title: Metabolic engineering of riboflavin production in Ashbya gossypii through pathway optimization.
abstract: BACKGROUND
  The industrial production of riboflavin mostly relies on the microbial fermentation of flavinogenic microorganisms and Ashbya gossypii is the main industrial producer of the vitamin. Accordingly, bioengineering strategies aimed at increasing riboflavin production in A. gossypii are highly valuable for industry.
  
  
  RESULTS
  We analyze the contribution of all the RIB genes to the production of riboflavin in A. gossypii. Two important metabolic rate-limiting steps that limit the overproduction of riboflavin have been found: first, low mRNA levels of the RIB genes hindered the overproduction of riboflavin; second, the competition of the AMP branch for purinogenic precursors also represents a limitation for riboflavin overproduction. Thus, overexpression of the RIB genes resulted in a significant increase in riboflavin yield. Moreover, both the inactivation and the underexpression of the ADE12 gene, which controls the first step of the AMP branch, also proved to have a positive effect on riboflavin production. Accordingly, a strain that combines both the overexpression of the RIB genes and the underexpression of the ADE12 gene was engineered. This strain produced 523 mg/L of riboflavin (5.4-fold higher than the wild-type), which is the highest titer of riboflavin obtained by metabolic engineering in A. gossypii so far.
  
  
  CONCLUSIONS
  Riboflavin production in A. gossypii is limited by a low transcription activity of the RIB genes. Flux limitation towards AMP provides committed substrate GTP for riboflavin overproduction without detrimental effects on biomass formation. A multiple-engineered Ashbya strain that produces up to 523 mg/L of riboflavin was generated.
authors.affiliation: Departamento de Microbiología y Genética, Universidad de Salamanca, Campus Miguel de Unamuno, 37007, Salamanca, Spain. rodrigoledesma@usal.es.
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Ledesma-Amaro
authors.affiliation: Departamento de Microbiología y Genética, Universidad de Salamanca, Campus Miguel de Unamuno, 37007, Salamanca, Spain. cserrano@usal.es.
authors.forename: Cristina
authors.initials: C
authors.lastname: Serrano-Amatriain
authors.affiliation: Departamento de Microbiología y Genética, Universidad de Salamanca, Campus Miguel de Unamuno, 37007, Salamanca, Spain. alji@usal.es.
authors.forename: Alberto
authors.initials: A
authors.lastname: Jiménez
authors.affiliation: Departamento de Microbiología y Genética, Universidad de Salamanca, Campus Miguel de Unamuno, 37007, Salamanca, Spain. revuelta@usal.es.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Revuelta
mesh_terms: D055169:Eremothecium / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D011687:Purines / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012256:Riboflavin / Q000096:biosynthesis*; D014158:Transcription, Genetic
chemical_list: D005656:Fungal Proteins; D011687:Purines; D012256:Riboflavin