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Oleaginous microalgae having an LPAAT ablation

A technology of microalgal cells and cell oil, applied in DNA/RNA fragments, microorganisms, single-celled algae, etc., can solve problems such as cell division inhibition

Inactive Publication Date: 2018-04-24
CORBION BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The oleaginous non-photosynthetic alga Prototheca moriformis stores large amounts of triacylglycerides under conditions of excess supply of nutrient carbon, but cell division is inhibited due to limitation of other essential nutrients

Method used

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  • Oleaginous microalgae having an LPAAT ablation
  • Oleaginous microalgae having an LPAAT ablation
  • Oleaginous microalgae having an LPAAT ablation

Examples

Experimental program
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Effect test

example 1

[0318] Example 1: Fatty acid analysis by fatty acid methyl ester detection

[0319] Prepare lipid samples from dried biomass. Resuspend 20-40mg of dry biomass in 2mL containing 5% H 2 SO 4 And add 200μl of toluene containing an appropriate amount of suitable internal standards (C19:0). The mixture was sonicated briefly to disperse the biomass, and then heated at 70°C-75°C for 3.5 hours. Add 2mL of heptane to extract fatty acid methyl esters, followed by 2mL of 6% K 2 CO 3 (Aqueous solution) to neutralize acid. The mixture was stirred vigorously, and a part of the upper layer was transferred to 2 SO 4 The (anhydrous) vial is used for gas chromatographic analysis using standard FAME GC / FID (Fatty Acid Methyl Ester Gas Chromatographic Flame Ionization Detection) method. This method is used to determine the fatty acid profile reported below.

example 2

[0320] Example 2: Engineering the microorganisms for fatty acid and sn-2 profiles to increase lauric acid through exogenous LPAAT expression

[0321] This example describes the use of multiple recombinant polynucleotides encoding coconut 1-acyl-sn-glycerol-3-phosphate acyltransferase (Cn LPAAT) to engineer a microorganism. The fatty acid profile and sn- 2 spectrum is already rich in lauric acid.

[0322] According to the bioprojectile transformation method described in PCT / US 2009 / 066141, PCT / US 2009 / 066142, PCT / US 2011 / 038463, PCT / US2011 / 038464 and PCT / US 2012 / 023696, the plasmid construct pSZ1283 was initially used. The classically mutagenic strain of transformed mulberry-type Prototheca (UTEX 1435), strain A. The pSZ1283 described in PCT / US 2011 / 038463, PCT / US2011 / 038464, and PCT / US 2012 / 023696 (incorporated herein by reference) comprises Wright calyx distance flower (Cuphea wrightii) FATB2 (CwTE2) thioesterase (SEQ ID NO: 10); the 5'(SEQ ID NO: 1) and 3'(SEQ ID NO: 2) homolog...

example 3

[0336] Example 3: Analysis of region-specific spectrum

[0337] LC / MS TAG distribution analysis was performed using a Shimadzu Nexera ultra-high performance liquid chromatography system coupled to a Shimadzu LCMS 8030 triple quadrupole mass spectrometer equipped with an APCI source. The system includes a SIL -30AC autosampler, two LC-30AD pumps, a DGU-20A5 online degasser and a CTO-20A column thermostat. The data was collected using a Q3 scan of m / z350-1050 at a scan speed of 1428u / sec in the positive ion mode where the CID gas (argon) pressure was set to 230kPa. The APCI, desolvation tube, and heating block temperature were set to 300°C, 250°C, and 200°C, respectively, the spray and drying gas flow rates were 3.0L / min and 5.0L / min, and the interface voltage was 4500V. The oil sample was dissolved in dichloromethane-methanol (1:1) to a concentration of 5 mg / mL, and 0.8 μL of the sample was injected into Shim-pack XR-ODS III maintained at 30°C (2.2μm, 2.0×200mm). At 0.48mL / min,...

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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, lysophosphatidic acid acyltransferase, ketoacyl-CoA reductase, hydroxyacyl-CoA dehydratase, and / or enoyl-CoA reductase. 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.

Description

[0001] Cross references to related applications [0002] This application claims the rights of U.S. Provisional Patent Application No. 62 / 143,711 filed on April 6, 2015 and U.S. Provisional Patent Application No. 62 / 145,723 filed on April 10, 2015 in accordance with 35 USC 119(e), these patents are incorporated by reference Combine here. [0003] Reference to Sequence Listing [0004] This application includes the sequence listing shown at the end of the detailed description. Invention field [0005] The embodiments of the present invention relate to the manufacture of oil / fat, fuel, food and oleochemicals, as well as the culture of genetically engineered cells. Specific embodiments relate to oils with high triglyceride content and fatty acyl groups in a specific region-specific pattern on the glycerol backbone, highly stable oils, oils with high levels of oleic acid or medium-chain fatty acids, and those made from these oils The manufactured product. Background of the invention [...

Claims

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Application Information

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IPC IPC(8): C12N1/13C12N9/10C12N9/12C12P7/64A23L33/115C11C1/00C12R1/89
CPCC12Y203/01199A23L33/115C11C1/002C12N1/12C12N9/1029C12N9/1288C12P7/64C12Y203/01051C12Y207/08C12Y207/08002C12Y203/01023C12N15/79C12N15/52
Inventor S·富兰克林R·巴特X·赵
Owner CORBION BIOTECH INC
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