HAL Id: hal-01269243
https://hal.archives-ouvertes.fr/hal-01269243
Submitted on 6 Jun 2020
HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
Developping a high-throughput screening method for new tools for lignocellulosic biomass conversion
Cédric Montanier, Marjorie Ochs, Régis Fauré, Michael O’Donohue
To cite this version:
Cédric Montanier, Marjorie Ochs, Régis Fauré, Michael O’Donohue. Developping a high-throughput screening method for new tools for lignocellulosic biomass conversion. Cellulosomes, cellulases and other carbohydrate modifying enzymes, Gordon Research Conferences (GRC). Andover, USA.; Uni- versity of Tokyo. JPN.; University of British Columbia (UBC). CAN., Aug 2015, Andover, United States. �hal-01269243�
LISBP • Laboratoire d’Ingénierie des Systèmes Biologiques et des Procédés
n = H bound/H free DP = n + 1
NMR analysis of the increased tranglycosylation abilities of mutants highlighted
Developping a high-throughput screening method for new tools for lignocellulosic biomass conversion
Background
Marjorie OCHS 1,2,3 , Cédric MONTANIER 1,2,3 , Régis FAURÉ 1,2,3 & Michael O'DONOHUE 1,2,3
Conclusions and outlook Experimental workflow
Generation of error prone PCR xylanase gene library
Use of 4-nitrocatechol β-
D-xylobioside as a substrate to screen yeast colonies in 2-step colorimetric HTS
1st step: donor alone
Donor:
4NTC β-D-xylobioside, colorless
Action of the enzyme:
Complexing of free 4-nitrocatechol (4NTC) with Fe3+ ions
→ Colored dimer
Digital image analysis For each colony:
Determination of the color intensity (T)
Calculation of the ratio R representing the variation of T between the two steps R = (T2nd step-T1st step)/T1st step
2nd step: donor + acceptor
Synthesis of
4-nitrocatechol β- D -xylobioside
Objectives
Develop a new generation of enzymes for the synthesis of commercially-valuable, xylose-based compounds. To achieve this, we use state-of-the-art enzyme engineering to improve xylanase-driven transglycosylation. In this work, our objectives were:
Develop a high-throughput screening (HTS) method to detect xylanases that better catalyze transglycosylation
Create and screen mutant (epPCR) xylanase gene libraries
Analyze and characterize first promising mutant xylanases, notably regarding their ability to catalyze transglycosylation
Results
About 15,000 colonies analyzed in 10 culture medium plates In each plate, 8 colonies having the highest
Transglycosylation/Hydrolysis ratios were selected
Analysis of the ratio distribution showed that among the 80 selected variants:
90% have a ratio >1σ (gaussian distribution) 60% have a ratio >3σ (gaussian distribution)
We have devised an original HTS method to select for endotransglycosylases.
Several potentially promising mutant xylanases displaying lowered hydrolysis were detected.
NMR analysis confirmed that 7 mutants display interesting target properties.
Tests using 1
stgeneration mutants are ongoing to explore their ability to synthesize high DP xylo-oligosaccharides using smaller xylooligosaccharides (obtained from biomass hydrolysates) as donors.
Acknowledgements
This work was supported by the european BIOCORE Project
1- Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France, 2- INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France, 3- CNRS, UMR5504, F-31400 Toulouse, France
Xylanase gene library
(epPCR) Selection medium + 3%
oleic acid
(protein secretion) Integration of library into Y.
lipolytica genome using homologous recombination
Cellulose membrane
The membrane is transferred successively onto two plates containing solid medium and substrates for the 2-step HTS procedure, which is adapted from Koné et al. [3].
[1] A. Ebringerovà, A. Macromolecular Symposia 2005, 232,1, 1-12.
[2] a) N. Kadi and J. Crouzet, Food Chem. 2008, 106, 466-474; b) M. Ochs, M. Muzard, R. Plantier-Royon, B. Estrine and C. Rémond, Green Chem. 2011, 13, 2380-2388.
[3] F. M. T. Koné, M. Le Bechec, J. P. Sine, M. Dion and C. Tellier, Protein Eng. Des. Sel. 2009, 22, 37-44.
References
Secondary plant cell walls are the repositories of renewable lignocellulosic biomass, which is composed of cellulose, hemicelluloses (HC) and lignins. Xylans are the major HC of cell walls of many plants, including grasses and broadleaved trees, representing up to 90% of the HC in cereals and 50% in soft wood[1]. Consequently xylans, which are composed of a main-chain built of D-xylosyl units that can be substituted by a variety of chemical moieties including acetyl groups, α-D-uronic acids and/or α-L-arabinose[1], represent a major source of pentoses in biorefineries. Therefore, the optimal valorization of these is vital for the sustainability of biorefinery processes. In this context, the development of new pentose-based products and the tools and processes to manufacture them is of considerable strategic value.
Xylans can be hydrolyzed by endo-β-1,4-xylanases. These enzymes cleave the glycosidic bond linking two D-xylosyl units in presence of a water molecule acting as an acceptor. However, in the presence of acceptors other than water, certain xylanases display the ability to catalyze transglycosylation reactions, leading to the synthesis of xylose-based products[2], such as xylooligosaccharides or alkylpolypentosides, both of which are potentially valuable molecules that have commercial value. Nevertheless, the yields of enzyme-driven transglycosylation reactions are often low, due to primary competition with hydrolysis and secondary hydrolysis of the synthetic product.
Digital image analysis of individual colonies
Detection of colonies
Determination of the duration of the different steps (time of incubation) Test performed with yeast containing wild-type xylanase gene
1st step after incubation
1 h at 37 °C Before
screening
2nd step after incubation
4 h at 37 °C
2nd step after incubation
15 h at room temperature
Observations and conclusions:
1. A quite uniform coloration of the different colonies is observed
Gaussian distribution
2. Over longer incubation periods
The greyness intensifies thus the R value and the distribution collapses
Screening of a library harboring 1.2 mutations/kilobase
Selected mutant (highest R value) : Test its transglycosylation abilities
Ratio > 3σ = less probability to have
wild-type (WT ) xylanase
0 50 100 150 200 250 300 350
0,25 0,75 1,25 1,75 2,25 2,75 3,25 3,75 4,25 4,75 5,25 5,75 6,25 R value
Number of colonies
Incubation times for the 1st step are fixed at 1 h, and 4 h for the 2nd step
For reactions catalyzed by 7 individual mutants, the average DP of the XOS synthesized was significantly higher compared to the WT xylanase
