Systematic Screening of All Signal Peptides from Bacillus subtilis: A Powerful Strategy in Optimizing Heterologous Protein Secretion in Gram-positive Bacteria

Brockmeier, Ulf; Caspers, Michael; Freudl, Roland; Jockwer, Alexander; Noll, Thomas; Eggert, Thorsten

doi:10.1016/j.jmb.2006.07.034

Cited by 232 publications

(239 citation statements)

References 41 publications

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“…In contrast, sequences with D-scores 0.7 (0.5 0.7) have a lower chance of functioning as a SP, and sequences with values 0.5 are commonly classified as not being a functional SP. According to Brockmeier et al (2006), D-score and other SP characteristics, including the numbers of charged amino acids in the N-terminal region and the overall hydrophobicity, do not correlate with secretion efficiencies. For example, for CelA secretion, three SPs showed high D-scores (pel, 0.804; yobB, 0.841; and ydjM, 0.828); however, SPs with D-scores of 0.5 0.7 (ywtF, 0.692; pbpD, 0.505; csn, 0.647; and ydbK, 0.499) also displayed high secretory efficiencies ( Table 2), implying that D-score may be suggestive but not completely reliable.…”

Section: Efficient Sp For Secretion Of the Four Heterologous Cellulasesmentioning

confidence: 87%

“…It is known that enzymes secreted through the Sec pathway are processed and folded into their active form only during or shortly after the translocation across the cytoplasmic membrane (Brockmeier et al, 2006;Degering et al, 2010). We did not determine the N-terminal residues of secreted cellulases, but the detected activities imply that they might be processed appropriately as designed.…”

Section: Efficient Sp For Secretion Of the Four Heterologous Cellulasesmentioning

confidence: 95%

“…Because different SPs mediated cellulase translocation across the cytoplasmic membrane by Sec translocase with different efficiencies as reported by Brockmeier et al (2006) and Degering et al (2010), the efficiency of SP cannot be predicted a priori. Previously, SPs of extracellular proteins secreted at high levels, such as SPs from NprE and AprE, have been intensively investigated (Fu et al, 2007;Nijland and Kuipers, 2008).…”

Section: Efficient Sp For Secretion Of the Four Heterologous Cellulasesmentioning

confidence: 96%

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Secretion of heterologous thermostable cellulases in Bacillus subtilis

Bien

Tsuji

Tanaka

et al. 2014

J. Gen. Appl. Microbiol.

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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...

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Section: Efficient Sp For Secretion Of the Four Heterologous Cellulasesmentioning

confidence: 87%

Section: Efficient Sp For Secretion Of the Four Heterologous Cellulasesmentioning

confidence: 95%

Section: Efficient Sp For Secretion Of the Four Heterologous Cellulasesmentioning

confidence: 96%

See 1 more Smart Citation

Secretion of heterologous thermostable cellulases in Bacillus subtilis

Bien

Tsuji

Tanaka

et al. 2014

J. Gen. Appl. Microbiol.

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“…Among the existing prediction methods, SignalP was the most effective and was widely used in many works to determine SPs (Brockmeier et al 2006). But each predictor has its limit, especially in cleavage site prediction.…”

Section: Improving Prediction By Combining Methodsmentioning

confidence: 99%

Evaluating signal peptide prediction methods for Gram-positive bacteria

Zhang

2009

Biologia

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Gram-positive bacteria have been widely investigated for their huge capability to secrete proteins, such as those involved in gene expression, bacterial surface display and bacterial pathogenesis. The N-terminal signal peptide of a secretory protein is responsible for the translocation of polypeptide through the cytoplasmic membrane. Recently, the signal peptide prediction has become a major task in bioinformatics, and many programs with different algorithms were developed to predict signal peptides. In this paper, five prediction programs (SignalP 3.0, PrediSi, Phobius, SOSUIsignal and SIG-Pred) were selected to evaluate their prediction accuracy for signal peptides and cleavage site using 509 unbiased and experimentally verified Gram-positive protein sequences. The results showed that SignalP was the most accurate program in signal peptide (96% accuracy) and cleavage site (83%) prediction. Prediction performance could further be improved by combining multiple methods into consensus prediction, which would increase the accuracy to 98%, and decrease the false positive to zero. When the consensus method was used to predict Bacillus's extracellular proteins identified by proteomics, more new signal peptides were successfully identified. It could be concluded that the consensus method would be useful to make prediction of signal peptides more reliable.

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“…Accordingly, the best signal peptide for the secretion of one target protein is not automatically the best, or even sufficient, for the secretion of a different target protein (Brockmeier et al, 2006).…”

Section: Signal Peptidementioning

confidence: 99%