Production of Industrial Enzymes in Trichoderma reesei

Paloheimo, Marja; Haarmann, Thomas; Mäkinen, Susanna; Vehmaanperä, Jari

doi:10.1007/978-3-319-27951-0_2

Cited by 48 publications

(40 citation statements)

References 166 publications

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“…DuPont, Novozyme, AB Enzyme) [9]. In some cases, antibiotics are used to prevent bacterial contamination [73,74,75], however, the use of antibiotics has been under increased scrutiny by the consumers and is not a well-accepted in the food industry. In this work, we report that the ptxD-expressing strain outcompetes a bacterial contaminant strain when cultured in liquid Phi-based media ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Assessment of the ptxD gene as a growth and selective marker in Trichoderma atroviride using Pccg6, a novel constitutive promoter

Carreras-Villaseñor

Rico-Ruiz

Chavez-Montes³

et al. 2020

Preprint

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Background Trichoderma species are among the most effective cell factories to produce recombinant proteins, whose productivity relies on the molecular toolkit and promoters available for the expression of the target protein. Although inducible promoter systems have been developed for producing recombinant proteins in Trichoderma , constitutive promoters are often a desirable alternative. Constitutive promoters are simple to use, do not require external stimuli or chemical inducers to be activated, and lead to purer enzyme preparations. Moreover, most of the promoters for homologous and heterologous expression reported in Trichoderma have been commonly evaluated by directly assessing production of industrial enzymes, requiring optimization of laborious protocols. Results Here we report the identification of P ccg6, a novel Trichoderma atroviride constitutive promoter, that has similar transcriptional strength as that of the commonly used pki1 promoter. P ccg6 displayed conserved arrangements of transcription factor binding sites between promoter sequences of Trichoderma ccg6 orthologues genes, potentially involved in their regulatory properties. The predicted ccg6 -encoded protein potentially belongs to the SPE1/SPI1 protein family and shares high identity with CCG6 orthologue sequences from other fungal species including Trichoderma reesei , Trichoderma virens , Trichoderma asperellum , and lo a lesser extent to that of Neurosposa crassa . We also report the use of the P ccg6 promoter to drive the expression of PTXD, a phosphite oxidoreductase of bacterial origin, which allowed T. atroviride to utilize phosphite as a sole source of phosphorus. We propose ptxD as a growth reporter gene that allows real-time comparison of the functionality of different promoters by monitoring growth of Trichoderma transgenic lines and enzymatic activity of PTXD. Finally, we show thatconstitutive expression of ptxD provided T. atroviride a competitive advantage to outgrow bacterial contaminants when supplied with phosphite as a sole source of phosphorus. Conclusions A new constitutive promoter, ccg6 , for expression of homologous and heterologous proteins has been identified and tested in T. atroviride by expressing PTXD, which resulted in an effective and visible phenotype to evaluate transcriptional activity of sequence promoters. Use of PTXD as a growth marker holds great potential for assessing activity of other promoters and for biotechnological applications as a contamination control system.

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Section: Discussionmentioning

confidence: 99%

Assessment of the ptxD gene as a growth and selective marker in Trichoderma atroviride using Pccg6, a novel constitutive promoter

Carreras-Villaseñor

Rico-Ruiz

Chavez-Montes³

et al. 2020

Preprint

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“…Trichoderma species are widely used at industrial level in the manufacture of a variety of pharmaceuticals, chemicals, and enzyme products [62]. Most of the enzymes required by the food industries, for example, are produced by recombinant strategies in T. reesei, typically cultured by submerged fermentation, and receive the generally recognized as safe (GRAS) status by the U.S. Food and Drug Administration (FDA) [62,63]. Enzyme preparations can be marketed as monocomponents (pure enzyme) or a mixture of enzymes that are produced by concentration (centrifugation, filtration) of the growth media when enzymes are secreted.…”

Section: Discussionmentioning

confidence: 99%

“…DuPont, Novozyme, AB Enzyme) [9]. In some cases, antibiotics are used to prevent bacterial contamination [62,63,64], however, the use of antibiotics has been under increased scrutiny in the last years by the consumers and is not a well-accepted practice when the product is intended for the food industry. In this work, we presented that the ptxD-expressing strain outcompetes a bacterial contaminant strain when cultured in liquid Phi-based media (Fig.…”

Section: Discussionmentioning

confidence: 99%

Assessment of the ptxD gene as a growth and selective marker in Trichoderma atroviride using Pccg6, a novel constitutive promoter

