Engineering of a protein translocation system in Rhodococcus jostii RHA1 for the secretion of ligninases

Lignin is an aromatic polymer in plant biomass that has considerable potential as a sustainable source for fuels and chemicals. One approach to transform lignin to target products is to develop bacterial biocatalyts. The soil bacterium Rhodococcus jostii RHA1 (RHA1) is an attractive candidate for developing lignin-transforming biocatalysts. A strain with engineered catabolic pathways could be used in a bioreactor to convert lignin to high-value chemicals. To achieve this, a strain which produces high amounts of extracellular lignin-modifying enzymes (ligninases) is required. Our aim is to engineer RHA1 to efficiently secrete ligninases using the twin-arginine translocation (Tat) system. The system has been engineered in strains related to Rhodococcus using signal peptides that lead to high-level secretion. As a first approach, we want to identify signal sequences that promote efficient secretion of ligninases inRHA1. We selected various sequences which were predicted as secretion signal in RHA1 and based on studies in related bacteria. We tested the secretion qualities of the signal sequences on three reporter proteins (green fluorescent protein, a phosphatase from RHA1, and the dye decolorizing peroxidase Dyp2). We have begun to evaluate the secretion of the reporter enzymes by measuring their activities in the culture medium. Four of the five tested Tat signal peptides mediated protein secretion in RHA1 compared to the controls without a signal peptide. Our preliminary data suggest that the effect of each signal peptide is dependent on the Tat substrate. Using a Tat knockout mutant, we will evaluate whether the observed secretion is truly dependent on the Tat system. Furthermore, we will test the secretion of various ligninases, and attempt to maximize secretion by optimizing gene expression and by overexpression of the Tat system machinery.