Genetic engineering microorganism for producing ethanol

A genetic engineering and genetic technology, applied in the field of ethanol production by genetically engineered microorganisms, can solve the problems of not producing ethanol

Inactive Publication Date: 2013-10-23
ZHEJIANG QICHENG TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The pyruvate decarboxylase gene pdc does not exist in the genome of cyanobacteria, so it does not have the ability to use sunlight and carbon dioxide to produce ethanol

Method used

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  • Genetic engineering microorganism for producing ethanol
  • Genetic engineering microorganism for producing ethanol
  • Genetic engineering microorganism for producing ethanol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1: Construction of vectors for expressing pyruvate decarboxylase and alcohol dehydrogenase

[0093] In order to exogenously introduce pyruvate decarboxylase into cyanobacteria and increase the production of pyruvate decarboxylase and alcohol dehydrogenase in cyanobacteria, the plasmid pCE03 capable of expressing the pyruvate decarboxylase gene ZmPDC derived from Zymomonas mobilis was constructed as follows And the plasmid pCE04 capable of expressing the endogenous alcohol dehydrogenase gene slr1192 derived from cyanobacterium PCC6803.

[0094] 1. Construction of plasmid pCE03

[0095] Using ZmPDC-F (5′-GCG GCA GCC ATA TGA GTT ATA CTG TCG-3′) and ZmPDC-R (5′-AGC TCG TCT CGA GTC TAG AGG AGC TTG-3′) as primers, motile Zymomonas Bacteria (Zymomonas Mobilis) genomic DNA was used as a template for PCR amplification, and according to the manufacturer's instructions, the PCR amplification product was cloned into pMD18-T vector (Takara, Catalog No.: D101A) to obtain pla...

Embodiment 2

[0098] Example 2: Construction of vectors for gene knock-in and gene knock-out

[0099] In order to confirm the role of pyruvate decarboxylase and alcohol dehydrogenase in the production of ethanol in cyanobacteria, and confirm that the production of ethanol in cyanobacteria can be improved by increasing the expression of the enzymes, the following constructions for the production of ethanol by the Prbc promoter The pyruvate decarboxylase gene (ZmPDC) and the alcohol dehydrogenase gene (slr1192) were integrated into the vector pCE09 at the slr0168 site of the cyanobacterial genome, and it was confirmed that by further increasing the pyruvate decarboxylase and alcohol dehydrogenase in ethanologenic cyanobacteria In order to increase the production of ethanol, a vector pCE11 for integrating the pyruvate decarboxylase gene (ZmPDC) and the alcohol dehydrogenase gene (slr1192) driven by the Prbc promoter into the cyanobacteria genome slr1193-1194 site was constructed as follows.

[0...

Embodiment 3

[0107] Embodiment 3: Transformation of cyanobacteria and screening of transformants

[0108] Transformation of cyanobacteria and selection of transformants were carried out as follows:

[0109] 1. Take 10 mL of cyanobacterial cells in the logarithmic growth phase (OD730 is about 0.5-1.0), and collect the cells by centrifugation; wash the cells twice with fresh BG11 medium, and then resuspend the cells in 1 mL of BG11 medium (1.5 g L -1 NaNO3, 40 mg L -1 K 2 HPO 4 ·3H 2 O, 36 mg L -1 CaCl2 2H2O, 6mg / L citric acid, 6mg / L ferric ammonium citrate, 1mg L -1 EDTA disodium salt, 20mg L -1 NaCO 3 , 2.9 mg L -1 h 3 BO 3 , 1.8 mg L -1 MnCl 2 4H 2 O, 0.22 mg L -1 ZnSO 4 7H2O, 0.39 mg L -1 NaMoO 4 2H 2 O, 0.079 mg L -1 CuSO 4 ·5H 2 O and 0.01 mg L -1 CoCl 2 ·6H 2 O).

[0110] 2. Take 0.2mL cell suspension into a new EP tube, add 2~3μg expression plasmid, mix well, and place at 30℃, 30μEm -2 the s -1 Incubate for 5 hours under light conditions.

[0111] 3. Spre...

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Abstract

The invention provides a genetic engineering blue-green alga for producing ethanol through photosynthesis. The genetic engineering blue-green alga contains exogenous pyruvate decarboxylase genes and ethanol dehydrogenase which are integrated in chromosomes. The invention further provides a construction body, a carrier and a preparation method for preparing the genetic engineering blue-green alga, and a method for producing ethanol by using the genetic engineering blue-green alga.

Description

technical field [0001] The invention relates to the fields of renewable energy and biotechnology. Specifically, it relates to a microorganism capable of efficiently producing ethanol through photosynthesis after being modified by genetic engineering, a method for modifying the microorganism by genetic engineering, and a method for preparing ethanol by using the genetically engineered microorganism. Background technique [0002] Since the beginning of the 21st century, the price of crude oil has remained high, the cost of development and utilization of solar energy, wind energy, and tidal energy has remained high, and the requirements for environmental protection in various countries are constantly improving. Therefore, the issue of how to develop alternative energy sources has attracted widespread attention from all countries. Many countries regard accelerating the development of renewable energy as an important strategy for the development of alternative energy, and bioener...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/13C12N15/60C12N15/53C12N15/63C12R1/89
Inventor 范文俊郑晓光
Owner ZHEJIANG QICHENG TECH
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