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Method for constructing recombinant bacteria for producing citral, recombinant bacteria constructed by method and application of recombinant bacteria

A technology of recombinant bacteria and citral, applied in the field of bioengineering, can solve problems such as harsh conditions, limitation of natural citral production capacity, and environmental pollution by chemical synthesis

Active Publication Date: 2021-09-07
SHENZHEN BLUEPHA BIOSCIENCES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of the plant extraction method is that the raw materials are affected by seasons and geography, which limits the production capacity of natural citral, while the chemical synthesis method has problems such as environmental pollution and harsh conditions

Method used

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  • Method for constructing recombinant bacteria for producing citral, recombinant bacteria constructed by method and application of recombinant bacteria
  • Method for constructing recombinant bacteria for producing citral, recombinant bacteria constructed by method and application of recombinant bacteria
  • Method for constructing recombinant bacteria for producing citral, recombinant bacteria constructed by method and application of recombinant bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: Construction of MVA expression vector

[0048] Construction of p15A-MVA plasmid: This plasmid includes the coding sequences of the enzymes that synthesize IPP and DMAPP, in which atoB, HMGS, and HMGR share a set of regulatory elements, which are regulated by the lac UV5 promoter (SEQ ID NO: 31); MK, PMK , PMD and idi share a set of regulatory elements, which are regulated by the pTac promoter (SEQ ID NO: 32).

[0049] Wherein atoB (SEQ ID NO: 1) is the coding sequence of acetoacetyl-CoA thiolase (SEQ ID NO: 38), HMGS (SEQ ID NO: 2) codes 3-Hydroxy-3-methylglutaryl-CoA synthase (3-hydroxy-3- Methylglutaryl-CoA synthase) (SEQ ID NO:39), HMGR (SEQ ID NO:3) encodes 3-Hydroxy-3-methylglutaryl-CoA reductase (3-hydroxy-3-methylglutaryl- CoA reductase) (SEQ ID NO:40), MK (SEQ ID NO:4) encodes MVA kinase (MVA kinase) (SEQ ID NO:41), PMK (SEQ ID NO:5) encodes phospho MVA kinase (phosphate MVA Kinase) (SEQ ID NO:42), PMD (SEQ ID NO:6) encodes MVAdiphosphate decarbo...

Embodiment 2

[0069] Embodiment 2: construct the expression vector that comprises the coding gene of the enzyme that is used for the synthesis of citral with IPP and DMAPP

[0070] Construction of pTALE-GPPS-GES-geoA-idi plasmid: the coding genes of four enzymes that synthesize citral with IPP and DMAPP as precursors, GPPS, GES, geoA and idi share a set of regulatory elements, promoters and ribosomes A sequence with an insulator function is inserted between the sites, wherein GPPS is the geranyl diphosphate synthase gene, GES is the sweet basil geraniol synthase gene, and idi is the isopentenyl pyrophosphate isomerase gene.

[0071] Select pTALE as the plasmid vector, and sp2 is the promoter sequence, which is a constitutive transcription promoter, and the functional sequence of the insulator function RiboJ (excluding the effect of other regulatory elements) is added between the promoter sequence and the ribosome binding site sequence, The arrangement order of the coding sequences of the fo...

Embodiment 3

[0082] Embodiment 3: the transformation of plasmid

[0083] The plasmid p15A-MVA constructed in Example 1 and the plasmid pTALE-GPPS-GES-geoA-idi constructed in Example 2 were co-transformed into Escherichia coli BW25113 (ATCC number: ATCC12435 purchased from American Type Culture Collection, the BW25113 strain is derived from E.coli K-12W1485, which is a derivative strain of K12, similar to MG1655, and also an engineering strain of E. coli that has undergone less modification and is closer to the "wild type".), through the following screening process Obtain citral-producing recombinant bacteria.

[0084] Screening process:

[0085] (1) Take 50 μL (100 μL) of competent cells and thaw slowly on ice;

[0086] (2) Add 100ng each of p15A-MVA and pTALE-GPPS-GES-geoA-idi, mix gently by pipetting, and place in ice bath for 20min;

[0087] (3) After heat shock at 42°C (metal bath) for 60s, quickly ice bath for 2min, do not shake the centrifuge tube during this process;

[0088] (4...

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Abstract

The invention relates to a method for constructing a recombinant bacterium for producing citral, the recombinant bacterium constructed by the method and application of the recombinant bacterium. In the invention, two plasmids are constructed, wherein one of the plasmids comprises coding genes for expressing various enzymes for generating IPP and DMAPP: atoB, HMGS, HMGR, MK, PMK, PMD and idi, and the other plasmid comprises coding genes for synthesizing citral enzymes by taking IPP and DMAPP as precursors: GPPS, GES, geoA and idi. The two plasmids are used for co-transfecting a conventional BW25113 bacterium to obtain the recombinant bacterium, and the recombinant bacterium can be used for producing citral in a classical LB culture medium in a high-yield fermentation manner.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular, the invention relates to a method for constructing a recombinant bacterium producing citral, the recombinant bacterium constructed therefrom and its application. Background technique [0002] Citral is one of the most important representatives of open-chain monoterpenes, and its molecular formula is C 10 h 16 O, a colorless or light yellow transparent liquid, which is usually composed of geranial (trans-citral, citral a, geranial, structural formula as follows) and neral (cis-citral, citral b) which are mutually isomers , neral, the structural formula is as follows) composition. [0003] [0004] The applications of citral include: a) Edible flavors: it can be directly used in baked goods, candies, cold drinks, chewing gum and soft drinks, and can also be used to prepare fruit flavors such as strawberries, apples, apricots, sweet oranges and lemons, Especially in the essence of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/54C12N15/53C12N15/60C12N15/61C12N1/21C12P7/24C12R1/19
CPCC12N15/70C12N15/52C12N9/1025C12N9/0006C12N9/88C12N9/1205C12N9/1229C12N9/90C12N9/1085C12N9/1029C12P7/24C12Y203/0301C12Y101/01088C12Y401/01033C12Y207/01036C12Y207/04002C12Y503/03002C12Y205/01029C12Y205/01001C12Y402/03C12Y101/03038C12Y203/01009
Inventor 杜德尧白超弦张倩李腾张浩千
Owner SHENZHEN BLUEPHA BIOSCIENCES CO LTD