STRUCTURING FATS AND METHODS OF PRODUCING STRUCTURING FATS

• US 20200032302A1
• Filed: 04/02/2019
• Published: 01/30/2020
• Est. Priority Date: 04/18/2012
• Status: Active Grant

• Current Assignee: Corbion Biotech, Inc. (Corbion NV)
• Original Assignee: Corbion Biotech, Inc. (Corbion NV)
• Inventors: Franklin, Scott, Somanchi, Aravind, Rudenko, George, Bhat, Riyaz, Zhao, Xinhua, Bond, Risha, Rakitsky, Walter, Marangoni, Alejandro, Braksmayer, Diza
• Granted Patent: US 10,683,522 B2
• Time in Patent Office: Days
• CPC Class Codes: A23D 9/00 Other edible oils or fats, e.g. shortenings, cooking oils
• CPC Class Codes: A23D 9/013 Other fatty acid esters, e.g. phosphatides
• CPC Class Codes: C07C 57/03 Monocarboxylic acids
• CPC Class Codes: C07J 9/00 Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
• CPC Class Codes: C11B 1/00 Production of fats or fatty oils from raw materials
• CPC Class Codes: C12N 1/125 Unicellular algae isolates
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• 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: C12N 9/2402 hydrolysing O- and S- glycosyl compounds (3.2.1)
• CPC Class Codes: C12P 33/00 Preparation of steroids
• CPC Class Codes: C12P 7/64 Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
• CPC Class Codes: C12P 7/6409 Fatty acids
• CPC Class Codes: C12P 7/6445 Glycerides
• 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 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12R 2001/89 : Algae ; Processes using algae
• CPC Class Codes: C12Y 203/01015 : Glycerol-3-phosphate O-acyltransferase (2.3.1.15)
• CPC Class Codes: C12Y 203/0102 : Diacylglycerol O-acyltransferase (2.3.1.20)
• CPC Class Codes: C12Y 203/01041 : Beta-ketoacyl-acyl-carrier-protein synthase I (2.3.1.41)
• CPC Class Codes: C12Y 203/01051 : 1-Acylglycerol-3-phosphate O-acyltransferase (2.3.1.51)
• CPC Class Codes: C12Y 203/01179 : Beta-ketoacyl-acyl-carrier-protein synthase II (2.3.1.179)
• CPC Class Codes: C12Y 203/0118 : Beta-ketoacyl-acyl-carrier-protein synthase III (2.3.1.180)
• CPC Class Codes: C12Y 302/01014 : Chitinase (3.2.1.14)
• 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: Y02P 20/52 : using catalysts, e.g. selective catalysts

Abstract

Recombinant DNA techniques are used to produce oleaginous recombinant cells that produce triglyceride oils having desired fatty acid profiles and regiospecific or stereospecific profiles. Genes manipulated include those encoding stearoyl-ACP desaturase, delta 12 fatty acid desaturase, acyl-ACP thioesterase, ketoacyl-ACP synthase, and lysophosphatidic acid acyltransferase. The oil produced can have enhanced oxidative or thermal stability, or can be useful as a frying oil, shortening, roll-in shortening, tempering fat, cocoa butter replacement, as a lubricant, or as a feedstock for various chemical processes. The fatty acid profile can be enriched in midchain profiles or the oil can be enriched in triglycerides of the saturated-unsaturated-saturated type.

Claims

1-98. -98. (canceled)

99. A microbial oil comprising triglycerides, wherein the microbial oil has an altered fatty acid profile relative to a microbial oil produced by a non-recombinant oleaginous microbe, the microbial oil comprising at least 45% of triglycerides being a combination of palmitic-oleic-palmitic triglyceride, stearic-oleic-stearic triglyceride, and palmitic-oleic-stearic triglyceride, and wherein the oleaginous microbe comprises an exogenous gene encoding ketoacyl-ACP synthase (KAS).

100. The microbial oil of claim 99, wherein the ketoacyl-ACP synthase is selected from the group consisting of KASI, KASII, KASIII or KASIV.

101. The microbial oil of claim 100, wherein the ketoacyl-ACP synthase is KASII.

102. The microbial oil of claim 101, wherein the KASII has at least 95% sequence identity to a protein encoded by nucleotides 4039 to 5328 of SEQ ID NO:

95.

103. The microbial oil of claim 99, wherein the oleaginous microbe further comprises an exogenous gene that encodes a fatty acyl-ACP thioesterase, lysophosphatidic acid acyltransferase (LPAAT), or sucrose invertase.

104. The microbial oil of claim 99, wherein the oleaginous microbe further comprises nucleic acids that is operable to reduce the expression of stearoyl-ACP desaturase or fatty acid desaturase, or fatty acyl-ACP thioesterase.

105. The microbial oil of claim 103, wherein the oleaginous microbe further comprises nucleic acids that are operable to reduce the expression of stearoyl-ACP desaturase or fatty acid desaturase, or fatty acyl-ACP thioesterase.

106. The microbial oil of claim 99, wherein the oleaginous microbe is an oleaginous microalgae.

107. The microbial oil of claim 106, wherein the oleaginous microalgae is of the genus Prototheca.

108. The microbial oil of claim 107, wherein the oleaginous microalge is Prototheca moriformis.

109. A microbial oil comprising triglycerides, wherein the microbial oil comprise:

a. palmitic-oleic-palmitic tryglyceride;

b. stearic-oleic-stearic triglyceride;

c. palmitic-oleic-stearic triglyceride; and

d. ergosterol,

wherein the sum of palmitic-oleic-palmitic tryglyceride, stearic-oleic-stearic triglyceride, and palmitic-oleic stearic triglyceride is at least 45% of the triglycerides in the microbial oil.

110. The microbial oil of claim 109, the microbial oil comprising additional sterols, wherein the most abundant sterol is ergosterol.

111. The microbial oil of claim 109, wherein the triglycerides comprise at least 50% C18:

0 fatty acids and less than 10% C16;

0.

112. The microbial oil of claim 109, wherein the ketoacyl-ACP synthase is selected from the group consisting of KASI, KASII, KASIII or KASIV.

113. The microbial oil of claim 112, wherein the ketoacyl-ACP synthase is KASII.

114. The microbial oil of claim 113, wherein the KASII has at least 95% sequence identity to a protein encoded by nucleotides 4039 to 5328 of SEQ ID NO:

95.

115. The microbial oil of claim 109, wherein the oleaginous microbe further comprises an exogenous gene that encodes a fatty acyl-ACP thioesterase, lysophosphatidic acid acyltransferase (LPAAT), or sucrose invertase.

116. The microbial oil of claim 109, wherein the oleaginous microbe further comprises nucleic acids that is operable to reduce the expression of stearoyl-ACP desaturase or fatty acid desaturase, or fatty acyl-ACP thioesterase.

117. The microbial oil of claim 116, wherein the oleaginous microbe further comprises nucleic acids that are operable to reduce the expression of stearoyl-ACP desaturase or fatty acid desaturase, or fatty acyl-ACP thioesterase.

118. The microbial oil of claim 109, wherein the oleaginous microbe is an oleaginous microalgae.

119. The microbial oil of claim 118, wherein the oleaginous microalgae is of the genus Prototheca.

120. The microbial oil of claim 119, wherein the oleaginous microalgae is Prototheca moriformis.