Increase in Xylanase Production by Streptomyces lividans through Simultaneous Use of the Sec- and Tat-Dependent Protein Export Systems

Abstract

Xylanase B1 (XlnB1) from Streptomyces lividans is a protein consisting of two discrete structural and functional units, an N-terminal catalytic domain and a C-terminal substrate binding domain. In the culture medium, two forms of xylanase B are present, namely, XlnB1 and XlnB2, the latter of which corresponds to the catalytic domain of XlnB1 deprived of the substrate binding domain. Both forms of the xylanase have the same activity on xylan. The enzyme is secreted through the Sec-dependent pathway with a better yield of XlnB1 than XlnB2. Interestingly, XlnB2 exhibits 80% identity with XlnC which is secreted exclusively through the Tatdependent pathway. To demonstrate whether XlnB1 and XlnB2 could also be secreted through the Tatdependent pathway, the Tat-targeting xlnC signal sequence was fused to the structural genes of xlnB1 and xlnB2. Both XlnB1 and XlnB2 were secreted through the Tat-dependent pathway, but XlnB2 was better produced than XlnB1. As XlnB1 and XlnB2 could be better secreted through the Sec-and Tat-dependent systems, respectively, a copy of the structural gene of xlnB1 fused to a Sec signal sequence and a copy of the structural gene of xlnB2 fused to a Tat signal sequence were inserted into the same plasmid under the control of the xlnA promoter. The transformant produced xylanase activity which corresponded approximately to the sum of activities of the individual strain producing xylanase by either the Sec-or Tat-dependent secretion system. This indicated that both secretion systems are functional and independent of each other in the recombinant strain. This is the first report on the efficient secretion of a protein using two different secretion systems at the same time. Assuming that the protein to be secreted could be properly folded prior to and after translocation via the Tat-and Sec-dependent pathways, respectively, the simultaneous use of the Sec-and Tat-dependent pathways provides an efficient means to increase the production of a given protein.

Monitoring Protein Secretion in Streptomyces Using Fluorescent Proteins

“…xyloglucanase of 100 kDa (Sianidis et al, 2006), an extremely thermostable cellulase (Hamed et al, 2017), phospholipase D (Ogino et al, 2004), transglutaminase, β-1,4-endoglucanase and β-glucosidase (Noda et al, 2010). In similar approaches, targeting to the Tat pathway has been achieved but generally with lower yields (Schaerlaekens et al, 2004a; Gauthier et al, 2005; Anné et al, 2012; Gullon et al, 2015a).…”

The Bacterial Twin-Arginine Translocation Pathway

“…Concomitant export of the respective B subunits via the Sec apparatus resulted in the formation of active and fluorescent holotoxin fusions in the periplasm. Other proteins of commercial interest have been translocated via the Tat pathway in E. coli and in S. lividans (49,76).…”

Differential translocation of green fluorescent protein fused to signal sequences ofRuminococcus albuscellulases by the Tat and Sec pathways ofEscherichia coli

“…In addition, it points out to the fact that R. albus might possess a Tat secretory pathway in addition to the Sec pathway. Streptomyces lividans , a soil Gram‐positive bacterium, was shown to simultaneously use the Sec‐ and Tat‐dependent pathways to export xylanases and thus increase the production of enzymes involved in polysaccharide degradation (Gauthier et al , 2005). The existence of both secretory pathways in R. albus might represent a functional and ecological advantage for the bacterium in a very competitive microbial ecosystem such as the rumen.…”