Recombinant microorganism for generating terpenoid and construction method thereof

一种重组菌、合成基因的技术，应用在生物领域，能够解决MEP途径效率低等问题

Active Publication Date: 2013-05-08
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Insufficient supply of cofactors leads to inefficiency of the MEP pathway

Method used

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  • Recombinant microorganism for generating terpenoid and construction method thereof
  • Recombinant microorganism for generating terpenoid and construction method thereof
  • Recombinant microorganism for generating terpenoid and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Embodiment 1, β-carotene synthesis gene is introduced into Escherichia coli to obtain recombinant Escherichia coli

[0105] 1. Construction of recombinant Escherichia coli ATCC8739 (pACYC184M-crt) by introducing β-carotene synthesis gene through plasmid

[0106]Escherichia coli itself can synthesize IPP, DMAPP and FPP through the MEP pathway and FPP synthetase. But Escherichia coli cannot synthesize β-carotene by itself. The β-carotene synthesis genes clustered under the same operon in Pantoea agglomerans, and the β-carotene synthesis gene cluster was composed of geranyl geranyl diphosphate (GGPP) synthase gene (crtE , SEQ ID NO: 1), β-carotene cyclase gene (crtX, SEQ ID NO: 2), lycopene β-cyclase gene (crtY, SEQ ID NO: 3), phytoene desaturase gene (crtI, SEQ ID NO: 4 ), phytoene synthase gene (crtB, sequence 5).

[0107] 1. Construction of recombinant Escherichia coli ATCC8739 (pACYC184-M-crt)

[0108] Introducing the β-carotene synthesis gene cluster into Escheric...

Embodiment 2

[0167] Example 2, construction and fermentation of recombinant Escherichia coli CAR001

[0168] The construction and fermentation of recombinant Escherichia coli CAR001 are divided into the following 8 steps:

[0169] 1. Improving the expression intensity of the β-carotene synthesis gene cluster in recombinant Escherichia coli QL002 and constructing recombinant Escherichia coli QL105

[0170]Recombinant Escherichia coli QL105 replaces the trc regulatory element (sequence 6 in the sequence listing) of the β-carotene synthesis gene cluster crtEXYIB in QL002 with the M1-12 artificial regulatory element (sequence 7 in the sequence listing) by two-step homologous recombination .

[0171] The artificial regulatory element is inserted in front of the gene to be regulated through two-step homologous recombination, and no resistance gene or FRT marker fragment is left after the operation. In the first step of homologous recombination, the original regulatory elements of the gene to b...

Embodiment 3

[0248] Example 3, Improving the expression intensity of α-ketoglutarate dehydrogenase gene of recombinant Escherichia coli CAR001

[0249] 1. Improve the expression intensity of α-ketoglutarate dehydrogenase gene of recombinant Escherichia coli CAR001 and construct recombinant bacteria SucAB46-FKF, SucAB37-FKF, SucAB93-FKF

[0250] Recombinant Escherichia coli SucAB46-FKF, SucAB37-FKF, and SucAB93-FKF are respectively replacing the original regulatory element (sequence 15) of the α-ketoglutarate dehydrogenase gene (sucAB) of recombinant Escherichia coli CAR001 with artificial regulatory element M1 -46 (Sequence 14), M1-37 (Sequence 10), M1-93 (Sequence 11).

[0251] Through a pair of universal primers, the original regulatory regions of genes on the chromosome of Escherichia coli are replaced by artificial regulatory elements with various strengths by using a one-step homologous recombination method. The upstream primer is gene-up-FRT, including 50 bases outside the original ...

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Abstract

The invention discloses a recombinant microorganism for generating terpenoid and a construction method of the microorganism. A recombinant stain 1 is provided by the invention to improve the activity of alpha-ketoglutarate dehydrogenase in colon bacillus or a mutant strain of the colon bacillus to obtain a recombinant strain. The method for improving the activity of the ketoglutarate dehydrogenase in the colon bacillus or the mutant strain of the colon bacillus comprises the following step of substituting any one of the following regulatory elements for the original regulatory element of a ketoglutarate dehydrogenase gene suc AB in the colon bacillus or the mutant strain of the colon bacillus: artificial regulatory elements M1-46, M1-37, and M1-93. Experiments prove that, according to the construction method disclosed by the invention, a plurality of recombinant strains are constructed, and NADPH (Nicotinamide Adenine Dinucleotide Phosphate) and ATP (Adenosine Triphosphate) synthesis capacity of a cell is improved by improving the activities of the alpha-ketoglutarate dehydrogenase, succinate dehydrogenase and transaldolase so as to improve efficiency of MEP (2-C-Methyl-D-Erythritol-4-Phosphate) pathway and terpenoid production capacity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant microorganism and a construction method for producing terpenoids. Background technique [0002] Terpenoids widely exist in higher plants, fungi, microorganisms, insects, and marine organisms in nature. More than 50,000 terpenes have been discovered so far, most of which are biologically active ingredients in medicine or health products, such as monoterpenes Monoterpenes (C10): menthol, linalool; sesquiterpene Sesquiterpenes (C15): Artemisinin (Artemissinin); Diterpenes (C20): Paclitaxel (Taxol), Tanshinone ⅡA (Tanshinone ⅡA); Triterpenes (C30): Ginsenosides (Ginsenosides), Notoginsenosides (Notoginsenosides) , Glycyrrhizine; Tetraterpenes (C40): Lycopene, β-carotene, Zeaxanthin, Cathaxanthin and Astaxanthin ) and other β-carotene (Carotenoides); Polyterpenes: Coenzyme Q10 (Coenzyme Q10); Terpenoid alkaloids: Dendrobine, Gentianine, Aconitine and Reserpine ( Reserpine...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N1/20C12N15/09C12P23/00C12R1/19
CPCC12N15/113C12P23/00C12R1/19C12N15/70C12N9/0008C12Y102/04002C12P5/007
Inventor 张学礼李清艳赵婧孙涛戴冠苹徐洪涛唐金磊马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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