Molecular Toolbox for Co-Expression of Chaperones for Heterologous Protein Production in Lactic Acid Bacteriametabolic Engineering XI: Design, Synthesis and System Integration for Metabolic Engineering

Lactic acid bacteria (LAB) are an important group of Gram positive genera traditionally used in the food industries due to its GRAS (generally recognized as safe) status. Members of LAB include Lactobacillus, Bifidobacterium, Lactococcus, Aerococcus, Leuconostoc, Oenococcus and Pediococcus. Considerable attention has been paid to them as commensal and probiotic bacteria in the gastrointestinal tract. To date, applications of LAB include acting as live vectors for targeted vaccine delivery and as microbial cell factories for production of recombinant proteins and bio-therapeutics. In its native environment, LAB are constantly exposed to environmental stresses such as heat, high salinity, organic acids and osmotic pressure. Consequently, they have evolved various adaptive mechanisms to response to these stresses. One of the classic response systems in LAB is the protein quality control system that consists of molecular chaperones and proteases that facilitate the quality control and folding of heterologous proteins.

Molecular chaperones are a group of proteins that mediates in the folding of other proteins. They are capable of binding to misfolded proteins with high affinity towards hydrophobic residues which are exposed in a non-native protein state. This causes a conformational change and subsequently releasing the correctly folded protein. Another group of chaperones namely the Clp family function as proteases that can help unfold protein aggregates. This adaptive mechanism adopted by LAB allows heterologous protein to be folded properly thus preventing aggregation. Currently, there have been little studies conducted on the co-expression of molecular chaperones in LAB to overcome protein aggregation during recombinant processes. This propose the need to examine chaperone co-expression to metabolically-engineer cells for production of difficult-to-express proteins. In this study, a strong and a moderate constituitive promoters (P59 and USP45) were cloned into a Lactococcal co-expression vector. Subsequenly, the chaperones (DnaK, GroES, GroEL, Trigger factor and ClpX) were cloned and transformed into Lactococcus lactis NZ900 strain. Nattokinase is chosen as the model protein. A comparision between the expression of Nattokinase in soluble fraction of recombinant L. lactis strains co-expressing various chaperones will be presented to detrmine their effect on Nattokinase production. The toolbox generated in this study will faciliate future applications to combat metabolic bottlenecks during heterologous protein production in LAB.