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Gibberella fujikuroi genetically engineered bacterium with high-yield gibberellin GA3, construction method and application

A technology of genetically engineered bacteria, Gibberella fujikura, applied in the field of metabolic engineering, can solve the problems such as the inability of the strain to synthesize uracil and the inability of the initial strain to survive, and achieve the effects of releasing transcription inhibition, weakening feedback inhibition and efficient fermentation production.

Pending Publication Date: 2021-12-24
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, orotic acid nucleoside-5'-phosphate decarboxylase can catalyze the synthesis of fluorine-containing nucleotide analogs from the fluorine-containing analog 5-fluoroorotic acid, which makes the initial strain containing the pyrG gene unable to survive
Knockout of the pyrG gene inactivates orotidine-5′-phosphate decarboxylase, and the strain cannot synthesize uracil

Method used

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  • Gibberella fujikuroi genetically engineered bacterium with high-yield gibberellin GA3, construction method and application
  • Gibberella fujikuroi genetically engineered bacterium with high-yield gibberellin GA3, construction method and application
  • Gibberella fujikuroi genetically engineered bacterium with high-yield gibberellin GA3, construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Acquisition of F. fujikuroi-ΔpyrG

[0032] Knockout of the pyrG gene:

[0033] In order to reduce the interference of negative transformants and improve the selection efficiency of transformation, the pyrG gene of Gibberella Fujikura was knocked out, see Van Hartingsveldt W, Mattern I E, van Zeijl C M J, etal.Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. Molecular and General Genetics MGG,1987,206(1):71-75.

[0034] The pyrG gene encoding orotidine-5'-phosphate decarboxylase in the genome of Gibberella fujikuroi (Fusarium fujikuroi) CCTCC NO: M2019378 (disclosed in CN110527630A) was knocked out by CRISPR-Cas9 system. Using primer 1 and primer 2 by PCR, the sgRNA-ΔpyrG-1 fragment was amplified using the sgRNA expression cassette as a template. The PCR reaction conditions were as follows: 98°C for 3min; 98°C for 15s, 60°C for 15s, and 72°C for 15s, repeating 30 cycles ; Continue at 72°C for 3 minutes.

[0035...

Embodiment 2

[0041] Example 2 F. Acquisition of Fujikuroi-RE:pyrGΔmeaB

[0042] (1) Completion of pyrG gene

[0043] The pyrG-deficient Gibberella fujikura cannot survive due to the inability to synthesize uracil. By complementing the pyrG gene, Gibberella can regain the ability to synthesize uracil, resulting in a reversion mutant strain that can survive on a plate without exogenous uracil . Using primers 11 and 12 by PCR, the complete pyrG expression cassette was amplified using the genome of Fusarium fujikuroi CCTCC NO: M2019378 as a template. The PCR reaction conditions were as follows: 98°C for 5min; 98°C for 30s, 60°C for 30s, 72°C for 1min30s, repeat 30 cycles; continue at 72°C for 5min. The PCR products were detected by 1.0% agarose gel electrophoresis and the purified fragments were recovered by cutting the gel.

[0044] (2) Amplification of upstream and downstream homology arms of meaB gene

[0045] The bZIP-type transcriptional repressor meaB represses the expression of the ...

Embodiment 3

[0053] Example 3: Acquisition of F.fujikuroi-RE:pyrG GpdA-tHmgRΔmeaB

[0054] (1) Enhanced expression of thmgR gene

[0055] 3-Hydroxy-3-methylglutaryl-CoA reductase encoded by hmgR gene in GA 3 In the synthesis of mevalonate, it acts as a rate-limiting enzyme in the mevalonate pathway. Since the C-terminus of 3-hydroxy-3-methylglutaryl-CoA reductase can bind to the endoplasmic reticulum membrane, the products of catalyzed synthesis trigger feedback inhibition. Using primers 23 and 24 by PCR, the gpdA promoter sequence was amplified using the plasmid pAN7-1 as a template, and the PCR reaction conditions were as follows: 98°C for 5 minutes; 98°C for 30s, 60°C for 30s, and 72°C for 30s, repeating 30 cycles; Continue at 72°C for 5 minutes.

[0056] The N-terminus of 3-hydroxy-3-methylglutaryl-CoA reductase responsible for catalysis was amplified by PCR using primers 25 and 26, using the genome of Fusarium fujikuroi CCTCC NO: M2019378 as a template For the coding region, the P...

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Abstract

The invention relates to a gibberella fujikuroi genetically engineered bacterium with high-yield gibberellin GA3, a construction method and application. A CRISPR-Cas9 gene editing technology is adopted to knock out related genes (pyrG) of a screening tag and regulatory genes (meaB) of a metabolic pathway, a catalytic group coding sequence thmgR of a precursor supply gene hmgR is over-expressed, so that a high-yield strain F.fujikuroi RE:pyrG GpdA-tHmgR delta meaB is constructed, and 2.35 g / L of a GA3 product can be accumulated after the strain is fermented by a fermentation medium for 168 hours. According to the gibberella fujikuroi genetically engineered bacterium with high-yield gibberellin GA3 and the construction method, the modified gibberella fujikuroi weakens feedback inhibition of precursor synthesis, improves precursor supply in a metabolic process, relieves transcription inhibition of GA3 related synthetic genes, and can perform fermentation production of GA3 more efficiently compared with an original strain; and the yield is increased from 2.0 g / L to 2.35 g / L, and a feasible route is provided for construction of engineering strains.

Description

(1) Technical field [0001] The invention belongs to the field of metabolic engineering, in particular to a high-yield gibberellin GA 3 Fujikura gibberella genetic engineering bacteria, construction method and application. (2) Background technology [0002] Gibberellin (Gibberellin Acid, GA 3 ) is a plant growth hormone synthesized by plants and some fungi. It is widely used in agriculture, horticulture and wine production because of its special ability to stimulate plant growth. Gibberella Fujikura was first discovered by Japanese plant pathologists in the study of rice bakanae disease. Subsequent studies have confirmed that Gibberella Fujikura naturally has a complete GA 3 synthetic ability. GA 3 Is a tetracyclic diterpene carboxylic acid, molecular formula C 19 h 22 o 6 , molecular weight 346.37, melting point 233-235°C, easily soluble in alcohols, acetone, ethyl acetate, sodium bicarbonate solution and phosphate buffer with pH 6.2, insoluble in water and ether. G...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/60C12N15/31C12N15/53C12N15/113C12N9/22C12N15/90C12P27/00C12R1/645
CPCC12N9/88C12N9/0006C12N15/113C12N15/1137C12N9/22C12N15/902C12N1/14C07K14/37C12P27/00C12Y401/01023C12Y101/01034C12N2310/20
Inventor 柳志强王浩南张博岑宇科翁春跃郑裕国
Owner ZHEJIANG UNIV OF TECH
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