ORCID Profile
0000-0001-6460-8718
Current Organisation
Queensland University of Technology
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In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Enzymes | Agricultural Biotechnology | Food Processing | Agricultural Biotechnology not elsewhere classified | Genetic Engineering And Enzyme Technology | Bioprocessing, Bioproduction and Bioproducts | Biochemistry and Cell Biology | Analytical Biochemistry |
Nutraceuticals and Functional foods | Barley | Aquaculture Oysters | Biological sciences
Publisher: Humana Press
Date: 2014
DOI: 10.1007/978-1-62703-712-9_1
Abstract: Filamentous fungi are important organisms for basic discovery, industry, and human health. Their natural growth environments are extremely variable, a fact reflected by the numerous methods developed for their isolation and cultivation. Fungal culture in the laboratory is usually carried out on agar plates, shake flasks, and bench top fermenters starting with an inoculum that typically features fungal spores. Here we discuss the most popular methods for the isolation and cultivation of filamentous fungi for various purposes with the emphasis on enzyme production and molecular microbiology.
Publisher: Elsevier BV
Date: 04-2017
Publisher: Elsevier BV
Date: 2016
DOI: 10.1016/J.NBT.2015.08.004
Abstract: The filamentous fungus Trichoderma reesei is an expression host widely exploited for the production of recombinant proteins. However, its capacity for expressing small peptides (<20 kDa) has remained largely uncharted to date. In this work, we have produced the hormone peptide obestatin fused to the hydrophobin I tag (Obe-HFBI), using the T. reesei cellobiohydrolase I core (CBHI) or xylanase 2 (XYN2) pro-region as a carrier and the cbh1 promoter for gene expression, in high protein-low protease producing mutant strains T. reesei Rut-C30 and HEPI. The yield of obestatin was improved from about 300 ng/ml to up to 5.5 μg/ml through adaptive laboratory evolution and modifications to the cultivation strategy, which included adjustments of the type and ratio of carbon and nitrogen sources used in the medium. The successful expression of Obe-HFBI demonstrated the potential of T. reesei as an expression host for small peptides and further enhancement of the recombinant yield through modification of culture conditions.
Publisher: Springer Science and Business Media LLC
Date: 2002
DOI: 10.1385/ABAB:98-100:1-9:165
Abstract: Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host-vector expression system critical. We have developed two systems for the bulk production of bleaching enzymes from thermophiles. Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2mu-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Fusion and nonfusion vectors have been constructed using the strong cellobiohydrolase 1 (cbh1) promoter. The KEX2 protease cleavage site and a 6 x HIS-tag have been incorporated to facilitate both cleavage and purification of the mature foreign proteins.
Publisher: American Society for Microbiology
Date: 25-02-2016
Abstract: The genome sequence of Caloramator mitchellensis strain VF08, a rod-shaped, heterotrophic, strictly anaerobic bacterium isolated from the free-flowing waters of a Great Artesian Basin (GAB) bore well located in Mitchell, an outback Queensland town in Australia, is reported here. The analysis of the 2.42-Mb genome sequence indicates that the attributes of the genome are consistent with its physiological and phenotypic traits.
