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Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris.

Haon M, Grisel S, Navarro D, Gruet A, Berrin JG, Bignon C - Front Microbiol (2015)

Bottom Line: We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol.Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed.The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR 1163 Biodiversité et Biotechnologie Fongiques Marseille, France ; Aix-Marseille Université, Polytech Marseille, UMR1163 Biodiversité et Biotechnologie Fongiques Marseille, France.

ABSTRACT
Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community.

No MeSH data available.


Related in: MedlinePlus

Comparative flowchart of Invitrogen (left) and our (right) protocol for expressing recombinant proteins in P. pastoris. In this example, constitutive expression was chosen. Numbers on the left and on the right are the estimated number of days required by the considered step, with their sum (total) at the bottom.
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Figure 7: Comparative flowchart of Invitrogen (left) and our (right) protocol for expressing recombinant proteins in P. pastoris. In this example, constitutive expression was chosen. Numbers on the left and on the right are the estimated number of days required by the considered step, with their sum (total) at the bottom.

Mentions: A comparative flowchart of the classical protocol set-up by Invitrogen (pgapz_man.pdf) and of the simplified protocol described here for expressing biomass-degrading fungal enzymes is provided in Figure 7. Overall, the simplified protocol allows dividing by two the time required to go from transformation to expression screening, and was used as a basis for setting-up a recombinant protein production facility for the heterologous expression of fungal CAZymes in P. pastoris (Figure 5). Table 1 summarizes all the results obtained using P. pastoris as host for expressing fungal biomass-degrading enzymes in replacement of native or recombinant expression in other fungi such a A. niger and T. reesei (Levasseur et al., 2004; Poidevin et al., 2009). Switching from the original Invitrogen protocol to the simplified protocol described in this report and now routinely used on the platform has resulted in an increased use of recombinant expression in yeast from a few coding sequences in 2011 to a total of more than 40 today, out of which more than 50% were expressed at high level (>100 mg/L). The fungal CAZymes functionally produced originate from several filamentous fungi and belong to different CAZy families within the GH, CE, and AA classes. The platform is accessible to the community of CAZyme users upon request.


Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris.

Haon M, Grisel S, Navarro D, Gruet A, Berrin JG, Bignon C - Front Microbiol (2015)

Comparative flowchart of Invitrogen (left) and our (right) protocol for expressing recombinant proteins in P. pastoris. In this example, constitutive expression was chosen. Numbers on the left and on the right are the estimated number of days required by the considered step, with their sum (total) at the bottom.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585289&req=5

Figure 7: Comparative flowchart of Invitrogen (left) and our (right) protocol for expressing recombinant proteins in P. pastoris. In this example, constitutive expression was chosen. Numbers on the left and on the right are the estimated number of days required by the considered step, with their sum (total) at the bottom.
Mentions: A comparative flowchart of the classical protocol set-up by Invitrogen (pgapz_man.pdf) and of the simplified protocol described here for expressing biomass-degrading fungal enzymes is provided in Figure 7. Overall, the simplified protocol allows dividing by two the time required to go from transformation to expression screening, and was used as a basis for setting-up a recombinant protein production facility for the heterologous expression of fungal CAZymes in P. pastoris (Figure 5). Table 1 summarizes all the results obtained using P. pastoris as host for expressing fungal biomass-degrading enzymes in replacement of native or recombinant expression in other fungi such a A. niger and T. reesei (Levasseur et al., 2004; Poidevin et al., 2009). Switching from the original Invitrogen protocol to the simplified protocol described in this report and now routinely used on the platform has resulted in an increased use of recombinant expression in yeast from a few coding sequences in 2011 to a total of more than 40 today, out of which more than 50% were expressed at high level (>100 mg/L). The fungal CAZymes functionally produced originate from several filamentous fungi and belong to different CAZy families within the GH, CE, and AA classes. The platform is accessible to the community of CAZyme users upon request.

Bottom Line: We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol.Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed.The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR 1163 Biodiversité et Biotechnologie Fongiques Marseille, France ; Aix-Marseille Université, Polytech Marseille, UMR1163 Biodiversité et Biotechnologie Fongiques Marseille, France.

ABSTRACT
Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community.

No MeSH data available.


Related in: MedlinePlus