Posttranslational processing of the xylanase Xys1L from Streptomyces halstedii JM8 is carried out by secreted serine proteases

Abstract

The xylanase Xys1L from Streptomyces halstedii JM8 is known to be processed extracellularly, to produce a protein of 33?7 kDa, Xys1S, that retains catalytic activity but not its cellulose-binding capacity. This paper demonstrates that at least five serine proteases isolated from Streptomyces spp. have the ability to process the xylanase Xys1L. The genes of two of these extracellular serine proteases, denominated SpB and SpC, were cloned from Streptomyces lividans 66 (a strain commonly used as a host for protein secretion), sequenced, and overexpressed in S. lividans; both purified proteases were able to process Xys1L in vitro. Three other previously reported purified Streptomyces serine proteases, SAM-P20, SAM-P26 and SAM-P45, also processed Xys1L in vitro. The involvement of serine proteases in xylanase processing-degradation in vivo was demonstrated by co-expression of the xylanase gene (xysA) and the gene encoding the serine protease inhibitor (SLPI) from S. lividans. Co-expression prevented processing and degradation of Xys1L and resulted in a threefold increase in the xylanase activity present in the culture supernatant. SpB and SpC also have the capacity to process other secreted proteins such as p40, a cellulose-binding protein from S. halstedii JM8, but do not have any clear effect on other secreted proteins such as amylase (Amy) from Streptomyces griseus and xylanase Xyl30 from Streptomyces avermitilis.

Cloning and expression of alkaline protease genes from two salt-tolerant alkaliphilic actinomycetes in E. coli

“…Similarly, the genes encoding chitinases were cloned and sequenced from alkaliphilic actinomycete, Nocardiopsis prasina OPC-131 into Escherichia coli [11]. FernandezAbalos et al [12] demonstrated the post translational processing of the xylanase Xys1L from Streptomyces halstedii JM8 by secreted alkaline serine protease. Recently, a novel calcium independent serine protease from an alkaliphilic bacterium, Nesterenkonia sp.…”

Post-translational processing of modular xylanases from Streptomyces is dependent on the carbohydrate-binding module

“…This processing was almost completely inhibited when the serine protease inhibitor slpI gene from S. lividans was co-expressed with the xylanase xysA gene in S. lividans. In contrast, none of these proteases was able to process Xyl30 in vitro [10].…”

“…plasmid, obtaining pSHA2vo, v1, v2, v3, v4 and v5. Then, all amplifications were cloned into the E. coli/Streptomyces shuttle vector pN702GEM3 [10] in a triple ligation to construct the xysA derivatives: HindIII/BglII fragment from pN702GEM3 ? BglII/XhoI fragment from pXHis1 [1] ?…”

Time-Resolved Transcriptomics and Constraint-Based Modeling Identify System-Level Metabolic Features and Overexpression Targets to Increase Spiramycin Production in Streptomyces ambofaciens

“…To construct pNGEM/OriT/P we utilized the E. coli – Streptomyces shuttle vector pN702GEM3. pN702GEM3 (Fernandez-Abalos et al, 2003) contains the pIJ702 origin of replication (for Streptomycetes), the bifunctional Neo/Kan resistance marker from Tn 5 (for both E. coli and Streptomycetes), the pUC origin of replication (for E. coli ) and a polylinker derived from pGEM3Zf(+) (Promega). This vector is replicative but not conjugative in Streptomycetes.…”