Publisher: Elsevier BV
Date: 11-2017
Publisher: Elsevier BV
Date: 11-2019
Publisher: Elsevier BV
Date: 02-2019
DOI: 10.1016/J.SCITOTENV.2018.10.127
Abstract: A combination of solubilization and degradation is essential for the bioremediation of environments contaminated with complex polychlorinated biphenyls (PCB) mixtures. However, the application of facultative anaerobic microorganisms that can both solubilize and breakdown hydrophobic PCBs in aqueous media under both anaerobic and aerobic conditions, has not been reported widely. In this comprehensive study, four bacteria discovered from soil and sediments and identified as Achromobacter sp. NP03, Ochrobactrum sp. NP04, Lysinibacillus sp. NP05 and Pseudomonas sp. NP06, were investigated for their PCB degradation efficiencies. Aroclor 1260 (50 mg/L), a commercial and highly chlorinated PCB mixture was exposed to the different bacterial strains under aerobic, anaerobic and two stage anaerobic-aerobic conditions. The results confirmed that all four facultative anaerobic microorganisms were capable of degrading PCBs under both anaerobic and aerobic conditions. The highest chlorine removal (9.16 ± 0.8 mg/L), PCB solubility (14.7 ± 0.93 mg/L) and growth rates as OD
Publisher: Elsevier BV
Date: 2014
DOI: 10.1016/J.NBT.2013.08.002
Abstract: Multiple copies of expression cassettes driven by the Trichoderma reesei xylanase 2 (xyn2) and cellobiohydrolase 2 (cbh2) promoters were introduced into the recombinant T. reesei EC-21 generated to express a thermostable Dictyoglomus thermophilum xylanase (XynB) under the egl2 promoter for further improvement of the enzyme yield. The transformants were screened based on increased XynB activity only. Multiple promoter transformant MPP-4 expressing the xynB gene under all the three promoters was found to be the highest producer of XynB, giving a 65% increase in yield compared to the parental single-promoter recombinant EC-21. The multiple-promoter transformant strains harboured six to nine copies of the xynB gene. Amongst the three promoters, egl2 seemed to have the strongest effect on XynB expression. The shotgun approach we used proved to be effective for rapid enhancement of protein expression using three promoters active at the near-neutral pH of the cultivation medium.
Publisher: Springer Science and Business Media LLC
Date: 19-06-2013
DOI: 10.1007/S00294-013-0394-X
Abstract: Cold-active lipases are of significant interest as biocatalysts in industrial processes. We have identified a lipase that displayed activity towards long carbon-chain-p-nitrophenyl substrates (C12-C18) at 25 °C from the culture supernatant of an Antarctic Penicillium expansum strain assigned P. expansum SM3. Zymography revealed a protein band of around 30 kDa with activity towards olive oil. DNA fragments of a lipase gene designated as lipPE were isolated from the genomic DNA of P. expansum SM3 by genomic walking PCR. Subsequently, the complete genomic lipPE gene was lified using gene-specific primers designed from the 5'- and 3'-regions. Reverse transcription PCR was used to lify the lipPE cDNA. The deduced amino acid sequence consisted of 285 residues that included a predicted signal peptide. Three peptides identified by LC/MS/MS analysis of the proteins in the culture supernatant of P. expansum were also present in the deduced amino acid sequence of the lipPE gene suggesting that this gene encoded the lipase identified by initial zymogram activity analysis. Full analysis of the nucleotide and the deduced amino acid sequences indicated that the lipPE gene encodes a novel P. expansum lipase. The lipPE gene was expressed in E. coli for further characterization of the enzyme with a view of assessing its suitability for industrial applications.
Publisher: Elsevier BV
Date: 09-2023
Publisher: Elsevier BV
Date: 09-2005
DOI: 10.1016/J.TIBTECH.2005.06.002
Abstract: Filamentous fungi are commonly used in the fermentation industry for the large-scale production of proteins--mainly industrial enzymes. Recent advances in fungal genomics and related experimental technologies such as gene arrays and proteomics are rapidly changing the approaches to the development and use of filamentous fungi as hosts for the production of both homologous and heterologous gene products. The emphasis is moving towards sourcing new genes of interest through database mining and unravelling the circuits related to fungal gene regulation, applying, for ex le, transcriptomics. However, although heterologous fungal proteins are efficiently expressed, expression of gene products from other organisms is subject to several bottlenecks that reduce yield. Current approaches emphasize the study of pathways involved in protein modification and degradation in general rather than gene-by-gene approaches.
Publisher: Springer Science and Business Media LLC
Date: 12-2003
DOI: 10.1007/S00294-003-0440-1
Abstract: We successfully isolated a lipase gene (designated lipPA) directly from the genomic DNA of an Antarctic isolate of Penicillium allii using PCR and a suite of degenerate primers specifically designed to target two conserved regions of fungal lipase genes. We applied the biolistic transformation system to successfully integrate the lipPA gene into a heterologous fungal host, Trichoderma reesei, one of the most powerful secretors of extracellular proteins, and induced the transformant to secrete an active lipase into the growth medium. The recombinant lipase had a temperature optimum of 25 degrees C at pH 7.9 and retained greater than 50% of the maximum activity from 10 degrees C to 35 degrees C and over a pH range from 4.0 to 8.5.
