantages while increasing glucose conversion to shikimate in an engineered Escherichia coli strain.
abstract: Metabolic engineering strategies applied over the last two decades to produce shikimate (SA) in Escherichia coli have resulted in a battery of strains bearing many expression systems. However, the effects that these systems have on the host physiology and how they impact the production of SA are still not well understood. In this work we utilized an engineered E. coli strain to determine the consequences of carrying a vector that promotes SA production from glucose with a high-yield but that is also expected to impose a significant cellular burden. Kinetic comparisons in fermentors showed that instead of exerting a negative effect, the sole presence of the plasmid increased glucose consumption without diminishing the growth rate. By constitutively expressing a biosynthetic operon from this vector, the more active glycolytic metabolism was exploited to redirect intermediates toward the production of SA, which further increased the glucose consumption rate and avoided excess acetate production. Fluxomics and metabolomics experiments revealed a global remodeling of the carbon and energy metabolism in the production strain, where the increased SA production reduced the carbon available for oxidative and fermentative pathways. Moreover, the results showed that the production of SA relies on a specific setup of the pentose phosphate pathway, where both its oxidative and non-oxidative branches are strongly activated to supply erythrose-4-phosphate and balance the NADPH requirements. This work improves our understanding of the metabolic reorganization observed in E. coli in response to the plasmid-based expression of the SA biosynthetic pathway. Biotechnol. Bioeng. 2017;114: 1319-1330. © 2017 Wiley Periodicals, Inc.
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos, Mexico.
authors.forename: Alberto
authors.identifier: 0000-0002-4045-0108
authors.initials: A
authors.lastname: Rodriguez
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos, Mexico.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Martínez
authors.affiliation: LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Millard
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos, Mexico.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Gosset
authors.affiliation: LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France.
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Portais
authors.affiliation: LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France.
authors.forename: Fabien
authors.initials: F
authors.lastname: Létisse
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos, Mexico.
authors.forename: Francisco
authors.initials: F
authors.lastname: Bolivar
mesh_terms: D003198:Computer Simulation; D004926:Escherichia coli / Q000502:physiology*; D024861:Genetic Enhancement / Q000379:methods*; D005947:Glucose / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods; D064688:Metabolic Flux Analysis; D008954:Models, Biological; D010957:Plasmids / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D012765:Shikimic Acid / Q000378:metabolism*; D014162:Transfection / Q000379:methods
chemical_list: D011994:Recombinant Proteins; D012765:Shikimic Acid; D005947:Glucose
keywords: Escherichia coli; metabolism; plasmid burden; shikimate