Use of genetically modified organisms to generate biomass degrading enzymes
View Patent ↗The present invention provides a method and compositions for high throughput screening of genetically modified photosynthetic organisms for plasmic state. The present invention provides methods of producing one or more proteins, including biomass degrading enzymes in a plant. Also provided are the methods of producing biomass degradation pathways in alga cells, particularly in the chloroplast. Single enzymes or multiple enzymes may be produced by the methods disclosed. The methods disclosed herein allow for the production of biofuel, including ethanol.
1. A composition comprising one or more non-vascular photosynthetic eukaryotic organisms having one or more exogenous biomass degrading enzymes selected from the group consisting of exo-β-glucanase, endo-β-glucanase, β-glucosidase, endoxylanase, and lignase.
2. The composition of claim 1 , wherein said composition comprises one or more crushed cells of said one or more non-vascular photosynthetic eukaryotic organisms.
3. The composition of claim 1 , wherein said one or more biomass degrading enzymes comprise 10% or higher of total protein in said composition.
4. A method for preparing an alcohol biofuel comprising: treating a biomass with a non-vascular photosynthetic eukaryotic organism having one or more exogenous biomass degrading enzymes selected from the group consisting of an exo-β-glucanase, endo-β-glucanase, β-glucosidase, endoxylanase, and lignase for a sufficient amount of time to degrade at least a portion of said biomass.
5. The method of claim 4 , wherein said eukaryotic organism is added to a saccharification tank.
6. The method of claim 4 , wherein said biofuel is ethanol.
7. The composition of claim 1 , wherein said one or more non-vascular photosynthetic eukaryotic organisms comprise photosynthetic algae.
8. The method of claim 4 , wherein said non-vascular photosynthetic eukaryotic organism comprises a photosynthetic alga.
9. The composition of claim 1 , wherein said one or more exogenous biomass degrading enzymes are selected from the group consisting of exo-β-glucanase from Trichoderma viride , exo-β-glucanase in from Trichoderma reesei , exo-β-glucanase from Aspergillus aculeatus , endo-β-glucanase from Trichoderma reesei , endo-β-glucanase from Aspergillus niger , β-glucosidase from Trichoderma reesei , β-glucosidase from Aspergillus niger endoxylanase from Trichoderma reesei , and endoxylanase from Aspergillus niger.
10. The method of claim 4 , wherein said one or more exogenous biomass degrading enzymes are selected from the group consisting of exo-β-glucanase from Trichoderma viride , exo-β-glucanase from Trichoderma reesei , exo-β-glucanase from Aspergillus aculeatus , endo-β-glucanase from Trichoderma reesei , endo-β-glucanase from Aspergillus niger , β-glucosidase from Trichoderma reesei , β-glucosidase in from Aspergillus niger , endoxylanase from Trichoderma reesei , and endoxylanase from Aspergillus niger.
11. The composition of claim 1 , wherein said photosynthetic non-vascular eukaryotic organism is selected from the group consisting of C. reinhardtii, D. salina, H. pluvialis, rhodophyta, chlorophyta, heterokontophyta, tribophyta, glaucophyta, chlorarachniophytes, euglenophyta, euglenoids, haptophyta, chrysophyta, cryptophyta, cryptomonads, dinophyta, dinoflagellata, pyrmnesiophyta, bacillariophyta, xanthophyta, eustigmatophyta, raphidophyta , and phaeophyta.
12. The method of claim 4 , wherein said photosynthetic non-vascular eukaryotic organism is selected from the group consisting of C. reinhardtii, D. salina, H. pluvialis, rhodophyta, chlorophyta, heterokontophyta, tribophyta, glaucophyta, chlorarachniophytes, euglenophyta, euglenoids, haptophyta, chrysophyta, cryptophyta, cryptomonads, dinophyta, dinoflagellata, pyrmnesiophyta, bacillariophyta, xanthophyta, eustigmatophyta, raphidophyta , and phaeophyta.
