Production of hydroxylated fatty acids in

• US 8,852,885 B2
• Filed: 07/12/2012
• Issued: 10/07/2014
• Est. Priority Date: 02/02/2011
• Status: Active Grant

• Current Assignee: Corbion Biotech, Inc. (Corbion NV)
• Original Assignee: Solazyme Inc.
• Inventors: Franklin, Scott, Somanchi, Aravind, Wee, Janice, Rudenko, George, Moseley, Jeffrey L., Rakitsky, Walt, Zhao, Xinhua, Bhat, Riyaz
• Primary Examiner(s): Fox, David T
• Assistant Examiner(s): KEOGH, MATTHEW R
• Application Number:
  US13/547,457
• Publication Number:
  US 20120277452A1
• Time in Patent Office: 817 Days
• Field of Search: None
• US Class Current: 435/58
• CPC Class Codes: C07C 69/52 Esters of acyclic unsaturated carboxylic acids having the esterified carboxyl group bound to an acyclic carbon atom
• CPC Class Codes: C10L 1/02 essentially based on components consisting of carbon, hydrogen, and oxygen only
• CPC Class Codes: C10L 2200/0476 Biodiesel, i.e. defined lower alkyl esters of fatty acids first generation biodiesel
• CPC Class Codes: C10L 2290/26 Composting, fermenting or anaerobic digestion fuel components or materials from which fuels are prepared
• CPC Class Codes: C10L 2290/30 Pressing, compressing or compacting
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12P 7/6463 obtained from glyceride producing microorganisms, e.g. single cell oil
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Methods and compositions for the production of oil, fuels, oleochemicals, and other compounds in recombinant microorganisms are provided, including oil-bearing microorganisms and methods of low cost cultivation of such microorganisms. Microalgal cells containing exogenous genes encoding, for example, a lipase, a sucrose transporter, a sucrose invertase, a fructokinase, a polysaccharide-degrading enzyme, a keto acyl-ACP synthase enzyme, a fatty acyl-ACP thioesterase, a fatty acyl-CoA/aldehyde reductase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, a fatty acid hydroxylase, a desaturase enzyme, a fatty aldehyde decarbonylase, and/or an acyl carrier protein are useful in manufacturing transportation fuels such as renewable diesel, biodiesel, and renewable jet fuel, as well as oleochemicals such as functional fluids, surfactants, soaps and lubricants.

Claims

1. A method for using sucrose to produce an oil comprising triacylglycerides that comprise ricinoleic acid, or a product produced from the oil, the method comprising:

cultivating a cell of a recombinant oleaginous microalga of the species Prototheca moriformis, the cell comprising;

nucleic acids operable to enhance oleic acid production so as to elevate the substrate levels for an exogenous oleate 12-hydroxylase by expressing a product of an exogenous gene encoding an active stearoyl ACP desaturase and decreasing or eliminating the expression of an enzyme encoded by one or more endogenous genes that encode a fatty acid desaturase by directly targeting said endogenous gene(s) or RNA products of said gene(s); and

an exogenous nucleic acid operable to express the active oleate 12-hydroxylase, so as to synthesize the ricinoleic acid; and

an exogenous nucleic acid operable to express an active invertase; and

feeding sucrose to the cell so as to produce the oil comprising triacylglycerides comprising ricinoleic acid, wherein the ricinoleic acid is detectable by gas chromatography-mass spectrometry.

2. The method of claim 1, wherein the cell produces at least 40% oleic acid.

4. The method of claim 1, wherein the oleate 12-hydroxylase is from Ricinus communis, Physaria lindheimeri, or Arabidopsis lyrata.

5. The method of claim 1, wherein the oleate 12-hydroxylase has an amino acid sequence corresponding to any of SEQ ID NOs 259-262.

3. A microalga cell of the species Prototheca moriformis, the cell comprising:

nucleic acids operable to enhance oleic acid production so as to elevate the substrate levels for an exogenous oleate 12-hydroxylase by expressing a product of an exogenous gene encoding an active stearoyl ACP desaturase and decreasing or eliminating the expression of an enzyme encoded by one or more endogenous genes that encode a fatty acid desaturase by directly targeting said endogenous gene(s) or RNA products of said gene(s);

an exogenous gene encoding the active oleate 12-hydroxylase; and

an exogenous gene encoding an invertase so as to enable the cell to use sucrose to synthesize ricinoleic acid in an amount detectable by gas chromatography-mass spectrometry.

6. The method of claim 3, wherein the oleate 12-hydroxylase is from Ricinus communis, Physaria lindheimeri, or Arabidopsis lyrata.

7. The method of claim 3, wherein the oleate 12-hydroxylase has an amino acid sequence corresponding to any of SEQ ID NOs 259-262.