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.


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.
Marreddy
Ravi K R
RK
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, Netherlands Proteomics Centre, Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands.
Geertsma
Eric R
ER
Permentier
Hjalmar P
HP
Pinto
Joao P C
JP
Kok
Jan
J
Poolman
Bert
B
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
D026905:Amino Acid Transport Systems; D000596:Amino Acids; D000597:Amino Acids, Branched-Chain; D001426:Bacterial Proteins; D011506:Proteins; D020543:Proteome