13. The composition of claim 9 , wherein said endoxylanase from Trichoderma reesei comprises the amino acid sequence:
(NCBI Accession No. CAA49293)
MVSFTSLLAASPPSRASCRPAAEVESVAVEKRQTIQPGTGYNNGYFYSYWN
DGHGGVTYTNGPGGQFSVNWSNSGNFVGGKGWQPGTKNKVINFSGSYNPNG
NSYLSVYGWSRNPLIEYYIVENFGTYNPSTGATKLGEVTSDGSVYDIYRTQ
RVNQPSIIGTATFYQYWSVRRNHRSSGSVNTANHFNAWAQQGLTLGTMDYQ
IVAVEGYFSSGSASITVS;
or
(NCBI Accession No. CAA49294)
MVAFSSLICALTSIASTLAMPTGLEPESSVNVTERGMYDFVLGAHNDHRRR
ASINYDQNYQTGGQVSYSPSNTGFSVNWNTQDDFVVGVGWTTGSSAPINFG
GSFSVNSGTGLLSVYGWSTNPLVEYYIMEDNHNYPAQGTVKGTVTSDGATY
TIWENTRVNEPSIQGTATFNQYISVRNSPRTSGTVTVQNHFNAWASLGLHL
GQMNYQVVAVEGWGGSGSASQSVSN.
14. The method of claim 10 , wherein said endoxylanase from Trichoderma reesei comprises the amino acid sequence:
(NCBI Accession No. CAA49293)
MVSFTSLLAASPPSRASCRPAAEVESVAVEKRQTIQPGTGYNNGYFYSYWN
DGHGGVTYTNGPGGQFSVNWSNSGNFVGGKGWQPGTKNKVINFSGSYNPNG
NSYLSVYGWSRNPLIEYYIVENFGTYNPSTGATKLGEVTSDGSVYDIYRTQ
RVNQPSIIGTATFYQYWSVRRNHRSSGSVNTANHFNAWAQQGLTLGTMDYQ
IVAVEGYFSSGSASITVS;
or
(NCBI Accession No. CAA49294)
MVAFSSLICALTSIASTLAMPTGLEPESSVNVTERGMYDFVLGAHNDHRRR
ASINYDQNYQTGGQVSYSPSNTGFSVNWNTQDDFVVGVGWTTGSSAPINFG
GSFSVNSGTGLLSVYGWSTNPLVEYYIMEDNHNYPAQGTVKGTVTSDGATY
TIWENTRVNEPSIQGTATFNQYISVRNSPRTSGTVTVQNHFNAWASLGLHL
GQMNYQVVAVEGWGGSGSASQSVSN.
15. The composition of claim 7 , wherein said photosynthetic algae is a member of the chlorophyta.
16. The composition of claim 15 , wherein said member of the chlorophyta is selected from the group consisting of C. reinhardtii, D. salina , and H. pluvialis.
17. The method of claim 8 , wherein said photosynthetic alga is a member of the chlorophyta.
18. The method of claim 17 , wherein said member of the chlorophyta is selected from the group consisting of C. reinhardtii, D. salina , and H. pluvialis.
19. A composition comprising one or more non-vascular photosynthetic plastid containing organisms having one or more exogenous biomass degrading enzymes selected from the group consisting of exo-β-glucanase, endo-β-glucanase, β-glucosidase, endoxylanase, and lignase.
20. The composition of claim 19 , wherein said composition comprises one or more crushed cells of said one or more non-vascular photosynthetic eukaryotic organisms.
21. The composition of claim 19 , wherein said one or more biomass degrading enzymes comprise 10% or higher of total protein in said composition.
22. The composition of claim 19 , wherein said one or more non-vascular photosynthetic plastid containing organisms comprise photosynthetic algae.
23. The composition of claim 19 , wherein said one or more exogenous biomass degrading enzymes are selected from the group consisting of exo-β-glucanase from Trichoderma viride , exo-β-glucanase from Trichoderma reesei , exo-β-glucanase from Aspergillus aculeatus , endo-β-glucanase from Trichoderma reesei , endo-β-glucanase from Aspergillus niger , β-glucosidase from Trichoderma reesei , β-glucosidase from Aspergillus niger , endoxylanase from Trichoderma reesei , and endoxylanase from Aspergillus niger.
24. The composition of claim 19 , wherein said photosynthetic non-vascular plastid containing organism is selected from the group consisting of C. reinhardtii, D. salina, H pluvialis, rhodophyta, chlorophyta, heterokontophyta, tribophyta, glaucophyta, chlorarachniophytes, euglenophyta, euglenoids, haptophyta, chrysophyta, cryptophyta, cryptomonads, dinophyta, dinoflagellata, pyrmnesiophyta, bacillariophyta, xanthophyta, eustigmatophyta, raphidophyta , and phaeophyta.
25. The composition of claim 19 , wherein said plastid is a chloroplast.
26. The composition of claim 24 , wherein said photosynthetic non-vascular plastid containing organism is a member of the chlorophyta.
27. The composition of claim 26 , wherein said member of the chlorophyta is selected from the group consisting of C. reinhardtii, D. salina , and H. pluvialis.
28. A method for preparing an alcohol biofuel comprising: treating a biomass with a non-vascular photosynthetic plastid containing organism having one or more exogenous biomass degrading enzymes selected from the group consisting of exo-β-glucanase, endo-β-glucanase, β-glucosidase, endoxylanase, and lignase for a sufficient amount of time to degrade at least a portion of said biomass.
29. The method of claim 28 , wherein said plastid containing organism is added to a saccharification tank.
30. The method of claim 28 , wherein said biofuel is ethanol.
31. The method of claim 28 , wherein said non-vascular photosynthetic plastid containing organism comprises a photosynthetic alga.
32. The method of claim 28 , wherein said photosynthetic alga is a member of the chlorophyta.
33. The method of claim 32 , wherein said member of the chlorophyta is selected from the group consisting of C. reinhardtii, D. salina, H. pluvialis.
34. The method of claim 28 , wherein said one or more exogenous biomass degrading enzymes are selected from the group consisting of exo-β-glucanase from Trichoderma viride , exo-β-glucanase from Trichoderma reesei , exo-β-glucanase from Aspergillus aculeatus , endo-β-glucanase from Trichoderma reesei , endo-β-glucanase from Aspergillus niger , β-glucosidase from Trichoderma reesei , β-glucosidase from Aspergillus niger , endoxylanase from Trichoderma reesei , and endoxylanase from Aspergillus niger.
35. The method of claim 28 , wherein said photosynthetic non-vascular plastid containing organism is selected from the group consisting of C. reinhardtii, D. salina, H. pluvialis, rhodophyta, chlorophyta, heterokontophyta, tribophyta, glaucophyta, chlorarachniophytes, euglenophyta, euglenoids, haptophyta, chrysophyta, cryptophyta, cryptomonads, dinophyta, dinoflagellata, pyrmnesiophyta, bacillariophyta, xanthophyta, eustigmatophyta, raphidophyta , and phaeophyta.
36. The method of claim 34 , wherein said endoxylanase from Trichoderma reesei comprises the amino acid sequence:
MVSFTSLLAASPPSRASCRPAAEVESVAVEKRQTIQPGTGYNNGYFYSYW
NDGHGGVTYTNGPGGQFSVNWSNSGNFVGGKGWQPGTKNKVINFSGSYNP
NGNSYLSVYGWSRNPLIEYYIVENFGTYNPSTGATKLGEVTSDGSVYDIY
RTQRVNQPSIIGTATFYQYWSVRRNHRSSGSVNTANHFNAWAQQGLTLGT
MDYQIVAVEGYFSSGSASITVS (NCBI Accession No.
CAA49293); or
MVAFSSLICALTSIASTLAMPTGLEPESSVNVTERGMYDFVLGAHNDHRR
RASINYDQNYQTGGQVSYSPSNTGFSVNWNTQDDFVVGVGWTTGSSAPIN
FGGSFSVNSGTGLLSVYGWSTNPLVEYYIMEDNHNYPAQGTVKGTVTSDG
ATYTIWENTRVNEPSIQGTATFNQYISVRNSPRTSGTVTVQNHFNAWASL
GLHLGQMNYQVVAVEGWGGSGSASQSVSN (NCBI Accession No.
CAA49294).