A method for synthesizing fatty alcohol acetate in microorganisms

一种醇乙酸酯、微生物体的技术，应用在微生物基因工程领域，能够解决合成成本高等问题，达到生长速度快、减少释放、遗传操作技术成熟的效果

Active Publication Date: 2019-02-22
GANNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical synthesis of sex pheromones involves multiple reaction steps, resulting in high synthesis costs

Method used

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  • A method for synthesizing fatty alcohol acetate in microorganisms
  • A method for synthesizing fatty alcohol acetate in microorganisms
  • A method for synthesizing fatty alcohol acetate in microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Construction of fatty alcohol acetate-producing Escherichia coli engineering bacteria based on fatty acyl ACP as a precursor molecule ( figure 1 shown)

[0025] A) Construction of Fatty Alcohol-Producing Engineering Bacteria

[0026] 1. Use primers AAR-XbaI (GTATCTAGAAAGAGGAGATATAATGTTCGGTCTTATCGGTCATCTC) and AAR-SpeI-BamHI (TGTGGATCCACTAGTTCAAATTGCCAATGCCAAGG) PCR amplification from Synechococcus elongates The AAR gene of bacteria, and then the amplified fragment was wxya and BamHI Insertion into pET28a(+) resulted in vector pDY01. Using primers AHR-XbaI (ATATCTAGAAAGAGGAGATATAATGTCGATGATAAAAAGCTATGCCG) and AHR-SpeI-BamHI (GTAGGATCCACTAGTTCAAAAATCGGCTTTCAACACCAC) to PCR amplify the AHR gene from Escherichia coli, using wxya and BamHI Insertion into pET28a(+) resulted in vector pDY02. The AAR expression cassette was obtained by double-digesting pDY01 with XbaI and XhoI, and inserted into the vector pDY02 double-digested with SpeI and XhoI to obta...

Embodiment 2

[0031] Example 2 Construction of Fatty Alcohol Acetate Escherichia coli Engineering Bacteria Based on Fatty Acids as Precursor Molecules ( figure 1 shown)

[0032] A) Construction of Fatty Alcohol-Producing Engineering Bacteria

[0033] 1. Using primers CAR-XbaI (ATATCTAGAAAGAGGAGATATAATGTCGCCAATCACGCGTGA) and CAR-SpeI-BamHI (AGAGGATCCACTAGTTCAGAGCAGGCCGAGTAGGC) PCR amplification from Mycobacterium marinum The CAR gene of bacteria, and then the amplified fragment was wxya and BamHI Insertion into pET28a(+) resulted in vector pDY06. The 'TesA gene from Escherichia coli was amplified by PCR with primers 'TesA-SacI (AGATGAGCTCATGG CGGACACGTTATTGATTCTGG) and 'TesA-SpeI (TGTACTAGTTTATGAGTCATGATTTACTAAAGGCTGC), and then the amplified fragment was double-digested with SacI and SpeI and inserted into pBBR1MCS1 to obtain vector pDY07. The AHR gene from Escherichia coli was amplified by PCR with primers AHR-BamHI (ACTGGATCCAA GAAGGAGATATAATGTCGATGATAAAAAAGCTATGCCG) and AHR-Xho...

Embodiment 3

[0038] Example 3 Construction of fatty alcohol acetate-producing Escherichia coli engineering bacteria based on fatty acyl-ACP and fatty acyl-CoA as precursor molecules ( figure 1 shown)

[0039] A) Construction of Fatty Alcohol-Producing Engineering Bacteria

[0040] 1. Using primers FAR-XbaI (CAATCTAGAAAGAGGAGATATAATGGCAATACAGCAGGTACATCAC) and FAR-SpeI-BamHI (CGAGGATCCACTAGTTCAGGCAGCTTTTTTGCGCT) PCR amplification from M. aquaolei Bacteria's FAR gene, and then the amplified fragment was wxya and BamHI Insertion into pET28a(+) resulted in vector pDY11.

[0041] 2. The recombinant vector pDY11 was introduced into Escherichia coli GM1655 to obtain GDY5 engineering bacteria. The engineering bacterium realizes the high expression of the FAR gene. The FAR enzyme was expressed to catalyze the direct reduction of fatty acid synthesis intermediates acyl-ACP and acyl-CoA to fatty alcohols.

[0042] B) Construction of Fatty Alcohol Acetate-Producing Engineering Bacteria

[004...

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Abstract

The invention discloses a method for synthesizing pheromone fatty alcohol acetate in microorganisms based on three intermediate products of fatty acid metabolism pathway as precursor molecules. The reductase CAR, fatty acyl-CoA reductase FAR and aldehyde reductase AHR are used to obtain engineering bacteria that can reduce fatty acyl-ACP, fatty acid or fatty acyl-CoA to produce fatty alcohol, respectively. Finally, alcohol acetyltransferase ATF1 was highly expressed in fatty alcohol-producing engineering bacteria, which was used to catalyze the reaction of fatty alcohol and acetyl-CoA to synthesize pheromone fatty alcohol acetate. The invention can realize the microbial transformation from glucose to fatty alcohol acetate, and provides a feasible way for large-scale biosynthesis of fatty alcohol acetate.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, and in particular relates to a method for directly synthesizing pheromone fatty alcohol acetate from monosaccharide by transforming microbial metabolic pathway. Background technique [0002] 1. Insect sex pheromones, also known as sex pheromones (artificially synthesized and used to control pests, are also called sex attractants), are secreted in vitro by special organs of individuals of a certain sex of the same species of insects, and can be absorbed by individuals of the same species. Trace chemical substances that are accepted by the receptors and cause corresponding behavioral responses or physiological effects (such as mate-seeking, directional courtship, mating, etc.). So far, about 2000 insect sex pheromones have been identified, most of which are various fatty alcohol acetate molecules. At present, a few insect sex pheromones have been chemically synthesized. Since the appl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/64C12R1/19
CPCY02E50/10
Inventor 郭道义潘虹李勋李永东范小林
Owner GANNAN NORMAL UNIV
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