Protein secretion in Lactococcus lactis: an efficient way to increase the overall heterologous protein production

Abstract

Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium. Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L. lactis. The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields. These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement (by signal peptide and propeptide optimization) results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins. The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed. The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L. lactis, but also in other LAB species.

RecombinantLactococcus lactisfails to secrete bovine chymosine

“…Considering that protein secretion in L. lactis occurs through a Secdependent pathway, which has been used to produce and successfully secrete different heterologous proteins, our results suggest that the transport of prochymosin B and chymosin A and B, via this Sec pathway, could be impaired. It is known that recombinant expression can be limited in L. lactis because expression of the target proteins can be subject to inadequate stability and/or solubility, leading to improper protein folding, inclusion body formation, protein degradation 17,18 and/or can be related to the inefficiency of the secretion process. 8 Thus, it is possible that a post-translational process, such as insoluble aggregate formation, could be limiting chymosin secretion.…”

Improvement of Heterologous Protein Secretion by Bacillus subtilis

“…In addition, in L. lactis, the nine-residue synthetic propeptide, LEISSTCDA, which is fused immediately after the signal peptide cleavage site, is known to enhance heterologous protein secretion (Le Loir et al, 1998;Le Loir et al, 2005;Zhuang et al, 2008;Zhang et al, 2010). Therefore, we evaluated whether the fusion of the AmyE signal peptide and the propeptide could improve the secretion of hIFNα-2b, compared to that with only AmyE signal peptide.…”

Engineering Signal Peptides for Enhanced Protein Secretion from Lactococcus lactis

“…Additional studies are needed to determine whether even greater improvements in secretion can be achieved solely by engineering the signal peptide, since other steps in the secretion process such as translocation efficiency (24), modification by chaperones (15), and transportation across the cell wall (42) might become limiting. Indeed, protein secretion in L. lactis has been improved by introduction of a synthetic propeptide (23) or by complementation with the SecDF machinery (24).…”

“…The signal peptide plays an important role in targeting the protein to the cytoplasmic membrane, where the protein precursor is subsequently translocated by the Sec machinery (14). Following cleavage of the signal peptide, the mature protein is released extracellularly (15). The lactococcal signal peptides follow the common tripartite structure, including a positively charged N terminus (n-region), a central hydrophobic core (h-region), and a more polar C terminus (c-region) containing the signal peptide cleavage site (16).…”