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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

Managing Zeocin consumption. The experiment was performed as described in the results section. After 4 days at 30°C, plates were photographed. In the upper row, Zeocin was added directly to the cell suspension before plating on Zeocin deficient plates. Numbers on top indicated the volume of Zeocin mother solution added to 150 μl of cell suspension. The lonely plate below the upper row is the reference plate. In that case, the cell suspension was not supplemented with Zeocin, but was plated on Zeocin-containing plate. This reference plate was located between plates “0” and “1” of the upper row because it roughly contained an intermediate number of clones between these two plates.
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Figure 3: Managing Zeocin consumption. The experiment was performed as described in the results section. After 4 days at 30°C, plates were photographed. In the upper row, Zeocin was added directly to the cell suspension before plating on Zeocin deficient plates. Numbers on top indicated the volume of Zeocin mother solution added to 150 μl of cell suspension. The lonely plate below the upper row is the reference plate. In that case, the cell suspension was not supplemented with Zeocin, but was plated on Zeocin-containing plate. This reference plate was located between plates “0” and “1” of the upper row because it roughly contained an intermediate number of clones between these two plates.

Mentions: In practice, X33 cells were electroporated with episomal Gateway plasmid pBGP1-DEST bearing GH5 coding sequence. After the sorbitol step, 100 out of 1000 μl cell suspension were spread on either a Zeocin plate made of 25 ml of YPDA supplemented with 25 μl of 100 mg/ml Zeocin solution, or on agar plates made of 25 ml of Zeocin-free YPDA. In the latter case, cell suspensions were supplemented with 0, 1, 5, 10, or 25 μl of 100 mg/ml Zeocin solution immediately before spreading on Zeocin-free plates. Plates were then incubated at 30°C for 4 days, and the number of colonies was compared. Results (Figure 3) indicate that adding 1 μl of Zeocin solution directly to the cell suspension just before plating provided roughly the same result as plating the same volume of the same cell suspension on an agar plate supplemented with 25 μl of Zeocin solution.


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)

Managing Zeocin consumption. The experiment was performed as described in the results section. After 4 days at 30°C, plates were photographed. In the upper row, Zeocin was added directly to the cell suspension before plating on Zeocin deficient plates. Numbers on top indicated the volume of Zeocin mother solution added to 150 μl of cell suspension. The lonely plate below the upper row is the reference plate. In that case, the cell suspension was not supplemented with Zeocin, but was plated on Zeocin-containing plate. This reference plate was located between plates “0” and “1” of the upper row because it roughly contained an intermediate number of clones between these two plates.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Managing Zeocin consumption. The experiment was performed as described in the results section. After 4 days at 30°C, plates were photographed. In the upper row, Zeocin was added directly to the cell suspension before plating on Zeocin deficient plates. Numbers on top indicated the volume of Zeocin mother solution added to 150 μl of cell suspension. The lonely plate below the upper row is the reference plate. In that case, the cell suspension was not supplemented with Zeocin, but was plated on Zeocin-containing plate. This reference plate was located between plates “0” and “1” of the upper row because it roughly contained an intermediate number of clones between these two plates.
Mentions: In practice, X33 cells were electroporated with episomal Gateway plasmid pBGP1-DEST bearing GH5 coding sequence. After the sorbitol step, 100 out of 1000 μl cell suspension were spread on either a Zeocin plate made of 25 ml of YPDA supplemented with 25 μl of 100 mg/ml Zeocin solution, or on agar plates made of 25 ml of Zeocin-free YPDA. In the latter case, cell suspensions were supplemented with 0, 1, 5, 10, or 25 μl of 100 mg/ml Zeocin solution immediately before spreading on Zeocin-free plates. Plates were then incubated at 30°C for 4 days, and the number of colonies was compared. Results (Figure 3) indicate that adding 1 μl of Zeocin solution directly to the cell suspension just before plating provided roughly the same result as plating the same volume of the same cell suspension on an agar plate supplemented with 25 μl of Zeocin solution.

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