Publisher: Elsevier
Date: 2003
Publisher: Wiley
Date: 19-07-2018
DOI: 10.1111/JPY.12758
Abstract: Euglena gracilis Z and a "sugar loving" variant strain E. gracilis var. saccharophila were investigated as producers of paramylon, a β-1,3-glucan polysaccharide with potential medicinal and industrial applications. The strains were grown under diurnal or dark growth conditions on a glucose-yeast extract medium supporting high-level paramylon production. Both strains produced the highest paramylon yields (7.4-8 g · L
Publisher: Elsevier BV
Date: 08-2010
DOI: 10.1016/J.FUNBIO.2010.05.005
Abstract: Metarhizium anisopliae is a well-characterized biocontrol agent of a wide range of insects including cane grubs. In this study, a two-dimensional (2D) electrophoresis was used to display secreted proteins of M. anisopliae strain FI-1045 growing on the whole greyback cane grubs and their isolated cuticles. Hydrolytic enzymes secreted by M. anisopliae play a key role in insect cuticle-degradation and initiation of the infection process. We have identified all the 101 protein spots displayed by cross-species identification (CSI) from the fungal kingdom. Among the identified proteins were 64-kDa serine carboxypeptidase, 1,3 beta-exoglucanase, Dynamin GTPase, THZ kinase, calcineurin like phosphoesterase, and phosphatidylinositol kinase secreted by M. ansiopliae (FI-1045) in response to exposure to the greyback cane grubs and their isolated cuticles. These proteins have not been previously identified from the culture supernatant of M. anisopliae during infection. To our knowledge, this the first proteomic map established to study the extracellular proteins secreted by M. ansiopliae (FI-1045) during infection of greyback cane grubs and its cuticles.
Publisher: Elsevier
Date: 2005
Publisher: Springer Science and Business Media LLC
Date: 16-02-2022
DOI: 10.1007/S00253-022-11817-1
Abstract: Heat haze-forming proteins are stable during winemaking and are typically removed via adsorption to bentonite. Proteolytic degradation is an alternative method to prevent wine-haze and offers the opportunity to reduce the environmental impacts and labor cost of the process. Herein, we describe the development of a production system for Botrytis cinerea proteases for the enzymatic degradation of heat haze-forming proteins. The effect of culture medium on the secretion of glucan by B. cinerea was investigated and methods to inactivate B. cinerea laccase in liquid culture medium were assessed. Protease production by B. cinerea was scaled up from 50 mL in shake flasks to 1 L in bioreactors, resulting in an increase in protease yield from 0.30 to 3.04 g L −1 . Glucan secretion by B. cinerea was minimal in culture medium containing lactose as a carbon source and either lactic or sulfuric acid for pH control. B. cinerea laccases were inactivated by reducing the pH of culture supernatant to 1.5 for 1 h. B. cinerea proteases were concentrated and partially purified using ammonium sulfate precipitation. SWATH-MS identified aspartic acid protease BcAP8 amongst the precipitated proteins. These results demonstrate a simple, affordable, and scalable process to produce proteases from B. cinerea as a replacement for bentonite in winemaking. • Isolates of B. cinerea that produce proteases with potential for reducing wine heat-haze forming proteins were identified. • Media and fermentation optimization increased protease yield tenfold and reduced glucan secretion. • Low pH treatment inactivated laccases but not proteases.