Carreras-Villaseñor

Rico-Ruiz

Lopez-Hernandez

et al. 2019

Preprint

View full text Add to dashboard Cite

BackgroundTrichoderma species are among the most effective cell factories to produce recombinant proteins, whose productivity relies on the molecular toolkit and promoters available for the expression of the target protein. Although inducible promoter systems have been developed for producing recombinant proteins in Trichoderma, constitutive promoters are often a desirable alternative. Constitutive promoters are simple to use, do not require external stimuli or chemical inducers to be activated, and lead to purer enzyme preparations. Moreover, most of the promoters for homologous and heterologous expression reported in Trichoderma have been commonly evaluated by directly assessing production of industrial enzymes, requiring optimization of laborious protocols. ResultsHere we report the identification of Pccg6, a novel Trichoderma atroviride constitutive promoter, that has similar transcriptional strength as that of the commonly used pki1 promoter. Pccg6 displayed conserved arrangements of transcription factor binding sites between promoter sequences of Trichoderma ccg6 orthologues genes, potentially involved in their regulatory properties. The predicted ccg6-encoded protein potentially belongs to the SPE1/SPI1 protein family and shares high identity with CCG6 orthologue sequences from other fungal species including Trichoderma reesei, Trichoderma virens, Trichoderma asperellum, and lo a lesser extent to that of Neurosposa crassa. We also report the use of the Pccg6 promoter to drive the expression of PTXD, a phosphite oxidoreductase of bacterial origin, which allowed T. atroviride to utilize phosphite as a sole source of phosphorus. We propose ptxD as a growth reporter gene that allows real-time comparison of the functionality of different promoters by monitoring growth of Trichoderma transgenic lines and enzymatic activity of PTXD. Finally, we show that constitutive expression of ptxD provided T. atroviride a competitive advantage to outgrow bacterial contaminants when supplied with phosphite as a sole source of phosphorus. ConclusionsA new constitutive promoter, ccg6, for expression of homologous and heterologous proteins has been 3 identified and tested in T. atroviride by expressing PTXD, which resulted in an effective and visible phenotype to evaluate transcriptional activity of sequence promoters. Use of PTXD as a growth marker holds great potential for assessing activity of other promoters and for biotechnological applications as a contamination control system. BackgroundTrichoderma is a fungal genus with high level of genetic diversity and great adaptability to different environmental conditions. These characteristics, together with factors such as their high capacity to produce and secrete proteins and bioactive compounds, as well as the availability of protocols and molecular tools for their genetic manipulation, have posed Trichoderma as a model organism to study fungal biology and to develop biotechnological applications for different industries [1].Some examples of the industrial applications of Tric...

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“…Currently, fungal enzymes from a handful of organisms, e.g., Trichoderma reesei or Aspergillus sp., are utilized on an industrial scale to break down plant biomass [8]. A recent report illustrates the utility of developing consortia between a cellulose degrader like T. reesei and the model bacterium Escherichia coli [9].…”

Section: Introductionmentioning

confidence: 99%

In Silico Identification of Microbial Partners to Form Consortia with Anaerobic Fungi

et al. 2018

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Abstract:Lignocellulose is an abundant and renewable resource that holds great promise for sustainable bioprocessing. However, unpretreated lignocellulose is recalcitrant to direct utilization by most microbes. Current methods to overcome this barrier include expensive pretreatment steps to liberate cellulose and hemicellulose from lignin. Anaerobic gut fungi possess complex cellulolytic machinery specifically evolved to decompose crude lignocellulose, but they are not yet genetically tractable and have not been employed in industrial bioprocesses. Here, we aim to exploit the biomass-degrading abilities of anaerobic fungi by pairing them with another organism that can convert the fermentable sugars generated from hydrolysis into bioproducts. By combining experiments measuring the amount of excess fermentable sugars released by the fungal enzymes acting on crude lignocellulose, and a novel dynamic flux balance analysis algorithm, we screened potential consortia partners by qualitative suitability. Microbial growth simulations reveal that the fungus Anaeromyces robustus is most suited to pair with either the bacterium Clostridia ljungdahlii or the methanogen Methanosarcina barkeri-both organisms also found in the rumen microbiome. By capitalizing on simulations to screen six alternative organisms, valuable experimental time is saved towards identifying stable consortium members. This approach is also readily generalizable to larger systems and allows one to rationally select partner microbes for formation of stable consortia with non-model microbes like anaerobic fungi.

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Production of Industrial Enzymes in Trichoderma reesei

Cited by 48 publications

References 166 publications

Assessment of the ptxD gene as a growth and selective marker in Trichoderma atroviride using Pccg6, a novel constitutive promoter

Assessment of the ptxD gene as a growth and selective marker in Trichoderma atroviride using Pccg6, a novel constitutive promoter

Assessment of the ptxD gene as a growth and selective marker in Trichoderma atroviride using Pccg6, a novel constitutive promoter

In Silico Identification of Microbial Partners to Form Consortia with Anaerobic Fungi

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