title: Amino acid accumulation limits the overexpression of proteins in Lactococcus lactis.
abstract: BACKGROUND
  Understanding the biogenesis pathways for the functional expression of recombinant proteins, in particular membrane proteins and complex multidomain assemblies, is a fundamental issue in cell biology and of high importance for future progress in structural genomics. In this study, we employed a proteomic approach to understand the difference in expression levels for various multidomain membrane proteins in L. lactis cells grown in complex and synthetic media.
  
  
  RESULTS
  The proteomic profiles of cells growing in media in which the proteins were expressed to high or low levels suggested a limitation in the availability of branched-chain amino acids, more specifically a too limited capacity to accumulate these nutrients. By supplying the cells with an alternative path for accumulation of Ile, Leu and/or Val, i.e., a medium supplement of the appropriate dipeptides, or by engineering the transport capacity for branched-chain amino acids, the expression levels could be increased several fold.
  
  
  CONCLUSIONS
  We show that the availability of branched chain amino acids is a critical factor for the (over)expression of proteins in L. lactis. The forward engineering of cells for functional protein production required fine-tuning of co-expression of the branched chain amino acid transporter.
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, Netherlands Proteomics Centre, Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands.
authors.forename: Ravi K R
authors.initials: RK
authors.lastname: Marreddy
authors.forename: Eric R
authors.initials: ER
authors.lastname: Geertsma
authors.forename: Hjalmar P
authors.initials: HP
authors.lastname: Permentier
authors.forename: Joao P C
authors.initials: JP
authors.lastname: Pinto
authors.forename: Jan
authors.initials: J
authors.lastname: Kok
authors.forename: Bert
authors.initials: B
authors.lastname: Poolman
mesh_terms: D026905:Amino Acid Transport Systems; D000596:Amino Acids / Q000378:metabolism*; D000597:Amino Acids, Branched-Chain / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D020543:Proteome / Q000032:analysis
chemical_list: D026905:Amino Acid Transport Systems; D000596:Amino Acids; D000597:Amino Acids, Branched-Chain; D001426:Bacterial Proteins; D011506:Proteins; D020543:Proteome