Methods and systems for chemoautotrophic production of organic compounds

• US 8,349,587 B2
• Filed: 10/31/2011
• Issued: 01/08/2013
• Est. Priority Date: 10/31/2011
• Status: Active Grant

• Current Assignee: Ginkgo Bioworks, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Ginkgo Bioworks, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Fischer, Curt R., Che, Austin J., Shetty, Reshma P., Kelly, Jason R.
• Primary Examiner(s): NOAKES, SUZANNE MARIE
• Application Number:
  US13/285,919
• Publication Number:
  US 20120064622A1
• Time in Patent Office: 435 Days
• Field of Search: None
• US Class Current: 435/41
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0051 acting on a sulfur group of donors (1.8)
• CPC Class Codes: C12P 5/00 Preparation of hydrocarbons or halogenated hydrocarbons
• CPC Class Codes: C12P 7/00 Preparation of oxygen-containing organic compounds
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12P 7/40 containing a carboxyl group including Peroxycarboxylic acids
• CPC Class Codes: C12Y 101/05006 Formate dehydrogenase-N (1.1.5.6)
• CPC Class Codes: C12Y 108/05004 Sulfide:quinone reductase (1.8.5.4)
• 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

Abstract

The present disclosure identifies pathways, mechanisms, systems and methods to confer chemoautotrophic production of carbon-based products of interest, such as sugars, alcohols, chemicals, amino acids, polymers, fatty acids and their derivatives, hydrocarbons, isoprenoids, and intermediates thereof, in organisms such that these organisms efficiently convert inorganic carbon to organic carbon-based products of interest using inorganic energy, such as formate, and in particular the use of organisms for the commercial production of various carbon-based products of interest.

Claims

1. An engineered cell for producing a carbon-based product, comprising:

an at least partially engineered energy conversion pathway having a recombinant formate dehydrogenase or a recombinant sulfide-quinone oxidoreductase introduced into a host cell, wherein

the recombinant formate dehydrogenase is encoded by SEQ ID NO;

1-4 or a homolog thereof having at least 80% sequence identity thereto, or comprises any one of SEQ ID NOs;

5-8 or a homolog thereof having at least 80% sequence identity thereto,

the recombinant sulfide-quinone oxidoreductase is encoded by any one of SEQ ID NOs;

9-16 or a homolog thereof having at least 80% sequence identity thereto, and

said energy conversion pathway is capable of using energy from oxidation to produce a reduced cofactor;

a carbon fixation pathway that is capable of converting inorganic carbon to a carbon-based central metabolite using energy from the reduced cofactor; and

optionally, a carbon product biosynthetic pathway that is capable of converting the carbon-based central metabolite into a carbon-based product of interest.

2. The engineered cell of claim 1, wherein the recombinant formate dehydrogenase reduces NADP⁺.

3. The engineered cell of claim 1, wherein the recombinant formate dehydrogenase reduces NAD⁺.

4. The engineered cell of claim 1, wherein the recombinant formate dehydrogenase reduces ferredoxin.

5. The engineered cell of claim 1, wherein the recombinant sulfide-quinone oxidoreductase reduces quinone.

6. The engineered cell of claim 1, wherein the energy conversion pathway includes the recombinant formate dehydrogenase and the energy from oxidation is from formate oxidation.

7. The engineered cell of claim 1, wherein the energy conversion pathway includes the recombinant sulfide-quinone oxidoreductase and the energy from oxidation is from hydrogen sulfide oxidation.

8. The engineered cell of claim 1, wherein the inorganic carbon is one or more of formate and carbon dioxide.

9. The engineered cell of claim 1, wherein said carbon fixation pathway is at least partially engineered and is derived from the 3-hydroxypropionate (3-HPA) bicycle.

