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Novel acyltransferases, variant thioesterases, and uses thereof

A technology of acyltransferase and construct, which is applied in the field of nucleic acid and protein, and can solve problems such as cell division inhibition

Inactive Publication Date: 2019-08-09
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 mornii stores large amounts of triglyceride oil under conditions of excess supply of nutrient carbon, but cell division is inhibited by other essential nutrients

Method used

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  • Novel acyltransferases, variant thioesterases, and uses thereof
  • Novel acyltransferases, variant thioesterases, and uses thereof
  • Novel acyltransferases, variant thioesterases, and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0127] Example 1: Fatty Acid Analysis Using Fatty Acid Methyl Esters Detection

[0128] Lipid samples were prepared from dried biomass. 20-40 mg of dry biomass was resuspended in 2 mL of 5% H in MeOH 2 SO 4 , and added 200 μl of toluene containing an appropriate amount of internal standard (C19:0). The mixture was sonicated briefly to disperse the biomass, then heated at 70-75°C for 3.5 hours. Add 2 mL of heptane to extract fatty acid methyl esters, then add 2 mL of 6% K 2 CO 3 (aqueous solution) to neutralize acid. Stir the mixture vigorously, and transfer a portion of the upper layer to a 2 SO 4(Anhydrous) vials were analyzed by gas chromatography using standard fatty acid methyl ester gaschromatography flame ionization detection (FAME GC / FID) method. The fatty acid profiles reported below were determined by this method.

example 2

[0129] Example 2: Analysis of regiospecific profiles

[0130] 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, which included a SIL-30AC autosampler, two LC-30AD pumps, a DGU-20A5 online degasser, and a CTO-20A column thermostat. Data were acquired using a Q3 scan of m / z 350-1050 at a scan speed of 1428 u / sec in positive ion mode with the CID gas (argon) pressure set to 230 kPa. The temperatures of APCI, desolvation tube and heating block were set at 300, 250 and 200 °C, respectively, the flow rates of atomizing and drying gases were 3.0 L / min and 5.0 L / min, respectively, and the interface voltage was 4500 V. The oil sample was dissolved in dichloromethane-methanol (1:1) to a concentration of 5 mg / mL, and Samples were injected into a Shimadzu gasket assembly XR-ODS III ( 2.0×200mm). From 30% dichloromet...

example 3

[0131] Example 3: Cultivating Microalgae

[0132] Standard lipid production conditions:

[0133] Cells scraped from the source plate with a toothpick were used to inoculate a pre-seeded culture with 0.5 mL of EB03, 0.5% glucose, IX DAS2 culture in 96-well blocks. The pre-seeded culture was grown in a Multitron shaker at 28°C, 900 rpm for 70-75 hours. 40 μL of the pre-seeded culture was used to inoculate a seed culture with 0.46 mL of H29, 4% glucose, 25 mM citrate pH 5 or 100 mM PIPES pH 7.3, 1X DAS2 (8% inoculum) and incubated at 28 °C, 900 rpm in a Multitron shaker. Leave to grow for 24-28 hours. 40 μL seed culture was used to inoculate lipid production cultures with 0.46 mL H43, 6% glucose, 25 mM citrate pH5, 1X DAS2 (8% inoculum) and grown in a Multitron shaker at 28 °C, 900 rpm for 70 -75 hours. Fatty acid profiling and lipid titer analysis were performed as disclosed in Examples 1 and 2.

[0134] 50mL shake flask type

[0135] Cells scraped from the source plate us...

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Abstract

Recombinant nucleic acids and vector constructs encoding acyltransferases and variant thioesterases, and the acyltransferases and variant thioesterases encoded by the nucleic acids are provided. The acyltransferases and variant thioesterases are useful in fatty acid synthesis and triacylglycerol production. Host cells that express the recombinant nucleic acids as well as methods of cultivating thehost cells, methods of producing oils from the host cells are provided. The recombinant host cells and the oils produced therefrom have altered fatty acid profiles and / or triacylglycerols with altered regiospecificity.

Description

[0001] Cross References to Related Applications [0002] This application is pursuant to 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Nos. 62 / 404,667 and 2017 filed October 5, 2016 and entitled "Novel Acyltransferases, Variant Thioesterases, and Uses Thereof" Priority to U.S. Patent Application Serial No. 15 / 725,222, filed October 4, 1999, and entitled "Novel Acyltransferases, Variant Thioesterases, and Uses Thereof," both of which are incorporated by reference in their entirety In this article. [0003] Reference Sequence Listing [0004] This application includes a Sequence Listing as shown at the end of the Examples. technical field [0005] Embodiments of the invention relate to oils / fats, fuels, food and oleochemicals and their production from cultures of genetically engineered cells. Examples relate to nucleic acids and proteins involved in fatty acid synthesis pathways; oils with high levels of triglycerides having fatty acyl groups on the glycerol ba...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10
CPCC12N9/1025C12N9/1029C12Y203/01051C12N15/82C12N2800/22C12P7/6463
Inventor J·L·莫斯利J·加索拉里X·赵A·俄温A·索曼奇S·富兰克林D·戴维斯
Owner CORBION BIOTECH INC
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