A Synechocystis pcc6803 algal strain with significantly improved tolerance to ethanol and its construction method

A technology of PCC6803 and Synechocystis, which is applied in microorganism-based methods, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problems of poor ethanol tolerance and reduced growth rate, and achieve the effect of wide application prospects.

Active Publication Date: 2019-06-18
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the ethanol content produced by transgenic Synechocystis PCC6803 can not meet the requirements of industrial production of ethanol, one of the key factors is the poor tolerance of Synechocystis PCC6803 to ethanol
In the medium containing 1.5% (v / v) ethanol, Synechocystis PCC6803 cells will aggregate and the growth rate will be reduced by more than 50%

Method used

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  • A Synechocystis pcc6803 algal strain with significantly improved tolerance to ethanol and its construction method
  • A Synechocystis pcc6803 algal strain with significantly improved tolerance to ethanol and its construction method
  • A Synechocystis pcc6803 algal strain with significantly improved tolerance to ethanol and its construction method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Homologous recombination double crossover plasmid pUC118-up-down-Sm r The build:

[0025] (1) Amplification of the insert:

[0026] Using SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing as upstream and downstream primers, using the wild-type Synechocystis PCC6803 genome as a template, the upstream sequence sigI-up of the sll0687 gene was obtained by PCR amplification, as shown in SEQ ID NO in the sequence listing Shown in ID NO: 7; using SEQ ID NO: 3 and SEQ ID NO: 4 as the upstream and downstream primers, using the wild-type Synechocystis PCC6803 genome as a template, the downstream sequence sigI-down of the sll0687 gene was obtained by PCR amplification, as shown in Shown in SEQ ID NO: 8; using SEQ ID NO: 5 and SEQ ID NO: 6 as upstream and downstream primers, and using pCDFDuet-1 plasmid as a template, the streptomycin gene and its upstream and downstream partial sequences were amplified by PCR SM r , as shown in SEQ ID NO:9. The genome extraction of Synecho...

Embodiment 2

[0033] Obtainment of Synechocystis PCC6803 strain with significantly improved tolerance to ethanol:

[0034] (1) Plasmid transformation

[0035] pUC118-up-down-Sm r After the plasmid was sterilized by filtration with a 0.22 μm microporous membrane, it was loaded into a 2 mL sterile centrifuge tube. A certain amount of BG11 medium (with HEPES buffer added) was added thereto, so that the final concentration of the plasmid was about 10 ng / μL. Take 30 mL of wild-type PCC6803 in logarithmic phase, centrifuge at 6000 rpm for 7 min, and remove the supernatant. Resuspend the algae mud with 20mL of fresh BG11 medium, centrifuge at 6000rpm for 7min, and remove the supernatant. Resuspend the algae mud with plasmid-containing medium. The resuspended algae liquid was cultured at 29°C, 150rpm, and 1400Lux continuous light for 6h. Apply the algae liquid to the solid medium covered with mixed fiber filter membrane and culture under light for 1 day (upright culture), then transfer the mem...

Embodiment 3

[0041] Analysis of the growth state of ISm5 strain under ethanol stress:

[0042] Measure the growth curve of the ISm5 algal strain obtained in Synechocystis PCC6803 wild type and Example 2 under 1.5% (v / v) ethanol stress: the algae grown to the logarithmic phase are used as seed liquid, inoculated into 50mL BG11 culture In the base, the starting OD of each bottle of algae 730 = 0.1. Continuous culture for 4 days, sampling OD every day 730 value to draw a growth curve. The culture conditions are 29°C, 150rpm, 1400Lux continuous light. At the beginning of the culture, ethanol was added to the medium of the wild-type and ISm5 experimental groups to a final concentration of 1.5% (v / v). The experimental group and the control group each had three parallels.

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Abstract

The invention discloses synechocystis PCC6803 alga for achieving remarkable improvement of tolerance to ethyl alcohol and a construction method thereof and belongs to the field of industrial microorganisms. Sll0687 genes in synechocystis PCC6803 are knocked out through a homologous recombination method, and the synechocystis PCC6803 alga ISm5 for remarkably improving tolerance to ethyl alcohol is obtained. Under stress of 1.5% (v / v) ethyl alcohol, the growth state of the alga is obviously superior to that of wild alga. The obtained ethyl-alcohol-tolerant alga has important theoretical and practical significance for constructing genetically engineered bacteria for producing fuel ethanol and has wide application prospects.

Description

technical field [0001] The invention belongs to the field of industrial microorganisms, and in particular relates to a Synechocystis PCC6803 algal strain with significantly improved ethanol tolerance and a construction method thereof. Background technique [0002] In response to the increasingly severe energy crisis, ethanol as a biofuel has become one of the options to replace petroleum fuels. Since ethanol produced by heterotrophic fermentation requires food crops as fuel, the increase in ethanol demand will exacerbate the food shortage problem. Using photosynthetic cyanobacteria as a bioreactor to convert CO 2 Conversion into ethanol is an important solution to this problem. Synechocystis sp. PCC6803 is easy to carry out transgenic operation, and it is a good genetic engineering bacterium for this research. For example, the pyruvate decarboxylase (pdc) gene and alcohol dehydrogenase II (adh) gene in Zymomonas mobilis are introduced into Synechocystis sp. Low concentra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74C12N15/66C12N15/65C12N1/21C12R1/01
CPCC07K14/195C12N15/65C12N15/66C12N15/74
Inventor 陈谷丁清龙
Owner SOUTH CHINA UNIV OF TECH
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