title: Efficient overproduction of membrane proteins in Lactococcus lactis requires the cell envelope stress sensor/regulator couple CesSR.
abstract: BACKGROUND
  Membrane proteins comprise an important class of molecules whose study is largely frustrated by several intrinsic constraints, such as their hydrophobicity and added requirements for correct folding. Additionally, the complexity of the cellular mechanisms that are required to insert membrane proteins functionally in the membrane and to monitor their folding state makes it difficult to foresee the yields at which one can obtain them or to predict which would be the optimal production host for a given protein.
  
  
  RESULTS
  We describe a rational design approach to improve the lactic acid bacterium Lactococcus lactis as a producer of membrane proteins. Our transcriptome data shows that the two-component system CesSR, which senses cell envelope stresses of different origins, is one of the major players when L. lactis is forced to overproduce the endogenous membrane protein BcaP, a branched-chain amino acid permease. Growth of the BcaP-producing L. lactis strain and its capability to produce membrane proteins are severely hampered when the CesSR system itself or particular members of the CesSR regulon are knocked out, notably the genes ftsH, oxaA2, llmg_2163 and rmaB. Overexpressing cesSR reduced the growth defect, thus directly improving the production yield of BcaP. Applying this rationale to eukaryotic proteins, some of which are notoriously more difficult to produce, such as the medically-important presenilin complex, we were able to significantly diminish the growth defect seen in the wild-type strain and improve the production yield of the presenilin variant PS1Δ9-H6 more than 4-fold.
  
  
  CONCLUSIONS
  The results shed light into a key, and perhaps central, membrane protein quality control mechanism in L. lactis. Modulating the expression of CesSR benefited the production yields of membrane proteins from different origins. These findings reinforce L. lactis as a legitimate alternative host for the production of membrane proteins.
authors.affiliation: Groningen Biomolecular Sciences and Biotechnology Institute, Department of Molecular Genetics, University of Groningen, Groningen, The Netherlands.
authors.forename: Joao P C
authors.initials: JP
authors.lastname: Pinto
authors.forename: Oscar P
authors.initials: OP
authors.lastname: Kuipers
authors.forename: Ravi K R
authors.initials: RK
authors.lastname: Marreddy
authors.forename: Bert
authors.initials: B
authors.lastname: Poolman
authors.forename: Jan
authors.initials: J
authors.lastname: Kok
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism*; D015536:Down-Regulation / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D005798:Genes, Bacterial / Q000235:genetics; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000378:metabolism; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis*; D026901:Membrane Transport Proteins / Q000096:biosynthesis; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D018085:Regulon / Q000235:genetics; D013312:Stress, Physiological; D013997:Time Factors; D014158:Transcription, Genetic; D059467:Transcriptome; D015854:Up-Regulation / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins