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Constructs, strains and methods for ethanol biosynthesis by cyanobacteria

A technology of cyanobacteria and microbial strains is applied in the field of high-efficiency biosynthesis of ethanol, and can solve the problems of increased production cost, high energy consumption, and inconsistent accumulation of microalgae biomass and oil esters.

Active Publication Date: 2018-02-09
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the main bottleneck problem it faces is that the biomass accumulation of microalgae is not consistent with the accumulation of oil and ester, and the energy consumption of the collection of algae cells, dehydration and removal of glycerol, a by-product of transesterification reaction, is high.
In a word, although cellulosic ethanol and microalgae biodiesel, two important development routes of bio-liquid fuels, have good development prospects, due to the existence of some bottleneck problems mentioned above, the production cost is greatly increased, which seriously restricts their promotion to industrialization. application

Method used

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  • Constructs, strains and methods for ethanol biosynthesis by cyanobacteria
  • Constructs, strains and methods for ethanol biosynthesis by cyanobacteria
  • Constructs, strains and methods for ethanol biosynthesis by cyanobacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Example 1: Construction of vectors for expressing pyruvate decarboxylase and alcohol dehydrogenase

[0109] In order to introduce pyruvate decarboxylase exogenously 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 that can express the endogenous alcohol dehydrogenase gene slr1192 derived from the cyanobacteria PCC6803.

[0110] 1. Construction of plasmid pCE03

[0111] Using ZmPDC-F (5′-GCG GCA GCC ATA TGA GTT ATA CTG TCG-3′) and ZmPDC-R (5′-AGCTCG TCT CGA GTC TAG AGG AGC TTG-3′) as primers, using Zymomonas mobilis (Zymomonas Mobilis) genomic DNA was used as a template for PCR amplification, and according to the manufacturer’s instructions, the PCR amplified product was cloned into pMD18-T vector (Takara, Catalog No.: D101A) to obtain plasmid pCE01. The ...

Embodiment 2

[0114] Example 2: Construction of vectors for gene knock-in and gene knock-out

[0115] In order to confirm the role of pyruvate decarboxylase and alcohol dehydrogenase in the production of ethanol by cyanobacteria, and to confirm that the production of ethanol in cyanobacteria can be increased by increasing the expression of the enzymes, the following constructs are used to drive 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 cyanobacteria genome, and it was confirmed that pyruvate decarboxylase and alcohol dehydrogenase could be further increased in ethanol-producing cyanobacteria The yield of cyanobacteria is to increase the yield of ethanol. The vector pCE11 for integrating the pyruvate decarboxylase gene (ZmPDC) and alcohol dehydrogenase gene (slr1192) driven by the Prbc promoter into the cyanobacteria genome slr1193-1194 site was constructed as follows.

[...

Embodiment 3

[0123] Example 3: Transformation of Cyanobacteria and Screening of Transformants

[0124] The transformation of cyanobacteria and the selection of transformants are carried out as follows:

[0125] 1. Take 10 mL of cyanobacterial cells in the logarithmic growth phase (OD730 is about 0.5~1.0), collect the cells by centrifugation; wash the cells twice with fresh BG11 medium, and then resuspend the cells in 1mL BG11 medium (1.5g L -1 NaNO3, 40mgL -1 K 2 HPO 4 ·3H 2 O, 36mg L -1 CaCl2·2H2O, 6mg / L citric acid, 6mg / L ferric ammonium citrate, 1mg L -1 EDTA disodium salt, 20mg L -1 NaCO3, 2.9mg L -1 H 3 BO 3 , 1.8mg L -1 MnCl 2 ·4H 2 O, 0.22mg L -1 ZnSO 4 ·7H2O, 0.39mg L -1 NaMoO 4 ·2H 2 O, 0.079mg L -1 CuSO 4 ·5H 2 O and 0.01mg L -1 CoCl 2 ·6H 2 O) In.

[0126] 2. Take 0.2mL cell suspension into a new EP tube, add 2~3μg expression plasmid, mix well, and place at 30℃, 30μEm -2 s -1 Incubate for 5 hours under light conditions.

[0127] 3. Spread the mixture of cyanobacteria cells and DNA ...

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Abstract

The invention provides a construction body for efficiently biosynthesizing ethanol in cyanobacteria, which comprises a first gene and a second gene operationally connected with a promoter having activity in cyanobacteria, wherein the first gene is selected from pyruvate decarboxylase genes; and the second gene is selected from alcohol dehydrogenase genes. The invention further provides a carrier comprising the construction body, cyanobacteria comprising the construction body or converted by using the carrier, and a method for efficiently biosynthesizing ethanol in cyanobacteria, wherein cyanobacteria is subjected to gene modification, and therefore, ethanol can be generated.

Description

Technical field [0001] The invention relates to the field of renewable energy and biomass energy. Specifically, the present invention relates to a construct for efficient biosynthesis of ethanol in cyanobacteria, a vector containing the construct, a cyanobacterium containing the construct or transformed with the vector, and an efficient A method of biosynthesis of ethanol, wherein the cyanobacteria have been genetically modified to produce ethanol. Background technique [0002] The continuous acceleration of energy demand and rising energy prices, the insufficient supply of petrochemical energy, and the environmental pollution and climate change caused by the large-scale use of petrochemical energy have made the development of renewable alternative energy urgent. As a renewable alternative energy source, biofuel is one of the important means to solve many problems currently faced. At present, the first-generation biofuel development route that uses food crops such as corn or ra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/60C12N15/53C12N1/13C12P7/06C12R1/89
CPCY02E50/10
Inventor 吕雪峰赵辉谈晓明范文俊郑晓光李志敏高政绪
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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