Heterotrophic microalgae expressing invertase

• US 8,497,116 B2
• Filed: 12/03/2010
• Issued: 07/30/2013
• Est. Priority Date: 06/01/2007
• Status: Active Grant

• Current Assignee: Corbion Biotech, Inc. (Corbion NV)
• Original Assignee: Solazyme Inc.
• Inventors: Trimbur, Donald E., Im, Chung-Soon, Dillon, Harrison F., Day, Anthony G., Franklin, Scott, Coragliotti, Anna
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US12/960,388
• Publication Number:
  US 20120122192A1
• Time in Patent Office: 970 Days
• Field of Search: None
• US Class Current: 435/257.1
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C10L 1/026 for compression ignition
• CPC Class Codes: C10L 1/06 for spark ignition
• CPC Class Codes: C10L 2200/0484 Vegetable or animal oils
• CPC Class Codes: C10L 2270/026 for diesel engines, e.g. automobiles, stationary, marine
• CPC Class Codes: C10L 2270/04 for turbines, planes, power generation
• CPC Class Codes: C11C 3/00 Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom sulfonated fats or oils C07C309/62; factice C08H; drying oils C09F
• CPC Class Codes: C12N 1/12 Unicellular algae; Culture media therefor as new plants A01H13/00
• CPC Class Codes: C12N 1/32 Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12N 9/14 Hydrolases (3)
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/20 Triglyceride splitting, e.g. by means of lipase
• CPC Class Codes: C12N 9/2408 acting on alpha -1,4-glucosidic bonds
• CPC Class Codes: C12N 9/2431 Beta-fructofuranosidase (3.2.1.26), i.e. invertase
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00
• CPC Class Codes: C12P 5/02 : acyclic C12P5/007 takes precedence
• CPC Class Codes: C12P 7/6409 : Fatty acids
• CPC Class Codes: C12P 7/6458 : by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
• CPC Class Codes: C12P 7/6463 : obtained from glyceride producing microorganisms, e.g. single cell oil
• CPC Class Codes: C12P 7/649 : Biodiesel, i.e. fatty acid alkyl esters
• CPC Class Codes: C12Y 302/01026 : Beta-fructofuranosidase (3.2.1.26), i.e. invertase
• CPC Class Codes: Y02E 50/10 : Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02E 50/30 : Fuel from waste, e.g. synthetic alcohol or diesel
• CPC Class Codes: Y02P 20/52 : using catalysts, e.g. selective catalysts
• CPC Class Codes: Y02T 50/678 : Aviation using fuels of non-fossil origin

Abstract

The invention provides methods of manufacturing oils and oil-based products such as transportation fuels, industrial chemicals, lubricants and plastics using sugar cane, sugar cane juice, corn steep liquor, corn starch or depolymerized cellulosic material as a feedstock for bioproduction processes. Oils are manufactured using microalgae expressing sucrose invertase, which allows the microalgae to produce oil using sucrose-based feedstocks, along or in combination with other feedstocks.

Claims

1. A composition comprising microalgae grown under heterotrophic conditions, wherein the microalgae comprise an exogenous sucrose invertase gene that is codon-optimized for expression in Chlorella protothecoides.

2. The composition of claim 1, wherein the microalgae comprise at least 45% lipid by dry cell weight.

3. The composition of claim 1, wherein the microalgae have a lipid profile of approximately 3% C18:

0, 71% C18;

1, 15% C18;

2, 1% C18;

3, and 8% C16;

0.

4. The composition of claim 1, wherein the microalgae are selected from Table 1.

5. The composition of claim 4, wherein the microalgae is a species of the genus Chlorella or Prototheca.

6. The composition of claim 5, wherein the microalgae is selected from the group consisting of Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella pyrenoidosa, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella, Chlorella zofingiensis, Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, and Prototheca zopfii.

7. The composition of claim 5, wherein the microalgae is Chlorella protothecoides or Prototheca moriformis.

8. The composition of claim 1, wherein the heterotrophic conditions comprise culturing the microalgae in a culture medium with at least one fixed carbon source selected from glycerol, sucrose, glucose, arabinose, galactose, xylose, fructose, mannose or acetate.

