Droplet microfluidic for high-throughput screening of microorganisms based on enzymatic activities From bacteria to fungi communities

Transcription

1 Droplet microfluidic for high-throughput screening of microorganisms based on enzymatic activities From bacteria to fungi communities

2 Introduction 1 Majdi Najah I Putu Mahendra Wijaya Prexilia Postros Pascal Leblond Angélique Couvent Estelle Mayot Antoine Drevelle Thomas Beneyton Raphaël Calbrix Andrew D. Griffiths Context Make use of cereal industry waste products Fermentation processes to generate highly valuable products Hydrolytic enzymes for the degradation of lignocellulosic biomass Xylanase, cellulase, amylase, protease

3 Introduction 2 Strategies to find new enzymatic activities High-Throughput Screening to go through large diversity Wheat bran

4 Introduction 3 High-throughtput screening (HTS) based on enzymatic activties Microtiter plates (HTS) Droplet microfluidics (HTS) Spatial confinement (Genotype/Phenotype) Wide choice of enzymatic assays ID tracking of the sample Volume (µl ml) Limited throughput (up to 1 Hz) High cost and space footprints Smaller volume (pl nl) Higher throughput (10 to Hz) Smaller cost and space footprints Adapt the enzymatic assay No ID tracking of the sample Larger libraries [Mayr and Bojanic, Curr. Opin. Pharmacol., 2009, 9, 580] [Theberge et al., Angew. Chem. Int. Ed. Eng., 2010, 49, 5846] [Abate et al., Lab. Chip., 2015, 15, 47]

5 Droplet-based microfluidics for HTS 4 Droplets production at khz frequencies 100 µm Fluorescence-Activated Droplet Sorting Fluorescent assay

6 From bacteria to fungi communities 5 Bacteria Yeast Filamentous fungi 1-5µm 5-20µm 100 µm to m pl droplets Bioprospection for cellulolytic microorganisms

7 Speeding up Bioprospection for cellulolytic microorganisms 6 [Najah and Calbrix et al., Chem. Biol., 2014, 21, 1722] Fluorogenic substrate Top 2% sorted 10 5 bacteria screened without prior cultivation 2000 bacteria selected Cellobiose Cellobiohydrolase 6,8-difluoro-7-hydroxycoumarin -4-methanesulfonate [Najah et al., Anal. Chem, 2013, 85, 9807]

8 Speeding up Bioprospection for cellulolytic microorganisms 7 [Najah and Calbrix et al., Chem. Biol., 2014, 21, 1722] 0.03% 0.43% 17-fold higher cellulase actvity

9 Speeding up Bioprospection for cellulolytic microorganisms 8 [Najah and Calbrix et al., Chem. Biol., 2014, 21, 1722] Analysis of the taxonomy diversity via 16S rdna deep sequencing Access to different biodivesity (cultivable cells <1% of the microbial diversity) [Torsvik and Ovreas, Curr. Opin. Microbiol., 2002, 5, 240]

10 From bacteria to fungi communities 9 Bacteria Yeast Filamentous fungi 1-5µm 5-20µm 100 µm to m pl droplets 10 nl droplets 10 µm

11 HTS of Aspergillus Niger for secreted a-amylase activity 10 Growing A. Niger in 18nL droplets for 32h 250pL [Beneyton et al., Submitted] a-amylase fluorescent assay 18nL 24h 18nL 32h 18nL 24h

12 HTS of Aspergillus Niger for secreted a-amylase activity 10 Growing A. Niger in 18nL droplets for 32h 250pL [Beneyton et al., Submitted] a-amylase fluorescent assay 18nL 24h 18nL 32h 18nL 24h Sorting 10-20nL droplets at 10Hz 500 µm

13 HTS of Aspergillus Niger for secreted a-amylase activity 11 HTS microfluidic platform [Beneyton et al., Submitted] 200 µm

14 HTS of Aspergillus Niger for secreted a-amylase activity 12 Screening of whole-genome UV-mutated libraries starting from 058 mother strain [Beneyton et al., Submitted] O fungi UV library fungi

15 HTS of Aspergillus Niger for secreted a-amylase activity fungi screened in 90min [Beneyton et al., Submitted] 620 fungi sorted 1262 fungi screened in 8 days 10 fungi screened in 6 days Sorted fungi in droplet (144) (1262) 196-fold enrichment 98.1% of active clones 2.3-fold improvement (M8) Library size Droplet microfluidics Microtiter plate Shake-flask

16 Conclusion 14 Droplet-based microfluidics to screen microorganisms based on industrially relevant enzymatic activities - Higher thoughputs, lower cost and space footprints: larger libraries accessible for functional screening - Bioprospection of bacteria: access to new diversity as uncultivable bacteria can be analyzed - Screening of filamentous fungi for secreted enzymatic activities Direct integration in industrial screening processes 1. Libraries construction: rapid analysis of phenotype distributions 2. Initial screening step of the process Library size Droplet microfluidics Microtiter plate Flask HITS

17 Acknowledgements 15 Majdi Najah I Putu Mahendra Wijaya Prexilia Postros Pascal Leblond Angélique Couvent Estelle Mayot Antoine Drevelle Raphaël Calbrix Andrew D. Griffiths Thank you for your attention