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Synechococcus genetic engineering bacteria for biosynthesizing caryophyllene, and construction method and application thereof

A technology of genetically engineered bacteria and biosynthesis, which is applied in the fields of genetic engineering, biochemical equipment and methods, plant genetic improvement, etc., and can solve the problems that the output cannot meet the industrial demand.

Active Publication Date: 2020-07-10
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the production of caryophyllene has been realized in cyanobacteria before, the output of only 46.4±2.9μg / L / week is difficult to meet the industrial demand

Method used

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  • Synechococcus genetic engineering bacteria for biosynthesizing caryophyllene, and construction method and application thereof
  • Synechococcus genetic engineering bacteria for biosynthesizing caryophyllene, and construction method and application thereof
  • Synechococcus genetic engineering bacteria for biosynthesizing caryophyllene, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Construct the basic vectors pSI-SPE, pSII-CM and pSIII-KAN respectively

[0056] The genome of Synechococcus sp. UTEX 2973 was extracted using the bacterial genome extraction kit as a template, and the sequences shown in SEQ ID NO.19 and SEQ ID NO.20 were used as upstream and downstream primers for PCR amplification to obtain the upstream homology arms of the neutral site NSI Gene (SEQ ID NO.3); with the sequences shown in SEQ ID NO.21 and SEQ ID NO.22 as the upper and lower primers, PCR amplification was performed to obtain the homology arm gene (SEQ ID NO. 4); with the sequences shown in SEQ ID NO.23 and SEQ ID NO.24 as the upper and lower primers, carry out PCR amplification to obtain the homology arm gene (SEQ ID NO.5) upstream of the neutral site NSII; with SEQ ID The sequences shown in NO.25 and SEQ ID NO.26 are the upper and lower primers, and PCR amplification is carried out to obtain the homology arm gene (SEQ ID NO.6) downstream of the neutral site NSII; with ...

Embodiment 2

[0068] Construct vector pSI-ispA-gpps-idi1 sc Using the basic vector pSI-SPE, using SEQ ID NO.41 and SEQ ID NO.42 as the upstream and downstream primers of the pSI-SPE plasmid backbone, respectively, PCR amplification was performed to obtain promoter-free P CPC560pSI-SPE plasmid backbone.

[0069] Synechococcus UTEX 2973 genome was used as a template, and SEQ ID NO.43 and SEQ ID NO.44 were used as P psba1 The upstream and downstream primers of the promoter were used for PCR amplification to obtain P psba1 Promoter (SEQ ID NO. 18).

[0070] The Escherichia coli MG1655 genome extracted by the bacterial genome extraction kit was used as a template, and SEQ ID NO.45 and SEQ ID NO.46 were respectively used as the upstream and downstream primers of the geranyl pyrophosphate / farnesyl pyrophosphate synthase gene ispA, Perform PCR amplification to obtain the gene ispA (SEQ ID NO.14).

[0071] The codon-optimized geranyl pyrophosphate gene gpps connected to the universal vector pUC5...

Embodiment 3

[0078] Construction of vector pSII-idi1 sc The basic vector pSII-CM was used, and SEQ ID NO.53 and SEQ ID NO.54 were respectively used as the upstream and downstream primers of the pSII-CM plasmid backbone for PCR amplification to obtain the pSII-CM plasmid backbone.

[0079] The Saccharomyces cerevisiae BY4741 genome extracted by the yeast genome extraction kit was used as a template, and SEQ ID NO.51 and SEQ ID NO.52 were respectively used as the upstream and downstream primers of the isopentenyl diphosphate δ-isomerase gene idi1 for PCR amplification. The gene idi1 (SEQ ID NO.15) was obtained.

[0080] The pSII-CM plasmid backbone and gene idi1 were assembled using the assembly kit to obtain the vector construction vector pSII-idi1 sc .

[0081] Dilute the obtained PCR products according to the ratio of 10ng / 1000bp / μl, take 1μl of each PCR product, 1.5μl of sterile water, 1μl of 5×CE MultiS Buffer, MultiS 0.5μl, react at 37°C for 30min. After the reaction was completed...

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Abstract

The invention discloses synechococcus genetic engineering bacteria for biosynthesizing caryophyllene, and a construction method and application thereof. The construction method is as follows: constructing basic vectors pSI-SPE, pSII-CM and pSIII-KAN; and then further constructing vectors pSI-ispA-gpps-idi1sc, pSII-idi1sc and pSIII-tps21, and transferring the vectors pSI-ispA-gpps-idi1sc, pSII-idi1sc and pSIII-tps21 into synechococcus UTEX 2973 to obtain the synechococcus genetic engineering bacteria for biosynthesizing caryophyllene. The method of the invention focuses on the speed-limiting problem of efficient biosynthesis of caryophyllene to systematically and pertinently reform synechococcus UTEX 2973, experiments show that the final output of caryophyllene of the genetic engineering bacteria of the invention reaches 212.37 [mu]g / L in 96 h, the output per unit time is 8 times that of the prior art, and thereby the genetic engineering bacteria has important theoretical and practicalsignificance for the production of caryophyllene by using photosynthetic microorganisms.

Description

technical field [0001] The invention belongs to the field of industrial microorganisms, and in particular relates to a Synechococcus genetically engineered bacterium for biosynthesizing caryophyllene, a construction method and application thereof. Background technique [0002] β-caryophyllene (beta-caryophyllene, referred to as caryophyllene) is a bicyclic sesquiterpene compound with the molecular formula C 15 h 24 , insoluble in water at room temperature, only soluble in ethanol, chloroform and other organic solvents, caryophyllene widely exists in various plants, and plays an important role in plant resistance to bacterial, fungal infection and insect pests. Caryophyllene is a colorless to slightly yellow liquid at room temperature, with a clove smell and a mild taste. Therefore, it has been approved as a food spice (GB 2760-1996) that is allowed to be used in China. It is also widely used in medicines and cosmetics. At the same time, because of its high density and heat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/65C12N15/61C12N15/54C12N1/21C12P5/00
CPCC12N15/74C12N15/65C12N9/90C12N9/1085C12P5/002C12Y503/03002C12Y205/0101C12Y205/01081C12N2800/22
Inventor 陈磊李树斌孙韬张卫文
Owner TIANJIN UNIV
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