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Method for improving ethanol tolerance of synechocystis PCC6803 and application

A technology of PCC6803 and Synechocystis, applied in the field of industrial microorganisms, can solve the problems of reduced growth rate and poor ethanol tolerance, and achieve the effect of improved tolerance and wide application prospects

Active Publication Date: 2017-02-15
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology can improve alcohol production from synechiacystis pneumoniae cells without causing damage or reducing their ability to produce other substances like sugar. By making certain changes on these cell lines, they are able to increase resistance against high levels of ethylene gas used during fermentations. These modifications make them more effective at controlling yeast metabolism while also improving its capacity to resist low concentrated gases such as nitrogen oxide emissions. Overall, this new technique makes it possible to creature biofuel crops called BACYCZS which could be useful for developing sustainable energy sources.

Problems solved by technology

The technical problem addressed in this patents relates to finding ways to improve the resistance or tolerant capacity of certain types of microorganisms used for producing industrially useful chemicals such as acetone from glucose-based syngases that have high concentrations of sugar compared with other sources like corn starch.

Method used

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  • Method for improving ethanol tolerance of synechocystis PCC6803 and application
  • Method for improving ethanol tolerance of synechocystis PCC6803 and application
  • Method for improving ethanol tolerance of synechocystis PCC6803 and application

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Experimental program
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Effect test

Embodiment 1

[0023] Homologous recombination double crossover plasmid pUC118-up-down-Km r The build:

[0024] (1) Amplification of the insert:

[0025] 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 the upstream and downstream primers, using the pET-30b plasmid as a template, the kanamycin gene and its upstream and downstream partial sequences were amplified by PCR Km r , as shown in SEQ ID NO:9. The genome extraction of Synechocy...

Embodiment 2

[0032] Obtainment of Synechocystis PCC6803 strain with significantly improved tolerance to ethanol:

[0033] (1) Plasmid transformation

[0034] pUC118-up-down-Km 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. After the algae solution was applied to the solid medium covered with mixed fiber filter membrane and cultured under light for 1 day (upright culture), the me...

Embodiment 3

[0040] Analysis of the growth status of IK2 strains under ethanol stress:

[0041] Measure the growth curve of the IK2 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 and inoculated to 50mL of BG11 medium containing , 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 IK2 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 a method for improving ethanol tolerance of synechocystis PCC6803 and an application, and belongs to the field of industrial microorganism. With the application of the method, a synechocystis PCC6803 strain with ethanol tolerance obviously improved is obtained, and the strain is applicable to the construction of genetically engineered bacteria for producing fuel ethanol. According to the method provided by the invention, an sll0687 gene in the synechocystis PCC6803 is knocked out through homologous recombination; and the ethanol tolerance of the synechocystis PCC6803 strain IK2 obtained through the application of the method is significantly improved. Under 1.5% (v/v) ethanol stress, the growth state of the strain is obviously better than that of a wild-type strain. The ethanol-tolerance strain prepared by the invention has important theoretical and practical significance for the construction of the genetically engineered bacteria for producing the fuel ethanol; and the strain has a broad application prospect.

Description

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Claims

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Application Information

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Owner SOUTH CHINA UNIV OF TECH
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