Production of Recombinant Bleaching Enzymes from Thermophilic Microorganisms in Fungal Hosts

Bergquist, Peter L.; Te’o, Junior; Gibbs, Moreland D.; Cziferszky, Angela; Faria, Fabrícia Paula de; Azevedo, M. O.; Nevalainen, Helena

doi:10.1007/978-1-4612-0119-9_13

Cited by 7 publications

(6 citation statements)

References 27 publications

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“…Several labs have attempted to improve the expression of cellulase enzymes in both microbial (Park et al, 1998;Pauly et al, 1999;Bergquist et al, 2004) and higher plant systems (Herbers et al, 1995;Dai et al, 1999Dai et al, , 2000aZiegler et al, 2001;Ziegelhoffer et al, 2001;Bailey et al, 2004). Previously, we examined E1 expression in tobacco and potato under the control of the Mac or RbcS-3C promoter combined with native E1, apoplastic, chloroplastic, or vacuolar targeting and a mannopine synthase (Mas) or T7/T5 transcriptional terminator (Dai et al, 2000a,b).…”

Section: Discussionmentioning

confidence: 99%

Optimization of Acidothermus cellulolyticus Endoglucanase (E1) Production in Transgenic Tobacco Plants by Transcriptional, Post-transcription and Post-translational Modification

et al. 2005

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An attempt was made to obtain a high-level production of intact Acidothermus cellulolyticus endoglucanase (E1) in transgenic tobacco plants. The E1 expression was examined under the control of the constitutive and strong Mac promoter or light-inducible tomato Rubisco small sub-unit (RbcS-3C) promoter with its original or Alfalfa Mosaic Virus (AMV) RNA4 5'-untranslated leader (UTL) and targeted to different sub-cellular compartments via transit peptides. The transit peptides included native E1, endoplasmic reticulum, vacuole, apoplast, and chloroplast. E1 expression and its stability in transgenic plants were determined via E1 activity, protein immunoblotting, and RNA gel-blotting analyses. Effects of sub-cellular compartments on E1 production and its stability were determined in transgenic tobacco plants carrying one of six transgene expression vectors, where the E1 was under the control of Mac promoter, mannopine synthase transcription terminator, and one of the five transit peptides. Transgenic tobacco plants with an apoplastic transit peptide had the highest average E1 activity and protein accumulation, which was about 0.25% of total leaf soluble proteins estimated via E1 specific activity and protein gel blots. Intercellular fluid analyses confirmed that E1 signal peptide functioned properly in tobacco cells to secret E1 protein into the apoplast. By replacing RbcS-3C UTL with AMV RNA4 UTL E1 production was enhanced more than twofold, while it was less effective than the mannopine synthase UTL. It was observed that RbcS-3C promoter was more favorable for E1 expression in transgenic plants than the Mac promoter. E1 activity in dried tobacco seeds stored one year at room temperature was 45% higher than that observed immediately after harvesting, suggesting that E1 protein can be stored at room temperature for a long period. E1 stability in different sub-cellular compartments and the optimal combination of promoter, 5'-UTL, and sub-cellular compartmentation for heterologous protein production in transgenic plants are discussed.

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Section: Discussionmentioning

confidence: 99%

Optimization of Acidothermus cellulolyticus Endoglucanase (E1) Production in Transgenic Tobacco Plants by Transcriptional, Post-transcription and Post-translational Modification

et al. 2005

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“…It would be advantageous to use a fungal protease as fungal expression systems are capable of producing larger quantities of enzymes than bacterial expression systems. Filamentous fungi, such as Aspergillus, have been the organism of choice for large scale production of bulk industrial enzymes, as the fungi can be grown on relatively inexpensive (agricultural waste) media and the fungi can secrete bulk quantities of enzymes [3]. A proteolytic enzyme that had been isolated from Aspergillus tamarii was used to dehair goat skins [7].…”

Section: Introductionmentioning

confidence: 99%

Isolation, characterization and optimization of culture parameters for production of an alkaline protease isolated from Aspergillus tamarii

Anandan

Marmer

Dudley

2007

J Ind Microbiol Biotechnol

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Aspergillus tamarii expresses an extracellular alkaline protease that we show to be eVective in removing hair from cattle hide. Large quantities of the enzyme will be required for the optimization of the enzymatic dehairing process so the growth conditions for maximum protease expression by A. tamarii were optimized for both solid-state culture on wheat bran and for broth culture. Optimal protease expression occurred, for both cultural media, at initial pH 9; the culture was incubated at 30°C for 96 h using a 5% inoculum. The crude enzyme was isolated, puriWed and characterized using MALDI TOF TOF. The alkaline protease was homologous to the alkaline protease expressed by Aspergillus viridinutans.

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“…Very few endogenous proteins are secreted by K. lactis. This is advantageous as extracellular proteases are also diminished decreasing the degradation of expression products and allowing for the easy isolation of recombinant proteins (van den Berg et al 1990;Hollenberg and Gellissen 1997;Dominguez et al 1998;Bergquist et al 2002).…”

Section: Introductionmentioning

confidence: 98%

Heterologous expression of cyan and yellow fluorescent proteins from the Kluyveromyces lactis KlMAL21–KlMAL22 bi-directional promoter

2007

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We have identified the Kluyveromyces lactis maltase (KlMAL22) and maltose permease (KlMAL21) intergenic region as a candidate bi-directional promoter for heterologous gene expression. The expressions of cyan and yellow fluorescent proteins from, respectively, the KlMAL22 and KlMAL21 orientations of the promoter, were compared between two promoter variants during growth in media containing glucose, galactose or glycerol. Expression from both orientations of the native promoter was repressed during growth in glucose and galactose and was induced during growth in glycerol. Disruption of a putative Mig1p binding site caused some de-repression of the maltase orientation of the promoter by 48 h of growth in glucose. The KlMAL21-KlMAL22 bi-directional promoter can be used to carry out regulated expression of heterologous gene products.

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Production of Recombinant Bleaching Enzymes from Thermophilic Microorganisms in Fungal Hosts

Cited by 7 publications

References 27 publications

Optimization of Acidothermus cellulolyticus Endoglucanase (E1) Production in Transgenic Tobacco Plants by Transcriptional, Post-transcription and Post-translational Modification

Optimization of Acidothermus cellulolyticus Endoglucanase (E1) Production in Transgenic Tobacco Plants by Transcriptional, Post-transcription and Post-translational Modification

Isolation, characterization and optimization of culture parameters for production of an alkaline protease isolated from Aspergillus tamarii

Heterologous expression of cyan and yellow fluorescent proteins from the Kluyveromyces lactis KlMAL21–KlMAL22 bi-directional promoter

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