Yarrowia lipolytica for producing farnesene

A technology for producing farnesene and bacteria, applied in the field of genetic engineering, can solve problems such as enhancing precursor flow, low fermentation level, and weakening branched metabolic pathways

Active Publication Date: 2019-03-19
ZHEJIANG MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to overcome the defect of low fermentation level of existing β-farnesene production strains, the present invention uses genetic engineering technology to transform a strain of Yarrowia lipolytica producing β-carotene, by introducing β-farnesene from Artemisia annua Synthase (β-farnesene synthase) gene, enhanced mevalonate pathway (MVA pathway) precursor flux, weakened branch metabolic pathway, and obtained a high-yielding β-farnesene production strain

Method used

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  • Yarrowia lipolytica for producing farnesene
  • Yarrowia lipolytica for producing farnesene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Construction of Backbone Plasmid B12408g-HUH

[0042] The primer sequences used in the construction of the backbone plasmid B12408g-HUH include:

[0043] 12408g-F: 5'-ATTTAAAT GATTGAGACGTGGCGATCCGGCAAC-3' (SEQ ID NO: 2),

[0044] 12408g-R: 5'-ATTTAAAT TAAGAAGCACACCACAGAACCGTAC-3' (SEQ ID NO: 3);

[0045] 408g-F:

[0046] 5'-CCCAAAGAGGTGAATTCGGCAAGCTTCTAGAGTAGGTGGTATGTACTCGTACTGTACT-3' (SEQ ID NO: 4),

[0047] 408g-R:

[0048] 5'-CTACTCTAGAAGCTTGCCGAATTCACCTCTTTGGGCCATTTCATTAGATCG-3' (SEQ ID NO: 5).

[0049] "-F" in the above names stands for forward; "-R" stands for reverse.

[0050] Using the CIBTS2112 genome as a template, the upstream and downstream homology arms were amplified by PCR using primer sets 12408g-F / 408g-R and 408g-F / 12408g-R, respectively. PCR conditions: Denaturation at 95°C for 5min, denaturation at 95°C for 30s, annealing at 58°C for 30s, extension at 68°C for 2min, 32 cycles, 68°C for 10min, and finally 16°C for 10min.

[0051] Usin...

Embodiment 2

[0053] Example 2: Construction of plasmid B12408g-HUH-FH

[0054] 1.1 Preparation of pTEF-FS-xpr2t expression module

[0055] Using the plasmid pUC57-FS as a template, the pTEF-FS-xpr2t expression module was obtained by PCR amplification using the following 408-FH-F / T-FS-X-R primer pair.

[0056] 408-FH-F:

[0057] 5'-TACCCTGATTGACTGGAACAGCCCCAAGAGACCGGGTTGGCGGCGTATTTGTG-3' (SEQ ID NO: 6),

[0058] T-FS-X-R:

[0059] 5'-GGTGCACCGAAGAGCCTGTGGAAGCGACGGGGACACGGGCATCTCACTTGC-3' (SEQ ID NO: 7).

[0060] PCR conditions: Denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 68°C for 3 minutes, 32 cycles, incubation at 68°C for 10 minutes, and finally incubation at 16°C for 10 minutes.

[0061] 1.2 Preparation of pEXP-tHmgR-pex20t expression module

[0062] The plasmid TAs-7h-GH was used as a template to obtain the pEXP-tHmgR-pex20t expression module by PCR amplification using the following E-H-P-F / 408-FH-R primer ...

Embodiment 3

[0069] Example 3: Construction of β-farnesene-producing bacteria

[0070] The plasmid B12408g-HUH-FH constructed in Example 2 was digested with SwaI to recover a large fragment, and the ZymogenFrozen-EZ Yeast Yransformation kit II kit (Zymo Research Corporation) was used to transform CIBTS2112 to obtain the genetically engineered strain CIBTS2617.

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Abstract

The invention constructs a beta-farnesene producing bacterium by a gene engineering. The beta-farnesene producing bacterium is collected in (China General Microbiological Culture Collection Center with the collection number of CGMCC No.16708. The beta-farnesene producing bacterium constructed by the invention has the beneficial effects that the effective accumulation of the beta-farnesene in fermentation liquid can be realized by fermentation, and the yield of the beta-farnesene is up to 400mg / L, so that the beta-farnesene producing bacterium has an industrial prospect.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a genetic engineering production bacterium with high yield of β-farnesene. Background technique [0002] When aphids are frightened, they can secrete a kind of "alarm pheromone" from the abdominal tube, so that other aphids around can perceive and escape quickly, thus stopping the damage to crops. Bowers et al. isolated and identified the aphid alarm pheromone trans-β-farnesene (E-β-farnesene, referred to as EβF or EBF) for the first time in 1972. EβF is a sesquiterpenoid. Francis et al. found that EBF is the main or only component of 16 aphid alarm pheromones (Bowers et al., 1972; Francis et al., 2005). The application of this type of pheromone in the field can control the population density of aphids within a certain range within the threshold value. At this time, the insects and diseases have little impact on the crops. EβF is not only present in aphid alarm ho...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P5/02C12R1/645
CPCC12N9/0006C12N9/88C12N15/815C12P5/007C12Y101/01032C12Y402/03047
Inventor 陈正杰蒋宇童阳阳许崇茂吴宇辉杨俊杰杨晟张芸赵梦凡朱丽
Owner ZHEJIANG MEDICINE CO LTD
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