Publisher: Wiley
Date: 04-2018
DOI: 10.1002/CPPS.52
Abstract: This unit describes production of a bacterial thermophilic xylanase enzyme in an industrially exploited filamentous fungus, Trichoderma reesei . Successful expression of a gene of interest in a heterologous host involves front‐end design of the expression constructs using bioinformatics tools, making the constructs in the laboratory, and introducing them into the expression host. This is followed by synthesis and characterization of the gene product on a laboratory scale and optimization of the cultivation parameters in a controlled, scaled‐up fermentation. The thermophilic xylanase B (XynB) enzyme from the bacterium Dictyoglomus thermophilum discussed here can be easily purified by heat‐precipitation from the culture supernatant of the mesophilic host. A functional XynB can also be produced in Escherichia coli , but at a lower yield compared to that obtained in T. reesei . The protocol provided here can be adapted to various other proteins and filamentous fungal hosts. © 2018 by John Wiley & Sons, Inc.
Publisher: Springer Science and Business Media LLC
Date: 23-12-2016
DOI: 10.1007/S00294-015-0555-1
Abstract: We have systematically analysed the ultrastructure of the early secretory pathway in the Trichoderma reesei hyphae in the wild-type QM6a, cellulase-overexpressing Rut-C30 strain and a Rut-C30 transformant BV47 overexpressing a recombinant BiP1-VenusYFP fusion protein with an endoplasmic reticulum (ER) retention signal. The hyphae were studied after 24 h of growth using transmission electron microscopy, confocal microscopy and quantitative stereological techniques. All three strains exhibited different spatial organisation of the ER at 24 h in both a cellulase-inducing medium and a minimal medium containing glycerol as a carbon source (non-cellulase-inducing medium). The wild-type displayed a number of ER subdomains including parallel tubular/cisternal ER, ER whorls, ER-isolation membrane complexes with abundant autophagy vacuoles and dense bodies. Rut-C30 and its transformant BV47 overexpressing the BiP1-VenusYFP fusion protein also contained parallel tubular/cisternal ER, but no ER whorls also, there were very few autophagy vacuoles and an increasing amount of punctate bodies where particularly the recombinant BiP1-VenusYFP fusion protein was localised. The early presence of distinct strain-specific features such as the dominance of ER whorls in the wild type and tub/cis ER in Rut-C30 suggests that these are inherent traits and not solely a result of cellular response mechanisms by the high secreting mutant to protein overload.
Publisher: Springer Science and Business Media LLC
Date: 03-09-2009
DOI: 10.1007/S00294-009-0270-X
Abstract: Metarhizium anisopliae is a naturally occurring cosmopolitan fungus infecting greyback canegrubs (Dermolepida albohirtum). The main molecular factors involved in the complex interactions occurring between the greyback canegrubs and M. anisopliae (FI-1045) were investigated by comparing the proteomes of healthy canegrubs, canegrubs infected with Metarhizium and fungus only. Differentially expressed proteins from the infected canegrubs were subjected to mass spectrometry to search for pathogenicity related proteins. Immune-related proteins of canegrubs identified in this study include cytoskeletal proteins (actin), cell communication proteins, proteases and peptidases. Fungal proteins identified include metalloproteins, acyl-CoA, cyclin proteins and chorismate mutase. Comparative proteome analysis provided a view into the cellular reactions triggered in the canegrub in response to the fungal infection at the onset of biological control.
Publisher: Oxford University Press (OUP)
Date: 02-2002
DOI: 10.1046/J.1472-765X.2002.01057.X
Abstract: To express a gene encoding a heterologous fungal xylanase in Trichoderma reesei. Humicola grisea xylanase 2 (xyn2) cDNA was expressed in Trichoderma reesei under the main cellobiohydrolase I (cbh1) promoter (i) as a fusion to the cellobiohydrolase I (CBHI) secretion signal and (ii) the mature CBHI core-linker. The recombinant xylanase (HXYN2) was secreted into the cultivation medium and processed in a similar fashion to the endogenous T. reesei xylanases, resulting in an active enzyme. HXYN2 was successfully processed in T. reesei. Composition of the culture medium affected the HXYN2 yields, favouring Avicel-lactose as a carbon source. Best yields (about 0.5 g l(-1)) in shake flask cultivations were obtained from a transformant where xyn2 was fused directly to the CBHI secretion signal.
