The Generally Recognized as Safe (GRAS) Process for Industrial Microbial Enzymes

SewaltVincent,; ShanahanDiane,; GreggLori,; MartaJames, La; CarrilloRoberto,

doi:10.1089/ind.2016.0011

Cited by 105 publications

(63 citation statements)

References 18 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%

“…Manufacture of safe and high-quality enzyme preparations requires the culture of pure strains throughout the process and therefore involve the implementation of expensive and complex contamination control measures (i.e. antibiotic dosage) and the use of sanitary stainless-steel tanks and piping, and steam sterilization of the growth media and accessories, which increase operation costs [63]. The implementation of these measures for the production of enzyme preparations intended for food processing at industrial scale in T. reesei is extremely important, as clearly manifested by large companies that state that any batch must be rejected if contamination is detected (i.e.…”

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%

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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

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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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“…The QPS status can be compared to the Generally Recognized as Safe (GRAS) status given to safe MO by the U.S. Food and Drug Administration (FDA) [61].…”

Section: Qualified Presumption Of Safety Statusmentioning

confidence: 99%

Genetically Modified Micro-Organisms for Industrial Food Enzyme Production: An Overview

Deckers

Deforce

Fraiture

et al. 2020

Foods

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The use of food enzymes (FE) by the industrial food industry is continuously increasing. These FE are mainly obtained by microbial fermentation, for which both wild-type (WT) and genetically modified (GM) strains are used. The FE production yield can be increased by optimizing the fermentation process, either by using genetically modified micro-organism (GMM) strains or by producing recombinant enzymes. This review provides a general overview of the different methods used to produce FE preparations and how the use of GMM can increase the production yield. Additionally, information regarding the construction of these GMM strains is provided. Thereafter, an overview of the different European regulations concerning the authorization of FE preparations on the European market and the use of GMM strains is given. Potential issues related to the authorization and control of FE preparations sold on the European market are then identified and illustrated by a case study. This process highlighted the importance for control of FE preparations and the consequent need for appropriate detection methods targeting the presence of GMM, which is used in fermentation products.

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The Generally Recognized as Safe (GRAS) Process for Industrial Microbial Enzymes

Cited by 105 publications

References 18 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

Genetically Modified Micro-Organisms for Industrial Food Enzyme Production: An Overview

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