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Corynebacterium glutamate for synthesizing geraniol and construction method and application of corynebacterium glutamate

A technology of glutamic acid sticks and construction methods, applied in the biological field, to achieve the effect of short fermentation time

Inactive Publication Date: 2019-11-12
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, Corynebacterium glutamicum has been successfully used in the biosynthesis of prodrugs such as valencene and carotenoids, but there is no report on the production of geraniol

Method used

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  • Corynebacterium glutamate for synthesizing geraniol and construction method and application of corynebacterium glutamate
  • Corynebacterium glutamate for synthesizing geraniol and construction method and application of corynebacterium glutamate
  • Corynebacterium glutamate for synthesizing geraniol and construction method and application of corynebacterium glutamate

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Experimental program
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Embodiment 1

[0044] Embodiment 1 Construction of geraniol synthesis expression vector

[0045] (1) Acquisition of related genes

[0046] Mutant geranyl pyrophosphate synthase gene ERG20 derived from Saccharomyces cerevisiae F96W-N127W The fragment is used as a template, and the sequences SEQ ID NO.7 and SEQ ID NO.8 are used as primers to amplify the mutant geranyl pyrophosphate synthase gene ERG20 derived from Saccharomyces cerevisiae F96W-N127W (SEQ ID NO. 1).

[0047] According to the amino acid sequence of geraniol synthase derived from valerian root with truncated 53 amino acid residues at the C-terminus, the 53 amino acid residues at the truncated C-terminus were synthesized by GenScript Biotechnology Co., Ltd. by chemical synthesis The geraniol synthase gene tVoGES (SEQ ID NO.2) derived from Valeriana officinalis was amplified using the sequences SEQ ID NO.9 and SEQ ID NO.10 as primers.

[0048] With wild-type Corynebacterium glutamicum ATCC13032 genome as template, with sequences...

Embodiment 2

[0059] The construction of the coryneform bacterium glutamicum of embodiment 2 synthesis geraniol

[0060] A method for constructing Corynebacterium glutamicum cells for synthesizing geraniol, expressing geranyl pyrophosphate synthase and geraniol synthase in Corynebacterium glutamicum at the same time to obtain recombinant bacterium 1.

[0061] The plasmid 1 capable of expressing geranyl pyrophosphate synthase and geraniol synthase is transformed into Corynebacterium glutamicum to obtain Corynebacterium glutamicum 1 capable of synthesizing geraniol.

[0062] In order to control the recombinant strains and facilitate the comparative analysis of metabolites, Corynebacterium glutamicum recombinant strain 3 containing pEC-XK99E empty plasmid was constructed according to the above transformation method, and pXMJ19 empty plasmid was introduced on the basis of strain 3 to obtain glutamic acid Coryneform bacteria recombinant 4.

[0063] The method for transforming the plasmid into C...

Embodiment 3

[0065] Construction of strains that increase the expression levels of deoxyxylulose-5-phosphate synthase and isopentenyl pyrophosphate isomerase in geraniol-synthesizing bacteria (Corynebacterium glutamicum 2 that synthesizes geraniol)

[0066] Improve the expression levels of deoxyxylulose-5-phosphate synthase and isopentenyl pyrophosphate isomerase in recombinant strain 1, and will be able to express deoxyxylulose-5-phosphate synthase and isopentenyl pyrophosphate isomerase The plasmid 2 was transformed into recombinant bacterium 1 to obtain recombinant bacterium 2.

[0067] The transformation method is as follows: take out the competent cells of the recombinant bacteria 1 preserved from the -80°C refrigerator, and place them on ice until they melt. Add a certain amount of plasmid 2, and gently pipette several times to mix evenly. Use a pipette to inhale the mixed DNA plasmid and competent cell system into a pre-cooled electric shock cup (aperture 1 mm) with toilet paper to...

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Abstract

The present invention discloses a corynebacterium glutamate for synthesizing geraniol and a construction method and application of corynebacterium glutamate. The construction method of corynebacteriumglutamate comprises the following steps: connecting a saccharomyces cerevisiae-derived mutant geranyl pyrophosphate synthase gene ERG20<F96W-N127W> and a valerian-derived geranol synthase gene tVoGEStruncated through cutting off of C-terminal 53 amino acid residues by fusion PCR, adding a ribosome binding site sequence to a homologous region, inserting SacI and XbaI enzyme cutting sites of a corynebacterium glutamate expression plasmid pEC-XK99E to obtain a plasmid 1; converting the plasmid 1 into corynebacterium glutamate to obtain corynebacterium glutamate 1 for synthesizing geraniol. Theconstruction method provides more precursors and short fermentation time (42 to 48 hours) for the synthesis of geraniol, and corresponding by-products are not detected out by GC-MS detection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a coryneform bacterium glutamicum for synthesizing geraniol, a construction method and application. Background technique [0002] Geraniol, also known as geraniol, is a colorless or yellow oily liquid at room temperature, with a mild rose scent and a bitter taste. It is often used as a food additive, the main ingredient of rose essence, and an indispensable flavoring raw material in various floral essences. It can also be used as a sweetener, and can also be used to prepare food, soap, and daily cosmetic essences. Products made from natural essential oils contain more than 90% geraniol when preparing food and daily cosmetic flavors. Industrial geraniol is the raw material for the manufacture of vanillyl alcohol, ionone and vitamin A. [0003] The raw material of the medicine is used for antibacterial and deworming. It is effective in the clinical treatment of chronic bronchitis. It...

Claims

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Application Information

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IPC IPC(8): C12N15/77C12N1/21C12P7/04C12R1/15
CPCC12N15/77C12N9/1085C12N9/88C12P7/04C12Y205/01029C12Y402/03
Inventor 卢文玉徐硕张传波李曼武玉芬
Owner TIANJIN UNIV
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