Publisher: Springer Science and Business Media LLC
Date: 06-1995
DOI: 10.1007/BF00172827
Abstract: In mammals, the acquisition of the germline from the soma provides the germline with an essential challenge: the need to erase and reset genomic methylation
Publisher: Portland Press Ltd.
Date: 04-2004
DOI: 10.1042/BST0320293
Abstract: Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host/vector expression system critical. We have tested two fungal systems for the bulk production of enzymes from thermophiles. The yeast Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2u-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Signal and protein fusion vectors have been constructed using the strong cellobiohydrolase 1 (cbh1) promoter and recombinant plasmid DNAs introduced into various high-secreting T. reesei strains using biolistic particle delivery. In some cases (e.g. the xynB gene of Dictyoglomus thermophilum) we have reconstructed the genes according to Trichoderma codon preferences and demonstrated a dramatic increase in the production of the enzymes. The heterologous XynB enzyme is glycosylated differently in different Trichoderma strains. A proteomics approach has been taken to identify strongly expressed proteins produced by T. reesei under various cultivation conditions in order to identify condition-specific promoters driving the production of these proteins. Analyses indicated that HEX1, the major protein of the fungal Woronin body, is a dominant protein under both cellulase-inducing and -repressing conditions. The hex1 gene together with its promoter and terminator sequences has been isolated and the promoter function studied relative to cultivation time and medium.
Publisher: Springer Science and Business Media LLC
Date: 09-05-2007
DOI: 10.1007/S10529-007-9380-Z
Abstract: Two novel genes, xyn5 and xyn6, coding for family 11 xylanases, were isolated from the thermotolerant filamentous fungus, Acrophialophora nainiana, by PCR using degenerate primers. The xyn6 gene was further expressed in Trichoderma reesei. DNA sequence analysis of xyn6 revealed an open reading frame (ORF) of 708 bp, interrupted by an intron of 58 bp. The xyn6 ORF encodes a predicted protein of 236 amino acid residues. The mature recombinant XynVI protein had a molecular mass of about 19 kDa, as estimated by gel electrophoresis. Analysis of the predicted amino acid sequence of XynVI paves the way for rational protein engineering by site-directed mutagenesis aiming to increase the thermostability of the heterologously-expressed xylanase.
Publisher: Elsevier BV
Date: 04-2009
DOI: 10.1016/J.SCITOTENV.2018.12.385
Abstract: Polychlorinated biphenyls (PCBs) are synthetic mixtures of chlorinated hydrocarbon compounds that were widely used in the past and still found in the environment due to their highly recalcitrant nature. A combination of anaerobic dechlorination and aerobic oxidation is essential to degrade these PCB mixtures into less toxic products. It was hypothesized that due to the complexity of PCB mixtures, a consortium of carefully selected suitable microbial species will perform better than the application of in idual microbes. In the present study, biodegradation of the commercial PCB mixture, Aroclor 1260, was studied under two different combined anaerobic-aerobic conditions, namely, alternating (AN) and two stage (TS). The facultative anaerobic bacterial consortium consisted of naturally occurring Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05. These bacteria were found capable as in iduals of solubilizing and degrading PCBs under both anaerobic and aerobic conditions. 49.2 ± 2.5% total reduction of the original 50 mg/L Aroclor 1260 mixture was achieved after 2 weeks in AN treatment whereas the reduction was only 24.44 ± 2.46% in TS treatment. At the end of week 6, a yield of 17.63 ± 0.91 mg/L chloride released was measured under AN condition compared to 11.79 ± 1.28 mg/L measured under TS condition. The overall results suggested that the microbial consortia capable of degrading and utilizing PCBs under both, anaerobic and aerobic conditions achieved better PCB degradation by repeated exposure to short periods of anaerobic and aerobic conditions alternatingly rather than the conventional two stage anaerobic-aerobic conditions.