9. The composition of claim 1, wherein the heterotrophic conditions comprise culturing the microalgae in a culture medium with at least one fixed carbon source selected from corn starch, corn steep liquor or depolymerized cellulosic material.

10. The composition of claim 1, wherein the heterotrophic conditions comprise culturing the microalgae in a culture medium comprising sucrose and a sucrose invertase enzyme.

11. The composition of claim 10, wherein the sucrose is provided by a carbon source selected from the group consisting of sorghum, sugar cane juice, sugar beet and molasses.

12. The composition of claim 1, wherein the sucrose invertase is codon optimized according to:

TTC (Phe) TAC (Tyr) TGC (Cys) TGA (Stop) TGG (Trp) CCC (Pro) CAC (His) CGC (Arg) CTG (Leu) CAG (Gln) ATC (Ile) ACC (Thr) GAC (Asp) TCC (Ser) ATG (Met) AAG (Lys) GCC (Ala) AAC (Asn) GGC (Gly) GTG (Val) GAG (Glu).

13. The composition of claim 1, wherein the invertase gene is SUC2.

14. The composition of claim 1, wherein the invertase gene comprises the nucleotide sequence of SEQ ID NO:

8.

15. The composition of claim 1, wherein the invertase comprises the amino acid sequence of SEQ ID NO:

19.

16. A method of producing a composition comprising microalgal biomass, wherein the method comprises the steps of:

(a) providing a microalgal cell capable of heterotrophic growth on sucrose as a sole carbon source by virtue of an active sucrose invertase gene codon optimized for expression in Chlorella protothecoides;

(b) placing the microalgal cell in a culture medium comprising at least one fixed carbon source; and

(c) culturing the microalgal cell under heterotrophic growth conditions until the microalgal cell comprises at least 40% of its dry cell weight as lipid.

17. The method of claim 16, wherein the microalgal cell comprises at least 45% lipid by dry cell weight.

18. The method of claim 16, wherein the microalgal cell has a lipid profile of approximately 3% C18:

0, 71% C18;

1, 15% C18;

2, 1% C18;

3, and 8% C16;

0.

19. The method of claim 16, wherein the microalgal cell is selected from Table 1.

20. The method of claim 19, wherein the microalgal cell is a species of the genus Chlorella or Prototheca.

21. The method of claim 20, wherein the microalgal cell is selected from the group consisting of Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella pyrenoidosa, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella, Chlorella zofingiensis, Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, and Prototheca zopfii.

22. The method of claim 20, wherein the microalgal cell is Chlorella protothecoides or Prototheca moriformis.

23. The method of claim 16, wherein the at least one fixed carbon source is selected from glycerol, sucrose, glucose, arabinose, galactose, xylose, fructose, mannose or acetate.

24. The method of claim 16, wherein the at least one fixed carbon source is selected from corn starch, corn steep liquor or depolymerized cellulosic material.

25. The method of claim 16, wherein the culture medium comprises sucrose.

26. The method of claim 25, wherein the sucrose is provided by a carbon source selected from the group consisting of sorghum, sugar cane juice, sugar beet and molasses.

27. The method of claim 16 further comprising the step of lysing the microalgal cell after step (c).

28. The method of claim 27, wherein the lysing of the microalgal cell is performed through mechanical lysis.

29. The method of claim 16 further comprising the step of drying the microalgal cell after step (c).

30. The method of claim 16 further comprising the step of washing the microalgal cell after step (c).

31. The method of claim 16 further comprising one or more of lysing, drying, and washing the microalgal cell after step (c).

32. The method of claim 16, wherein the sucrose invertase is codon optimized according to:

TTC (Phe) TAC (Tyr) TGC (Cys) TGA (Stop) TGG (Trp) CCC (Pro) CAC (His) CGC (Arg) CTG (Leu) CAG (Gln) ATC (Ile) ACC (Thr) GAC (Asp) TCC (Ser) ATG (Met) AAG (Lys) GCC (Ala) AAC (Asn) GGC (Gly) GTG (Val) GAG (Glu).

33. The method of claim 16, wherein the invertase gene is SUC2.

34. The method of claim 16, wherein the invertase gene comprises the nucleotide sequence of SEQ ID NO:

8.

35. The method of claim 16, wherein the invertase comprises the amino acid sequence of SEQ ID NO:

19.