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Application of glycometabolism and lipid metabolism for improving synthetic yield of fatty acid derivatives by yarrowia

A technology of Yarrowia lipolytica, fatty acid derivatives, applied in the field of synthetic biology

Active Publication Date: 2019-06-25
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] On the other hand, most of the current metabolic engineering methods are steady-state adaptation of the upstream and downstream expression intensity of the metabolic flow (the expression intensity is not dynamically regulated with the fermentation process)

Method used

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  • Application of glycometabolism and lipid metabolism for improving synthetic yield of fatty acid derivatives by yarrowia
  • Application of glycometabolism and lipid metabolism for improving synthetic yield of fatty acid derivatives by yarrowia
  • Application of glycometabolism and lipid metabolism for improving synthetic yield of fatty acid derivatives by yarrowia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Construction of different alcohologenic lipolytic yeast strains, and use of transcriptome comparison to determine the main response pathways

[0060] Experimental Materials:

[0061] 1. Gene

[0062] 1) FAR: fatty acyl-CoA reductase, derived from Marinobacter aquaeolei VT8, codon optimized according to Yarrowia lipolytica, the optimized DNA encoding FAR has the nucleotides shown in SEQ ID No.1 Acid sequences were synthesized at GenScript Company.

[0063] 2. Gene expression vector information

[0064] The integration plasmid PUC57-IntF-HUM was synthesized from GenScript Company.

[0065] The integrated plasmid PUC57-RS8d-HUM was synthesized from GenScript Company.

[0066] 3. Reagents required for genetic manipulation

[0067] 1) Frozen EZ Yeast Transformation Kit II (Zymo Research Corporation) was purchased from Beijing Tianmo Technology Development Co., Ltd.;

[0068] 2) KOD FX DNA polymerase and dNTP (2.0mM each) were purchased from Toyobo Shanghai C...

Embodiment 2

[0118] Example 2: Using glycolytic promoters to synergistically enhance alcohol production in lipolytic yeast

[0119] Experimental Materials:

[0120] 1. Strain information: See Table 4 for the plasmid information used in this example, and Table 5 for the strain name, genotype information, and source.

[0121] 2. Other genetic information, reagents and culture medium are the same as in Example 1.

[0122] experimental method:

[0123] 1. Construction of recombinant plasmids related to glycolysis promoters

[0124] Use the primers shown in Table 5 to amplify the sequences of various glycolysis-related promoters using the genome of lipolytica ATCC201249 as a template, and perform amplification with the FAR fragment and the vector PUC57-K8Zero-HUM using BsaI, BsmbI or BbsI and T4 ligase respectively. The GoldenGate gene was assembled to obtain the recombinant plasmids PUC57-P1-K8Zero-FAR~PUC57-P11-K8Zero-FAR, and then digested with NotI and recovered the recombinant fragments...

Embodiment 3

[0162] Example 3: Increasing the carbon source concentration of the medium to further "push" the synergistic strains to produce alcohol

[0163] Experimental Materials:

[0164] The strain information, reagents, etc. are the same as those in Examples 1 and 2.

[0165]In addition to the YPD medium involved in Example 1 (in order to facilitate the distinction, the medium in Example 1 is marked as YPDx1 here), a glucose-doubled YPDx2 medium was also configured. The main components of YPDx2 medium are as follows: image 3 As shown in A.

[0166] experimental method:

[0167] 1. First, the JLFL-13 strain was image 3 A Each medium is fermented, and the optimal N source ratio of YPDx2 medium is determined.

[0168] 2. Comparatively ferment JLFL-1, JLFL-2 and JLFL-13 in the optimized YPDx2 and YPDx1 medium respectively, and the process is the same as in Example 1.

[0169] 3. the extraction of product, method process is with embodiment 1.

[0170] 4. The detection of product, ...

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Abstract

The invention relates to the technical field of synthetic biology, in particular to an application of glycometabolism and lipid metabolism for improving synthetic yield of fatty acid derivatives by yarrowia. The expression of fatty alcohol and a glycometabolism path can achieve balance and coordination of lipid production and other related pathways to promote rational distribution of cellular metabolism and improve lipid synthesis efficiency. The application finds through experiments that when glycolysis and lipid product production pathways are synergistic, the effective pull-push effect canbe achieved, and the production of the lipid product can up-regulate the expression of the glycolysis, the glycolysis can synergistically further promote the expression of lipid products for gene synthesis, the overall metabolic flux can be greatly improved, and efficient production of the lipid products can be achieved. on the other hand, synergistic expression is also conducive to the realization of dynamic regulation, so that the production of the product can be adjusted in real time according to the different metabolic states of the cells, and further the production efficiency of a cell factory is optimized.

Description

technical field [0001] The invention relates to the technical field of synthetic biology, in particular to the application of sugar metabolism and lipid metabolism to synergistically improve the yield of fatty acid derivatives synthesized by Yarrowia lipolytica. Background technique [0002] Medium and long chain fatty acid derivatives (C8-C18) - fatty alcohol (Fatty alcohol, FOH), fatty acid ethyl ester (fatty acid ethyl ester, FAEE), special fatty acid (unusual fatty acid), etc. , chemical raw materials, nutrition and health products and other uses, the market demand is increasing, and it has high economic value. [0003] Since fatty acids are the main energy storage substances of microorganisms and important components of biofilms, most cells naturally contain a complete synthesis pathway of medium- and long-chain (C16, C18) fatty acids and fatty acyl-CoA, and their corresponding fatty acid derivatives The production of is mainly realized by introducing the corresponding...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/90C12N1/19C12P7/04C12P7/64C12P7/62C12R1/645
Inventor 元英进张金来曹英秀晋聪聪白秋艳张若思彭洋子刘夺
Owner TIANJIN UNIV
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