Publisher: Elsevier BV
Date: 11-2002
DOI: 10.1016/S0167-7012(02)00126-4
Abstract: Effective biolistic transformation of intact conidia from the filamentous fungus Trichoderma reesei was achieved using the Bio-Rad Hepta Adaptor system with seven barrels for particle launch. Transformation frequencies of up to 39 colonies per microg of circular DNA and 37 colonies per microg of linear DNA were obtained at an optimal target distance of 3 cm and a helium pressure of 1350 psi. These values are about 3.5- to 6-fold higher than transformant yields reported earlier for T. reesei using the hygromycin phosphotransferase (hph) gene conferring resistance to the antibiotic hygromycin B as a selectable marker in combination with the PDS-1000/He single barrel system. High mitotic stability of the transformants (98-100%) was demonstrated. The Hepta Adaptor device allowing bombardment of seven lots of conidia in a single plate offers clear advantage in terms of transformant numbers over the single barrel system where target cells are restricted to the center of the plate.
Publisher: Elsevier
Date: 2002
Publisher: Wiley
Date: 04-02-2019
Publisher: Elsevier BV
Date: 12-2006
DOI: 10.1016/J.GENE.2006.07.017
Abstract: UV mutagenesis was applied to improve protein secretion in Ophiostoma floccosum. Amylase activity was used as an indicator for enhanced protein production after repeated rounds of mutagenic treatment. The amylase activity in the culture supernatant of the best mutant (MQ.5.1) was increased by more than 240-fold compared to the initial parental strain. At the same time, the increase in total secreted protein was about six times greater than the parental strain. Secreted proteinase and lipase activities of the parental strain and four key mutants were also investigated. N-terminal sequencing of the five dominant protein bands separated by SDS-PAGE from the culture supernatant was conducted. Two of the proteins identified were subtilisin-like proteinases and one was a pepsin-like proteinase. In addition, one protein was identified as an alpha-amylase and one remained unidentified. A 6.5 kb DNA fragment was isolated by Genomic Walking PCR using primers based on the alpha-amylase amino acid sequence. The lified fragment contained the entire gene encoding alpha-amylase (amy1) and its regulatory sequences. Analysis showed that multiple transcripts were generated from the single alpha-amylase gene locus.
Publisher: Elsevier BV
Date: 04-2004
Publisher: Springer Science and Business Media LLC
Date: 06-2002
DOI: 10.1007/S00792-001-0252-5
Abstract: Bulk production of xylanases from thermophilic microorganisms is a prerequisite for their use in industrial processes. As effective secretors of gene products, fungal expression systems provide a promising, industrially relevant alternative to bacteria for heterologous enzyme production. We are currently developing the yeast Kluyveromyces lactis and the filamentous fungus Trichoderma reesei for the extracellular production of thermophilic enzymes for the pulp and paper industry. The K. lactis system has been tested with two thermophilic xylanases and secretes gram amounts of largely pure xylanase A from Dictyoglomus thermophilum in chemostat culture. The T. reesei expression system involves the use of the cellobiohydrolase I (CBHI) promoter and gene fusions for the secretion of heterologous thermostable xylanases of both bacterial and fungal origin. We have reconstructed the AT-rich xynB gene of Dictyoglomus thermophilum according to Trichoderma codon preferences and demonstrated a dramatic increase in expression. A heterologous fungal gene, Humicola grisea xyn2, could be expressed without codon modification. Initial amounts of the XYN2 protein were of a gram per liter range in shake-flask cultivations, and the gene product was correctly processed by the heterologous host. Comparison of the expression of three thermophilic heterologous microbial xylanases in T. reesei demonstrates the need for addressing each case in idually.
