1887

Abstract

The facultatively anaerobic, thermophilic bacterium is being developed as an industrial micro-organism for cellulosic bioethanol production. Process improvement would be gained by enhanced secretion of glycosyl hydrolases. Here we report the construction of a modular system for combining promoters, signal peptide encoding regions and glycosyl hydrolase genes to facilitate selection of the optimal combination in . Initially, a minimal three-part sp. shuttle vector pUCG3.8 was constructed using Gibson isothermal DNA assembly. The three PCR amplicons contained the pMB1 origin of replication and multiple cloning site (MCS) of pUC18, the sp. origin of replication pBST1 and the thermostable kanamycin nucleotidyltransferase gene (), respectively. could be transformed with pUCG3.8 at an increased efficiency [2.8×10 c.f.u. (µg DNA)] compared to a previously reported shuttle vector, pUCG18. A modular cassette for the inducible expression and secretion of proteins in designed to allow the simple interchange of parts, was demonstrated using the endoglucanase Cel5A from as a secretion target. Expression of was placed under the control of a cellobiose-inducible promoter (P) together with a signal peptide encoding sequence from a C56-YS93 endo-β-1,4-xylanase. The interchange of parts was demonstrated by exchanging the gene with the 3′ region of a gene with homology to from and substituting P for the synthetic, constitutive promoter P, which increased Cel5A activity five-fold. Cel5A and CelA activities were detected in culture supernatants indicating successful expression and secretion. N-terminal protein sequencing of Cel5A carrying a C-terminal FLAG epitope confirmed processing of the signal peptide sequence.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.066332-0
2013-07-01
2019-10-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/159/7/1267.html?itemId=/content/journal/micro/10.1099/mic.0.066332-0&mimeType=html&fmt=ahah

