Construction method of oil-producing yeast engineering strain by using methyl alcohol and application

An oil-producing yeast and engineering strain technology, applied in the fields of biotechnology and bioenergy, can solve problems such as inability to use methanol, and achieve the effects of reducing CO2 emissions and reducing costs

Active Publication Date: 2018-06-29
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Natural methanol-utilizing bacteria do not accumulate oil, and natural oil-producing strains cannot utilize methanol as a substrate
After searching, there is currently no report on the use of metabolically engineered oleaginous yeast to use methanol as a substrate to produce oil

Method used

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  • Construction method of oil-producing yeast engineering strain by using methyl alcohol and application
  • Construction method of oil-producing yeast engineering strain by using methyl alcohol and application
  • Construction method of oil-producing yeast engineering strain by using methyl alcohol and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Recombinant Strain M-R1 Utilizes Methanol to Produce Oil

[0027] The oleaginous yeast Rhodosporidium toruloides CGMCC 2.1389 was purchased from China General Microorganism Culture Collection and Management Center. The recombinant strain M-R1 is an R. toruloides strain co-overexpressing MDH, HPS and PHI, and its construction process is as follows.

[0028] Step 1 Design the following primers according to the nucleotide sequence of GPD-p to construct the constitutive cloning vector pZPK (the underlined part of primer Pgpd-Xbal I-F is the XbaI restriction site, and the underlined part of primer Pgpd-Eco V-R is the EcoRV enzyme cutting site) wherein the specific primers used for cloning GPD-p are:

[0029] Pgpd-Xbal I-F 5'-CTAG TCTAGA TCCATGCTGCTGCGATCTGGGAGTG-3'

[0030] Pgpd-Eco V-R 5'-CCATGG GATATC TGTGACTGATCTGGTGTTGTTCTGA-3'

[0031] According to the nucleotide sequences of MDH, HPS and PHI, the following primers were respectively designed to construct the corr...

Embodiment 2

[0055] Recombinant Strain M-R2 Utilizes Methanol to Produce Oil

[0056] The oleaginous yeast R. toruloides CGMCC 2.1389 was purchased from China General Microorganism Culture Collection and Management Center. The recombinant strain M-R2 is an R. toruloides strain co-overexpressing MDH3, HPS and PHI, and its construction process is basically the same as in Example 1. The difference lies in the selection of methanol dehydrogenase MDH3 during the construction of M-R2.

[0057] According to the nucleotide sequence of MDH3, the following primers were respectively designed to construct the corresponding constitutive expression vector pZPK-GDP-MDH3 (the underlined part with EcoR V in the primer name is the EcoR V restriction site, and the primer name with The underlined part of SpeI is the SpeI restriction site):

[0058] The specific primers used to clone MDH3 are:

[0059] MDH3-EcoR V-F 5’-CCATGG GATATC ATGAAGAACACCCAGTCGGC-3'

[0060] MDH3-Spe I-R 5'-GG ACTAGT CTACTTGATGA...

Embodiment 3

[0075] Recombinant Strain M-R3 Utilizes Methanol to Produce Oil

[0076] The oleaginous yeast R. toruloides CGMCC 2.1389 was purchased from China General Microorganism Culture Collection and Management Center. The recombinant strain M-R3 is an R. toruloides strain co-overexpressing MDH3ACT, HPS and PHI, and its construction process is basically the same as in Example 1. The difference is that methanol dehydrogenase MDH3-ACT with activator protein is expressed during the construction of M-R2.

[0077] According to the nucleotide sequence of MDH3-ACT, the following primers were respectively designed to construct the corresponding constitutive expression vector pZPK-GDP-MDH3 (the underlined part with EcoR V in the primer name is the EcoR V restriction site, and the primer name in The underlined part with SpeI is the SpeI restriction site):

[0078] The specific primers used for cloning MDH3 are: MDH3-EcoR V-F and MDH3-ACT-Spe I-R, wherein

[0079] MDH3-ACT-Spe I-R 5'-GG ACTA...

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Abstract

The invention discloses a construction method of an oil-producing yeast engineering strain by using methyl alcohol and application. The strain construction method disclosed by the invention relates tosimultaneous overexpression of methanol dehydrogenase, hexose phosphatase and oxoisomerase which are key enzymes used by the methyl alcohol in oil-producing yeast. An obtained gene engineering straincan efficiently make use of the methyl alcohol to produce grease, so as to provide a core technology for conversion of the methyl alcohol into long carbon chain fuel and a functional chemical. The construction method has a wide industrial application prospect.

Description

technical field [0001] The invention relates to a method for constructing a methanol utilization pathway in oleaginous yeast and the application of the obtained genetic engineering strain, belonging to the fields of biotechnology and bioenergy. Background technique [0002] Oil is a kind of natural renewable resource with low oxygen content, high energy density, and rich in hydrocarbon elements. It is the basic raw material for the production of liquid biofuels, oleochemicals, food and materials, and is widely used in daily production and all aspects of life. (Jin MJ et al. Trends Biotechnol, 2015, 33(1):43-54.). The production of traditional animal and vegetable oils is limited, and it is difficult to meet the growing demand. It is urgent to develop new technologies for oil production. In order to solve the problem of oil source, industrial biotechnology is used to adopt oleaginous yeasts such as Rhodosporidium toruloides (R.toruloides), Lipoyeast starkeyi (L.starkeyi), Tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12P7/64
CPCC12N9/0006C12N9/88C12N9/90C12N15/815C12N2800/102C12N2800/22C12P7/6463C12Y101/01244C12Y401/02043Y02E50/10Y02T50/678
Inventor 赵宗保杨晓兵张素芳
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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