Publisher: Elsevier BV
Date: 2013
DOI: 10.1016/J.NBT.2012.07.005
Abstract: Trichoderma reesei Rut-C30 is used widely as an expression host for various gene products. We have explored cellular effects caused by the expression of a mutant form of cellobiohydrolase I (CBHI), the major secreted protein of T. reesei using biochemical and transcriptomic analyses and confocal laser scanning microscopy. The mutated CBHI was tagged fluorescently with Venus to establish the subcellular location of the fusion protein and its potential association with the proteasome, an organelle assigned for the disposal of misfolded proteins. Expression of the mutant CBHI in the high protein-secreting host Rut-C30 caused physiological changes in the fungal hyphae, affected protein secretion and elicited ER stress. A massive upregulation of UPR- and ERAD-related genes sec61, der1, uba1, bip1, pdi1, prp1, cxl1 and lhs1 was observed by qRT-PCR in the CBHIΔ4-Venus strain with four mutations introduced in the DNA encoding the core domain of CBHI. Further stress was applied to this strain by inhibiting function of the proteasome with MG132 (N-benzoylcarbonyl(Cbz)-Leu-Leu-leucinal). The effect of MG132 was found to be specific to the proteasome-associated genes. There are no earlier reports on the effect of proteasome inhibition on protein quality control in filamentous fungi. Confocal fluorescence microscopy studies suggested that the mutant CBHI accumulated in the ER and colocalized with the fungal proteasome. These results provide an indication that there is a limit to how far T. reesei Rut-C30, already under secretion stress, can be pressed to produce higher protein yields.
Publisher: Elsevier BV
Date: 06-2013
DOI: 10.1016/J.NBT.2013.02.005
Abstract: Expression vectors were constructed for Trichoderma reesei using the promoters, secretion signals and the modular structure of the efficiently expressed and secreted cellulase enzymes EGL2 (Cel5A) and CBH2 (Cel6A) as a prelude to establishing a platform where a gene of interest can be expressed under several promoters simultaneously. The designs featured (i) EGL2sigpro (egl2 promoter and secretion signal), (ii) EGL2cbmlin (egl2 promoter, secretion signal, EGL2 cellulose binding module and linker), (iii) CBH2sigpro (cbh2 promoter and secretion signal) and (iv) CBH2cbmlin (cbh2 promoter, secretion signal, CBH2 cellulose binding module and linker). Recombinant vectors were introduced in idually into the high protein-secreting T. reesei RUT-C30 strain to generate single-promoter transformants expressing the Dictyoglomus thermophilum xynB gene that encodes a thermophilic xylanase enzyme (XynB). Ten transformants producing XynB representing each of the four different types of vectors were selected for further testing and the highest XynB production was achieved from a transformant containing 1-2copies of the EGL2cbmlin vector. Best xylanase producers did not show any particular pattern in terms of the number of gene copies and their mode of integration into the chromosomal DNA. Transformants generated with the cbmlin-type vectors produced multiple forms of XynB which were decorated with various N- and O-glycans. One of the O-glycans was identified as hexuronic acid, whose presence had not been observed previously in the glycosylation patterns of T. reesei.
Publisher: American Chemical Society (ACS)
Date: 07-2020
Publisher: Springer Science and Business Media LLC
Date: 29-05-2018
DOI: 10.1038/S41598-018-26227-5
Abstract: Production of ethanol by the yeast Saccharomyces cerevisiae is a process of global importance. In these processes, productivities and yields are pushed to their maximum possible values leading to cellular stress. Transient and lasting enhancements in tolerance and performance have been obtained by genetic engineering, forced evolution, and exposure to moderate levels of chemical and/or physical stimuli, yet the drawbacks of these methods include cost, and multi-step, complex and lengthy treatment protocols. Here, plasma agitation is shown to rapidly induce desirable phenotypic changes in S . cerevisiae after a single treatment, resulting in improved conversion of glucose to ethanol. With a complex environment rich in energetic electrons, highly-reactive chemical species, photons, and gas flow effects, plasma treatment simultaneously mimics exposure to multiple environmental stressors. A single treatment of up to 10 minutes performed using an atmospheric pressure plasma jet was sufficient to induce changes in cell membrane structure, and increased hexokinase 2 activity and secondary metabolite production. These results suggest that plasma treatment is a promising strategy that can contribute to improving metabolic activity in industrial microbial strains, and thus the practicality and economics of industrial fermentations.
Start Date: 05-2009
End Date: 05-2012
Amount: $138,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 05-2013
End Date: 09-2017
Amount: $2,100,000.00
Funder: Australian Research Council
View Funded Activity