Comparison of expression of the endo-?-1,3-1,4-glucanase gene from Bacillus subtilis in Saccharomyces cerevisiae from the CYC1 and ADH1 promoters

Cantwell, B. A.; Brazil, G.; Murphy, Noel B.; McConnell, David J.

doi:10.1007/bf00389427

Cited by 26 publications

(10 citation statements)

References 16 publications

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“…The endo-␤-1,3-1,4-glucanase gene from Bacillus subtilis was expressed in S. cerevisiae under its own promoter and signal sequences (269,628). The synthesis of high levels of ␤-glucanase in brewing yeast strains was achieved by placing this gene under the control of the S. cerevisiae ADH1 promoter on a high-copy-number 2m-based plasmid vector (92). The fact that no extracellular endo-␤-1,3-1,4-glucanase activity could be detected in cultures of S. cerevisiae was attributed to the inability of yeast to process the protein so as to promote secretion.…”

Section: Heterologous Cellulase Expression In Bacteriamentioning

confidence: 99%

Microbial Cellulose Utilization: Fundamentals and Biotechnology

Lynd

Weimer

Zyl³

et al. 2002

Microbiol Mol Biol Rev

4,009

2,868

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Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for “consolidated bioprocessing” (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts

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Section: Heterologous Cellulase Expression In Bacteriamentioning

confidence: 99%

Microbial Cellulose Utilization: Fundamentals and Biotechnology

Lynd

Weimer

Zyl³

et al. 2002

Microbiol Mol Biol Rev

4,009

2,868

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“…Therefore many brewing laboratories have been interested in the construction of recombinant brewer's yeasts secreting fl-glucanase (reviewed by Penttil~i et al 1989). fl-Glucanase genes from Bacillus subtilis (Cantwell et al 1985;…”

Section: Introductionmentioning

confidence: 99%

Construction and analysis of recombinant glucanolytic brewer's yeast strains

Suihko

Lehtinen

Zurbriggen

et al. 1991

Appl Microbiol Biotechnol

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Four different types of endo-fl-l,4-glucanase active bottom-fermenting brewer's yeast strains were constructed using recombinant DNA technology. To study the effects of different promoters, copy numbers and integration sites, the eoll gene of the filamentous fungus Trichoderma reesei was inserted between the promoter and terminator regions of either the PGK1 or ADH1 gene of yeast. The eoll gene was transferred to the industrial brewer's yeast on a multicopy plasmid or alternatively integrated into the LEU2, PGK1 or A D H 1 locus of the yeast. Integration into the PGK1 or A D H 1 locus did not affect the brewing properties of the yeast or the quality of the finished beer. Integration into the LEU2 locus, however, decreased the metabolic activity of yeast and prolonged fermentation was needed. In pilot brewing conditions the PGK1 promoter was stronger than that of ADH1. Even a single copy of the eoH gene in the PGK1 integrant strains gave rise to sufficient enzyme activity for the hydrolysis even of unusually high total amounts of fl-glucans in worts.

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“…Although incapable of degrading crystalline forms of cellulose individually, bacterial P-1,4-glucanases (most likely endo acting) may be able to act synergistically with cellulases of other specificities, such as exo-acting P-1,4-glucanases or P-glucosidases, or both, to achieve the enzymatic bioconversion of cellulose to more commercially useful products. A Bacillus P-1,4-glucanase may also have a role to play in the brewing industry (5,11,20).…”

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confidence: 99%

Endo-beta-1,4-glucanase gene of Bacillus subtilis DLG

Robson¹,

Chambliss²

1987

J Bacteriol

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The DNA sequence of the BaciUus subtlis DLG endo-,B-1,4-glucanase gene was determined, and the in vivo site of transcription initiation was located. Immediately upstream from the transcription start site were sequences closely resembling those recognized by B. subtilis &43-RNA polymerase. Two possible ribosomebinding sites were observed downstream from the transcription start site. These were followed by a long open reading frame capable of encoding a protein of ca. 55,000 daltons. A signal sequence, typical of those present in gram-positive organisms, was observed at the amino terminus of the open reading frame. Purification of the mature exocellular 0-1,4-glucanase and subsequent amino-terminal protein sequencing defined the site of signal sequence processing to be between two alanine residues following the hydrophobic portion of the signal sequence. The probability of additional carboxy-terminal processing of the 0-1,4-glucanase precursor is discussed. Si nuclease protection studies showed that the amount of ,B-1,4-glucanase mRNA in cells increased significantly as the culture entered the stationary phase. In addition, glucose was found to dramatically stimulate the amount of ,B-1,4-glucanase mRNA in vivo. Finally, the specffic activities of purified B. subtilis DLG endo-0-1,4-glucanase and Trichoderma reesei QM9414 endo-f-1,4-glucanase (EC 3.2.1.4) were compared by using the noncrystalline cellulosic substrate trinitrophenyl-carboxymethyl cellulose.Many members of the family Bacillaceae produce extracellular ,B-glucanases. The linkage specificities of these enzymes are varied, the most common being ,-1,3-1,4-glucanases and 13-1,3-glucanases (6,20,32,41,57). There are relatively few reports of members of the family Bacillaceae producing ,B-1,4-glucanases (13,21,41,44). The two relatively well characterized examples, the alkalophilic Bacillus strain N-4 (21, 48) and Bacillus subtilis DLG (44, 45), produce cellulolytic activity in the form of a carboxymethyl cellulase (CMC) (i.e., P-1,4-glucanase). Although incapable of degrading crystalline forms of cellulose individually, bacterial P-1,4-glucanases (most likely endo acting) may be able to act synergistically with cellulases of other specificities, such as exo-acting P-1,4-glucanases or P-glucosidases, or both, to achieve the enzymatic bioconversion of cellulose to more commercially useful products. A Bacillus P-1,4-glucanase may also have a role to play in the brewing industry (5,11,20). For B. subtilis DLG, ,B-1,4-glucanase production begins at the onset of the stationary phase and is not repressed by either glucose or cellobiose (44), as are many other cellulolytic systems (12,30,53,55). In fact, glucose stimulates enzyme production in some fashion. Additionally, the enzyme is initially translated as a large (ca. 51,500-dalton), enzymatically active intracellular precursor in B. subtilis before undergoing efficient secretion to the exterior of the cell (45). The mature exocellular ,B-1,4-glucanase is 35,200 ± 400 daltons.Cloning the ,3-1,4-glucanase gene ha...

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Comparison of expression of the endo-?-1,3-1,4-glucanase gene from Bacillus subtilis in Saccharomyces cerevisiae from the CYC1 and ADH1 promoters

Cited by 26 publications

References 16 publications

Microbial Cellulose Utilization: Fundamentals and Biotechnology

Microbial Cellulose Utilization: Fundamentals and Biotechnology

Construction and analysis of recombinant glucanolytic brewer's yeast strains

Endo-beta-1,4-glucanase gene of Bacillus subtilis DLG

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