References

  1. Assareh R., Shahbani Zahiri H., Akbari Noghabi K., Aminzadeh S., Bakhshi Khaniki G.. ( 2012;). Characterization of the newly isolated Geobacillus sp. T1, the efficient cellulase-producer on untreated barley and wheat straws. . Bioresour Technol 120:, 99–105. [CrossRef][PubMed]
    [Google Scholar]
  2. Bartosiak-Jentys J., Eley K., Leak D. J.. ( 2012;). Application of pheB as a reporter gene for Geobacillus spp., enabling qualitative colony screening and quantitative analysis of promoter strength. . Appl Environ Microbiol 78:, 5945–5947. [CrossRef][PubMed]
    [Google Scholar]
  3. Brautaset T., Lale R., Valla S.. ( 2009;). Positively regulated bacterial expression systems. . Microb Biotechnol 2:, 15–30. [CrossRef][PubMed]
    [Google Scholar]
  4. Brockmeier U., Caspers M., Freudl R., Jockwer A., Noll T., Eggert T.. ( 2006;). Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria. . J Mol Biol 362:, 393–402. [CrossRef][PubMed]
    [Google Scholar]
  5. Brown N. L., Stoyanov J. V., Kidd S. P., Hobman J. L.. ( 2003;). The MerR family of transcriptional regulators. . FEMS Microbiol Rev 27:, 145–163. [CrossRef][PubMed]
    [Google Scholar]
  6. Chhabra S. R., Shockley K. R., Ward D. E., Kelly R. M.. ( 2002;). Regulation of endo-acting glycosyl hydrolases in the hyperthermophilic bacterium Thermotoga maritima grown on glucan- and mannan-based polysaccharides. . Appl Environ Microbiol 68:, 545–554. [CrossRef][PubMed]
    [Google Scholar]
  7. Cripps R. E., Eley K., Leak D. J., Rudd B., Taylor M., Todd M., Boakes S., Martin S., Atkinson T.. ( 2009;). Metabolic engineering of Geobacillus thermoglucosidasius for high yield ethanol production. . Metab Eng 11:, 398–408. [CrossRef][PubMed]
    [Google Scholar]
  8. Deutscher J.. ( 2008;). The mechanisms of carbon catabolite repression in bacteria. . Curr Opin Microbiol 11:, 87–93. [CrossRef][PubMed]
    [Google Scholar]
  9. Deutscher J., Francke C., Postma P. W.. ( 2006;). How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria. . Microbiol Mol Biol Rev 70:, 939–1031. [CrossRef][PubMed]
    [Google Scholar]
  10. Gibson D. G., Benders G. A., Andrews-Pfannkoch C., Denisova E. A., Baden-Tillson H., Zaveri J., Stockwell T. B., Brownley A., Thomas D. W. et al. ( 2008;). Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. . Science 319:, 1215–1220. [CrossRef][PubMed]
    [Google Scholar]
  11. Gibson D. G., Young L., Chuang R.-Y., Venter J. C., Hutchison C. A. III, Smith H. O.. ( 2009;). Enzymatic assembly of DNA molecules up to several hundred kilobases. . Nat Methods 6:, 343–345. [CrossRef][PubMed]
    [Google Scholar]
  12. Lai X. K., Ingram L. O.. ( 1993;). Cloning and sequencing of a cellobiose phosphotransferase system operon from Bacillus stearothermophilus XL-65-6 and functional expression in Escherichia coli.. J Bacteriol 175:, 6441–6450.[PubMed]
    [Google Scholar]
  13. Liao H., McKenzie T., Hageman R.. ( 1986;). Isolation of a thermostable enzyme variant by cloning and selection in a thermophile. . Proc Natl Acad Sci U S A 83:, 576–580. [CrossRef][PubMed]
    [Google Scholar]
  14. Marciniak B. C., Pabijaniak M., de Jong A., Duhring R., Seidel G., Hillen W., Kuipers O. P.. ( 2012;). High- and low-affinity cre boxes for CcpA binding in Bacillus subtilis revealed by genome-wide analysis. . BMC Genomics 13:, 401. [CrossRef][PubMed]
    [Google Scholar]
  15. McKenzie T., Hoshino T., Tanaka T., Sueoka N.. ( 1986;). The nucleotide sequence of pUB110: some salient features in relation to replication and its regulation. . Plasmid 15:, 93–103. [CrossRef][PubMed]
    [Google Scholar]
  16. Natale P., Brüser T., Driessen A. J. M.. ( 2008;). Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane–distinct translocases and mechanisms. . Biochim Biophys Acta 1778:, 1735–1756. [CrossRef][PubMed]
    [Google Scholar]
  17. Pereira J. H., Chen Z., McAndrew R. P., Sapra R., Chhabra S. R., Sale K. L., Simmons B. A., Adams P. D.. ( 2010;). Biochemical characterization and crystal structure of endoglucanase Cel5A from the hyperthermophilic Thermotoga maritima. . J Struct Biol 172:, 372–379. [CrossRef][PubMed]
    [Google Scholar]
  18. Sargent F., Berks B. C., Palmer T.. ( 2006;). Pathfinders and trailblazers: a prokaryotic targeting system for transport of folded proteins. . FEMS Microbiol Lett 254:, 198–207. [CrossRef][PubMed]
    [Google Scholar]
  19. Stoll R., Goebel W.. ( 2010;). The major PEP-phosphotransferase systems (PTSs) for glucose, mannose and cellobiose of Listeria monocytogenes, and their significance for extra- and intracellular growth. . Microbiology 156:, 1069–1083. [CrossRef][PubMed]
    [Google Scholar]
  20. Taylor M. P., Esteban C. D., Leak D. J.. ( 2008;). Development of a versatile shuttle vector for gene expression in Geobacillus spp. . Plasmid 60:, 45–52. [CrossRef][PubMed]
    [Google Scholar]
  21. Tjalsma H., Bolhuis A., Jongbloed J. D. H., Bron S., van Dijl J. M.. ( 2000;). Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. . Microbiol Mol Biol Rev 64:, 515–547. [CrossRef][PubMed]
    [Google Scholar]
  22. Wood I. P., Elliston A., Ryden P., Bancroft I., Roberts I. N., Waldron K. W.. ( 2012;). Rapid quantification of reducing sugars in biomass hydrolysates: Improving the speed and precision of the dinitrosalicylic acid assay. . Biomass Bioenergy 44:, 117–121. [CrossRef]
    [Google Scholar]
  23. Yanisch-Perron C., Vieira J., Messing J.. ( 1985;). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. . Gene 33:, 103–119. [CrossRef][PubMed]
    [Google Scholar]
  24. Zambare V. P., Bhalla A., Muthukumarappan K., Sani R. K., Christopher L. P.. ( 2011;). Bioprocessing of agricultural residues to ethanol utilizing a cellulolytic extremophile. . Extremophiles 15:, 611–618. [CrossRef][PubMed]
    [Google Scholar]
  25. Zeng L., Burne R. A.. ( 2009;). Transcriptional regulation of the cellobiose operon of Streptococcus mutans. . J Bacteriol 191:, 2153–2162. [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.066332-0
Loading
/content/journal/micro/10.1099/mic.0.066332-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error