title: Recombinant Lactococcus lactis fails to secrete bovine chymosine.
abstract: Bovine chymosin is an important milk-clotting agent used in the manufacturing of cheeses. Currently, the production of recombinant proteins by genetically modified organisms is widespread, leading to greatly reduced costs. Lactococcus (L.) lactis, the model lactic acid bacterium, was considered a good candidate for heterologous chymosin production for the following reasons: (1) it is considered to be a GRAS (generally regarded as safe) microorganism, (2) only one protease is present on its surface, (3) it can secrete proteins of different sizes, and (4) it allows for the direct production of protein in fermented food products. Thus, three genetically modified L. lactis strains were constructed to produce and target the three different forms of bovine chymosin, prochymosin B, chymosin A and chymosin B to the extracellular medium. Although all three proteins were stably produced in L. lactis, none of the forms were detected in the extracellular medium or showed clotting activity in milk. Our hypothesis is that this secretion deficiency and lack of clotting activity can be explained by the recombinant protein being attached to the cell envelope. Thus, the development of other strategies is necessary to achieve both production and targeting of chymosin in L. lactis, which could facilitate the downstream processing and recovery of this industrially important protein.
authors.affiliation: a Department of General Biology; Institute of Biological Sciences ; Federal University of Minas Gerais (UFMG-ICB) ; Belo Horizonte , Brazil.
authors.forename: Tessália Diniz
authors.initials: TD
authors.lastname: Luerce
authors.forename: Marcela Santiago Pacheco
authors.initials: MS
authors.lastname: Azevedo
authors.forename: Jean Guy
authors.initials: JG
authors.lastname: LeBlanc
authors.forename: Vasco
authors.initials: V
authors.lastname: Azevedo
authors.forename: Anderson
authors.initials: A
authors.lastname: Miyoshi
authors.forename: Daniela Santos
authors.initials: DS
authors.lastname: Pontes
mesh_terms: D000818:Animals; D002417:Cattle; D012085:Chymosin / Q000235:genetics / Q000378:metabolism*; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D013294:Lactococcus lactis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004792:Enzyme Precursors; D011994:Recombinant Proteins; C032443:prorennin; D012085:Chymosin
keywords: Lactococcus lactis; bovine chymosin; bovine prochymosin; heterologous expression