Efficient expression of a Paenibacillus barcinonensis endoglucanase in Saccharomyces cerevisiae

Abstract

The endoglucanase coded by celA (GenBank Access No. Y12512) from Paenibacillus barcinonensis, an enzyme with good characteristics for application on paper manufacture from agricultural fibers, was expressed in Saccharomyces cerevisiae by using different domains of the cell wall protein Pir4 as translational fusion partners, to achieve either secretion or cell wall retention of the recombinant enzyme. Given the presence of five potential N-glycosylation sites in the amino acid sequence coded by celA, the effect of glycosylation on the enzymatic activity of the recombinant enzyme was investigated by expressing the recombinant fusion proteins in both, standard and glycosylation-deficient strains of S. cerevisiae. Correct targeting of the recombinant fusion proteins was confirmed by Western immunoblot using Pir-specific antibodies, while enzymatic activity on carboxymethyl cellulose was demonstrated on plate assays, zymographic analysis and colorimetric assays. Hyperglycosylation of the enzyme when expressed in the standard strain of S. cerevisiae did not affect activity, and values of 1.2 U/ml were obtained in growth medium supernatants in ordinary batch cultures after 24 h. These values compare quite favorably with those described for other recombinant endoglucanases expressed in S. cerevisiae. This is one of the few reports describing the expression of Bacillus cellulases in S. cerevisiae, since yeast expressed recombinant cellulases have been mostly of fungal origin. It is also the first report of the yeast expression of this particular endoglucanase.

An Efficient Genome-Wide Fusion Partner Screening System for Secretion of Recombinant Proteins in Yeast

“…Cell wall proteins have been used as fusion partners for incorporation of recombinant proteins into the yeast cell wall; however, most of the studies conducted to date have focused on the anchoring domain 14 32 . Two of our selected TFPs containing pre- and pro-peptides ( CIS3 and HSP150 ) have already been reported as fusion partners for the production of xylanase, β-lactamase, rat nerve growth factor receptor, the VP8 rotavirus antigen, lipase, human IL-1β, endoglucanase, glycosyltransferases, rat alpha 2,3-sialyltransferase and laccase 14 33 34 35 36 37 38 39 40 41 . These fusion partners were used to produce target proteins as fusion proteins, while our TFPs were removed at the Golgi complex via an artificially introduced KEX2 cleavage site, resulting intact proteins.…”

How to achieve high-level expression of microbial enzymes

“…An endoglucanase from Paenibacillus barcinonensis has been expressed in S. cerevisiae by using different domains of the cell wall protein Pir4 as translational fusion partners. 145 The manipulation of the unfolded protein response (UPR) pathway regulator Hac1p can stimulate the secretory pathway of S. cerevisiae to improve the secretion of recombinant enzymes. Overexpression of S. cerevisiae HAC1 yielded a 2.0-fold enhancement in the secretion of the endogenous invertase.…”