Amino Acid Accumulation Limits the Overexpression of Proteins in Lactococcus lactis

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. Methodology/Principal Findings 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.