Assessing the fast development of production platforms for renewable chemicals

Transcription

1 Assessing the fast development of production platforms for renewable chemicals Bio World Congress on Industrial Biotechnology 2017, 26 th July, 2017 Kari Koivuranta VTT Technical Research Centre of Finland Ltd

2 VTT Technical Research Centre of Finland Ltd, a leading Technology provider RTO in Europe Customer & VTT Customer Target identification R&D&I Technology development Industrial production Go to market Excellence Impact World-class R&D competence & technologies Long tradition working with industry R&D targeted to respond to customer needs Development of own strategic IPR 2

3 Production platform development from design to pilot Organism selection High throughput screening and selection of the host organism based on desired properties. Organism development Construction of metabolic pathways to product by introducing optimal (heterologous) enzymes. Process development Testing and optimizing productivity, yield and titre by the organism in bioreactor conditions. Scale-up and piloting Piloting at 1200 L. Down-stream processing for purification. Feasibility evaluation. 08/08/2017 3

4 Challenge: faster development of production platforms for bio-chemicals needed Need for speed Lactic Succinic Itaconic Citric Butanol Diols Isoprene Others Organism selection High throughput screening and selection of the host organism based on desired properties. Organism development Construction of metabolic pathways to product by introducing optimal (heterologous) enzymes. Process development Testing and optimizing productivity, yield and titre by the organism in bioreactor conditions. Scale-up and piloting Piloting at 1200 L. Down-stream processing for purification. Feasibility evaluation. 08/08/2017 4

5 Challenge: faster development of production platforms for bio-chemicals needed Need for speed Lactic Succinic Itaconic Citric Butanol Diols Isoprene Others Organism selection High throughput screening and selection of the host organism based on desired properties. Organism development Construction of metabolic pathways to product by introducing optimal (heterologous) enzymes. Process development Production organisms development faster Advanced tools Automation Testing and optimizing productivity, yield and titre by the organism in bioreactor conditions. Scale-up and piloting Piloting at 1200 L. Down-stream processing for purification. Feasibility evaluation. 08/08/2017 5

6 Advanced tools for the development of bio-chemical production organisms Genome engineering of increasing number of organisms is needed Novel organisms are not necessarily well characterized VTT has established A novel gene expression system - SES CRISPR/Cas9 technology for a broad range of eukaryotic microbes 08/08/2017 6

7 A novel synthetic expression system for fungi - SES Modular and tunable expression system Higher expression achieved than by native promoters Growth condition insensitive system 08/08/2017 7

8 SES Modular and tunable expression system Gene expression level can be controlled by the number of binding sites Rantasalo et al., 2016, patent applied 8

9 With SES higher expression achieved than by native promoter S. cerevisiae A. niger T. reesei P. kudriavzevii P. pastoris Y. lipolytica 08/08/2017 9

10 SES is also working in plants: mcherry expression in tobacco 0 BS 1 BS 2 BS 4 BS 8 BS 35S_p 08/08/

11 SES Growth conditions insensitive system Example: T. reesei CBH1 expression No induser needed cheaper process Purer product cost savings in DSP µg Coomassie Total secreted proteins CBH1 11

12 SES also speed up novel strains construction Non-conventional yeast No genome data No genetic modification so far 4 weeks (highly dependent from yeast growth speed) SES system established Exogenous gene expressed 08/08/

13 Advanced tools for the development of bio-chemical production organisms Genome engineering of increasing number of organisms is needed DNA sequence information offers a huge potential VTT has established A novel gene expression system - SES CRISPR/Cas9 technology for a broad range of eukaryotic microbes 08/08/

14 Examples of CRISPR/Cas9 use at VTT in fungal hosts Organism Success S. cerevisiae (polyploid) + Kluyveromyces lactis + Yarrowia lipolytica yeast + Kazachstania exigua (diploid) + Pichia kudriavzevii + Aspergillus niger filamentous fungi Trichoderma reesei filamentous fungi

15 Impact of CRISPR/Cas9 S. cerevisiae 29 days Traditional gene knockout method for 4 genes 7 days CRISPR-mediated quadruple integrants/knock-out simultaneously 2-3 days When removal of the Cas9 plasmid is not required T. reesei 9 months Traditional gene knock-out method for 3 genes 3 months CRISPR-mediated tripple integrants/knock-out simultaneously CRISPR/Cas9 system enables >10X faster engineering of S. cerevisiae strains (4-plex) 3x faster engineering of T. reesei (3-plex) 5-fold improved transformation efficiency for T. reesei 15

16 Advanced strain engineering tools combined with automated screening 16

17 Faster Host Engineering > 20 fungal species engineered at VTT Customer s strains OR Genetic modifications Screening Robot workflow Selection of best producers Validation MTP Bioreactors VTTCC strains Strain selection Modification Testing months > Example of development timeline for a new strain starting from cratch 17

18 To take with you: VTT developed expression system that is independent of growth phase or induction works in wide range of industrially relevant host organisms enables fine tuning of metabolic pathways for biochemicals enables protein production with purer product and lower cost Novel tools shorten the strain development times significantly - up to 10 fold The robotics system established enables screening of strains in a week New tools save time and money! 08/08/

19 Contact us! Kari Koivuranta Principal Scientist Mervi Toivari Principal Scientist Jari Rautio Key Account Manager /08/