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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

Plating is dispensable for expressing recombinant GH in Pichia pastoris. Liquid and solid phase selections were performed as described in the results section. For the solid phase selection, three individual clones (numbered 1, 2, 3 on the x axis) were grown each in triplicate in individual wells of a 24-wells deep-well plate. For the liquid phase selection, three individual wells (numbered 1, 2, 3 on the x axis) of a 24-wells deep-well were seeded each with 200 out of 1000 μl of the cell suspension in sorbitol solution after electroporation and assayed in triplicate. Results show the mean enzymatic activity assayed in culture supernatants, with standard deviations of the triplicate assay.
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Figure 2: Plating is dispensable for expressing recombinant GH in Pichia pastoris. Liquid and solid phase selections were performed as described in the results section. For the solid phase selection, three individual clones (numbered 1, 2, 3 on the x axis) were grown each in triplicate in individual wells of a 24-wells deep-well plate. For the liquid phase selection, three individual wells (numbered 1, 2, 3 on the x axis) of a 24-wells deep-well were seeded each with 200 out of 1000 μl of the cell suspension in sorbitol solution after electroporation and assayed in triplicate. Results show the mean enzymatic activity assayed in culture supernatants, with standard deviations of the triplicate assay.

Mentions: We compared both approaches to assess whether having individual clones was critical for our project. X33 cells were transformed with linearized pGAPZαA constructs bearing coding sequences for GH5, GH11, or GH45. After the sorbitol step, 200 μl of cell suspension in sorbitol were plated and then incubated at 30°C for 3 days as usual, and another 200 μl of the same cell suspension in sorbitol were used for liquid phase selection as described in Section “Materials and Methods”. When clones began to appear on agar, they were individually used to inoculate 5 ml YPD-Zeocin and then allowed to grow and express the recombinant protein for 4 days. Expression of recombinant proteins of all solid and liquid phase experimental points was assessed by assaying GH5, GH11, and GH45 enzymatic activities. Results (Figure 2) definitely show that, at least for our three reference proteins, there was no difference between either selection mean. In addition to using less Zeocin than solid phase selection [2 μl in 2 ml YPD instead of 25 μl in 25 ml YPD containing 1.2% agar (YPDA; i.e., the volume poured per plate)], liquid phase selection does not preclude individual clones to be selected on plate if necessary since it uses only 200 out of 1000 μl of the sorbitol suspension of transformed cells and so an additional 200 μl aliquot can be plated and grown in parallel with the liquid phase selection. In conclusion, the liquid phase selection was chosen as default option for our facility because it allows a yes/no answer to be obtained more quickly than solid state screening. In case of negative or poor response, plated clones can be used as a rescue.


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)

Plating is dispensable for expressing recombinant GH in Pichia pastoris. Liquid and solid phase selections were performed as described in the results section. For the solid phase selection, three individual clones (numbered 1, 2, 3 on the x axis) were grown each in triplicate in individual wells of a 24-wells deep-well plate. For the liquid phase selection, three individual wells (numbered 1, 2, 3 on the x axis) of a 24-wells deep-well were seeded each with 200 out of 1000 μl of the cell suspension in sorbitol solution after electroporation and assayed in triplicate. Results show the mean enzymatic activity assayed in culture supernatants, with standard deviations of the triplicate assay.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Plating is dispensable for expressing recombinant GH in Pichia pastoris. Liquid and solid phase selections were performed as described in the results section. For the solid phase selection, three individual clones (numbered 1, 2, 3 on the x axis) were grown each in triplicate in individual wells of a 24-wells deep-well plate. For the liquid phase selection, three individual wells (numbered 1, 2, 3 on the x axis) of a 24-wells deep-well were seeded each with 200 out of 1000 μl of the cell suspension in sorbitol solution after electroporation and assayed in triplicate. Results show the mean enzymatic activity assayed in culture supernatants, with standard deviations of the triplicate assay.
Mentions: We compared both approaches to assess whether having individual clones was critical for our project. X33 cells were transformed with linearized pGAPZαA constructs bearing coding sequences for GH5, GH11, or GH45. After the sorbitol step, 200 μl of cell suspension in sorbitol were plated and then incubated at 30°C for 3 days as usual, and another 200 μl of the same cell suspension in sorbitol were used for liquid phase selection as described in Section “Materials and Methods”. When clones began to appear on agar, they were individually used to inoculate 5 ml YPD-Zeocin and then allowed to grow and express the recombinant protein for 4 days. Expression of recombinant proteins of all solid and liquid phase experimental points was assessed by assaying GH5, GH11, and GH45 enzymatic activities. Results (Figure 2) definitely show that, at least for our three reference proteins, there was no difference between either selection mean. In addition to using less Zeocin than solid phase selection [2 μl in 2 ml YPD instead of 25 μl in 25 ml YPD containing 1.2% agar (YPDA; i.e., the volume poured per plate)], liquid phase selection does not preclude individual clones to be selected on plate if necessary since it uses only 200 out of 1000 μl of the sorbitol suspension of transformed cells and so an additional 200 μl aliquot can be plated and grown in parallel with the liquid phase selection. In conclusion, the liquid phase selection was chosen as default option for our facility because it allows a yes/no answer to be obtained more quickly than solid state screening. In case of negative or poor response, plated clones can be used as a rescue.

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