Secretion of heterologous thermostable cellulases in Bacillus subtilis

Abstract

Bacillus subtilis is used industrially for the production of secreted enzymes. The most characteristic feature of the secreted enzymes is variation in the N-terminal signal peptides that is recognized by secretion machinery, which is one of the determinants of efficiency and must be customized in each case. Culturing cellulolytic B. subtilis to secrete heterologous cellulases combined with customized signal peptides would be beneficial for producing biocommodities from cellulosic biomass. Four Clostridium thermocellum genes, encoding endoglucanases (celA and celB) and exoglucanases (celK and celS) were cloned to construct random libraries of combinations with 173 different signal peptides originating from the B. subtilis genome. The libraries were successfully screened to identify the signal peptides most efficient in secretion of each of the four cellulases, which were theoretically unpredictable. The secreted cellulases were assayed on carboxymethyl cellulose, phosphoric acid swollen cellulose, and microcrystalline cellulose to determine the possible effects of the signal peptides on substrate specificity. The customized signal peptides for CelA, CelB, and CelS did not affect enzyme performance but those for CelK might influence its substrate specificity.Key words: Bacillus subtilis; cellulase; Clostridium thermocellum; secretion; signal peptide Introduction Cellulose, the main structural component of plant cell walls, is the most abundant carbohydrate polymer in nature. Despite its abundance, it is extremely difficult to degrade because it is insoluble and is present as hydrogen-bonded crystalline fibers (Shawn et al., 1999). Cellulase is an important commercial enzyme that is widely used in food, animal feed, textiles, pulp and paper, alcohol fermentation, and other applications (Oksanen et al., 2000). Complete enzymatic hydrolysis of cellulose requires the combined action of three types of cellulase enzyme, including endoglucanase (1,4-β-D-glucan glucanohydrolase), exoglucanase (1,4-β-D-glucan cellobiohydrolase), and cellobiase (β-glucosidase) (Ryu and Mandels, 1980). These enzymes work synergistically. First, endoglucanase randomly cleaves the ce llulose chain to produce cello-oligosaccharides. Second, exoglucanase acts on exposed chain ends by splitting off cellobiose. Finally, cellobiose is hydrolyzed by cellobiase to release glucose. Many cellulolytic microorganisms possess a cellulosome, a multicellulase complex associated with the cell surface, which mediates cell attachment to the insoluble cellulosic substrate and efficiently degrades it (Karmakar and Ray, 2011).Use of different substrates allows the modes of action of cellulases to be determined (Ghose, 1987). Soluble cellulose substrates, such as carboxymethyl cellulose (CMC), can be used to measure endoglucanase activity because CMC has a high degree of polymerization and is readily accessible to endocellulolytic cleavage, although methylation may block enzyme action (Ghose, 1987;Wood and Bhat, 1988;Xiao et al., 2005;Zhang et al., 2006). Microcry...

Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi

“…Eventually, the signal peptide-cleaved proteins are transported from Golgi to the cell surface, and the mature proteins are released extracellularly. The choice of different signal peptides has been shown to significantly affect the yield of secreted proteins, including cellulases [40]. Thus, screening signal peptides is an important strategy for optimizing the secretion of heterologous proteins in bacteria and yeasts [50–52].…”

“…In this study, a total of four signal peptides were selected and used; these signal peptides can be divided into three types based on their cleavage-site motifs (Table 2): LRR/DCT of L. starkeyi amylase, VLG/AAV of L. starkeyi dextranase I and II, and VLA/APL protease of Y. lipolytica. It was reported that some customized signal peptides used for thermostable cellulase secretion have no observable effect on enzyme performance, whereas others might influence its substrate specificity in Bacillus subtilis [40]. …”

“…Another possible reason for the poor cellulase performance observed so far is that suitable signal peptides are also important for cellulase expression in yeast, as they guide proteins to and through the endoplasmic reticulum and the secretory pathway [33–35]. Indeed, some specific signal peptides have been shown to enhance the secretion efficiency of targeted proteins, including heterologous cellulases [36–39], whereas others can affect the functionality of secreted cellulases [40]. More recently, some cellulase and hemicellulase genes introduced to oleaginous yeasts resulted in relatively high activity and yield of secreted heterologous proteins [41–45].…”

Lignocellulolytic capacities of Geobacillus thermodenitrificans: towards consolidated bioprocessing

“…In our study, we used mainly the (Sec dependent) signal peptide originating from the extracellular endoxylanase gtXynA (GTHT_01347; GenBank: KX962565.1) as this signal peptide was shown to be useful for hydrolytic protein secretion (Daas et al 2016;Bartosiak-Jentys et al 2013;. Recently, a library of 173 genome-derived signal peptides of B. subtilis were screened for their influence on the heterologous expression of cellulases from C. thermocellum (Thanh Bien et al 2014). It was found that these signal peptides caused negligible differences in the amounts of enzyme secreted and in their activity towards cellulose.…”

“…Heterologous production of C. thermocellum cellulases has been demonstrated in Bacillus subtillis (Lan Thanh Bien et al 2014). Here, the production of CelK (reducing end exoglucanase; GH 9) and CelS (non-reducing end exoglucanase; GH48) was demonstrated by the clearing zones of mutant colonies on CMC plates.…”