10. The engineered cell of claim 9, wherein said carbon fixation pathway includes one or more of:

acetyl-CoA carboxylase, malonyl-CoA reductase, propionyl-CoA synthase, propionyl-CoA carboxylase, methylmalonyl-CoA epimerase, methylmalonyl-CoA mutase, succinyl-CoA;

(S)-malate CoA transferase, succinate dehydrogenase, fumarate hydratase, (S)-malyl-CoA/β

-methylmalyl-CoA/(S)-citramalyl-CoA lyase, mesaconyl-C1-CoA hydratase or β

-methylmalyl-CoA dehyratase, mesaconyl-CoA C1-C4 CoA transferase and mesaconyl-C4-CoA hydratase.

11. The engineered cell of claim 1, wherein said carbon fixation pathway is at least partially engineered and is derived from the ribulose monophosphate (RuMP) cycle.

12. The engineered cell of claim 11, wherein said carbon fixation pathway includes one or more of:

hexylose-6-phosphate synthase, 6-phospho-3-hexyloisomerase, hexylose-6-phosphate synthase/6-phospho-3-hexyloisomerase fusion enzyme, phosphofructokinase, fructose bisphosphate aldolase, transketolase, transaldolase, transketolase, ribose 5-phosphate isomerase and ribulose-5-phosphate-3-epimerase.

13. The engineered cell of claim 1, wherein said carbon fixation pathway is at least partially engineered and is derived from the Calvin-Benson-Bassham cycle or the reductive pentose phosphate (RPP) cycle.

14. The engineered cell of claim 13, wherein said carbon fixation pathway includes one or more of:

ribulose bisphosphate carboxylase, phosphoglycerate kinase, glyceraldehyde-3P dehydrogenase (phosphorylating), triose-phosphate isomerase, fructose-bisphosphate aldolase, fructose-bisphosphatase, transketolase, sedoheptulose-1,7-bisphosphate aldolase, sedoheptulose bisphosphatase, transketolase, ribose-5-phosphate isomerase, ribulose-5-phosphate-3-epimerase and phosphoribulokinase.

15. The engineered cell of claim 1, wherein said carbon fixation pathway is at least partially engineered and is derived from the reductive tricarboxylic acid (rTCA) cycle.

16. The engineered cell of claim 15, wherein said carbon fixation pathway includes one or more of:

ATP citrate lyase, citryl-CoA synthetase, citryl-CoA lyase, malate dehydrogenase, fumarate dehydratase, fumarate reductase, succinyl-CoA synthetase, 2-oxoglutarate;

ferredoxin oxidoreductase, isocitrate dehydrogenase, 2-oxoglutarate carboxylase, oxalosuccinate reductase, aconitate hydratrase, pyruvate;

ferredoxin oxidoreductase, phosphoenolpyruvate synthetase and phosphoenolpyruvate carboxylase.

17. The engineered cell of claim 1, wherein the host cell is selected from the group consisting of Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Acetobacter aceti, Actinobacillus succinogenes, Mannheimia succiniciproducens, Mesoplasma florum, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Cupriavidus necator (formerly Ralstonia eutropha), Streptomyces coelicolor, Clostridium ljungdahlii, Clostridium thermocellum, Clostridium acetobutylicum, Pseudomonas fluorescens, Pseudomonas putida, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Penicillium chrysogenum and Pichia pastoris.

18. The engineered cell of claim 1, wherein the host cell is Escherichia coli.

19. The engineered cell of claim 12, wherein

the hexylose-6-phosphate synthase is encoded by SEQ ID NO:

46 or 47 or a homolog thereof having at least 80% sequence identity thereto;

or

the 6-phospho-3-hexyloisomerase is encoded by SEQ ID NO;

48 or a homology thereof having at least 80% sequence identity thereto;

or

the hexylose-6-phosphate synthase/6-phospho-3-hexyloisomerase fusion enzyme is encoded by SEQ ID NO;

49 or a homolog thereof having at least 80% sequence identity thereto, or comprises SEQ ID NO;

50 or a homolog thereof having at least 80% sequence identity thereto.