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Application of sll0528 gene in improving oxidative stress tolerance of Synechocystis pcc6803

A PCC6803, oxidative stress technology, applied in the field of industrial microorganisms, can solve problems such as low tolerance, and achieve the effect of wide application prospects

Active Publication Date: 2022-05-24
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to overcome the disadvantages and deficiencies of the low oxidative stress tolerance of Synechocystis PCC6803 in the prior art, the object of the present invention is to provide an application of the sll0528 gene in improving the oxidative stress tolerance of Synechocystis PCC6803

Method used

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  • Application of sll0528 gene in improving oxidative stress tolerance of Synechocystis pcc6803
  • Application of sll0528 gene in improving oxidative stress tolerance of Synechocystis pcc6803
  • Application of sll0528 gene in improving oxidative stress tolerance of Synechocystis pcc6803

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Embodiment 1

[0045] The acquisition of the sll0528 gene overexpression strain Osll0528, including the construction of the homologous recombination double-crossover plasmid P3031, the homologous exchange between the homologous recombination double-crossover plasmid and the wild-type genome of Synechocystis sp. PCC6803, and the screening and verification of Osll0528:

[0046] (1) Construction of homologous recombination double-crossover plasmid P3031

[0047] Take wild-type Synechocystis PCC6803 genome as a template, design primers, take SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence table as the upstream and downstream primers of the target fragment slr2030, SEQ ID NO: 7 and SEQ ID NO: 8 are The upstream and downstream primers of the psbA2 promoter, SEQ ID NO: 9 and SEQ ID NO: 10 are the upstream and downstream primers of s110528, SEQ ID NO: 11 and SEQ ID NO: 12 are the upstream and downstream primers of slr2031, amplified by PCR. The target gene slr2030, psbA2 promoter, sll0528, slr2031 wer...

Embodiment 2

[0067] The gene knockout strain Δsll0528 is disclosed in the patent "201811591708.3, the application of sll0528 gene in improving the ethanol tolerance of Synechocystis PCC6803".

[0068] Phenotypic analysis of Synechocystis PCC6803 wild-type WT, gene knockout strain Δsll0528, and gene overexpression strain Osll0528 under menadione oxidative stress:

[0069]The wild-type WT, knockout strain Δsll0528, and overexpression strain Osll0528 grown to log phase were used as seed solutions to inoculate with 0 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 12.5 μM, 15 μM, and 17.5 μM menadione, respectively. The starting OD of each bottle of algae in 50 mL of BG11 medium 730 = 0.1. Measure Day 3 OD 730 , with 0 μM menadione as the control, the OD of the algae under each stress 730 OD with the control group 730 For comparison, the OD 730 (% Control) represents the survival rate of WT, Δs110528 and Os110528 under the oxidative stress of menadione at each concentration, and the survival rate curve ...

Embodiment 3

[0077] Phenotypic analysis of Synechocystis PCC6803 wild-type WT, gene knockout strain Δsll0528, and gene overexpression strain Osll0528 under hydrogen peroxide stress:

[0078] The wild-type WT, knockout strain Δs110528, and overexpression strain Os110528 grown to log phase were used as seed solution, inoculated into cells containing 0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM H, respectively. 2 O 2 The starting OD of each bottle of algae in 30 mL of BG11 medium 730 = 0.24. Measure 12h OD 730 , with 0mM H 2 O 2 The middle algae was the control, and the algae OD under each stress was 730 OD with the control group 730 For comparison, the OD 730 (% Control) represents the survival rate of WT, Δs110528 and Os110528 under the stress of hydrogen peroxide at each concentration, and the survival rate curve is drawn.

[0079] The wild-type WT, knockout strain Δs110528, and overexpression strain Os110528 grown to log phase were used as seed solutions, inoculated into 2.5 mM or 3 m...

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Abstract

The invention discloses the application of a sll0528 gene in improving the oxidative stress tolerance of Synechocystis sp. PCC6803, which belongs to the field of industrial microbes. The present invention overexpresses the s110528 gene in Synechocystis PCC6803 through homologous recombination, and obtains a Synechocystis PCC6803 strain Os110528 with significantly improved tolerance to oxidative stress. In the BG11 medium supplemented with different concentrations (2.5μM-15μM) of menadione or different concentrations (1mM-3mM) of hydrogen peroxide, the growth state of Osll0528 was significantly better than that of the wild-type strain. The oxidative stress-tolerant algae strain obtained by the invention has important theoretical and practical significance and broad application prospects for constructing genetically engineered bacteria tolerant to high oxidative stress and genetically engineered algal strain chassis tolerant to various environmental stresses.

Description

technical field [0001] The invention belongs to the field of industrial microorganisms, in particular to the application of a sll0528 gene in improving the oxidative stress tolerance of Synechocystis PCC6803. Background technique [0002] Reactive Oxygen Species (ROS) include singlet oxygen ( 1 O 2 ), superoxide anion (O 2 · - ), hydrogen peroxide (H 2 O 2 ) and hydroxyl radicals (OH·), etc., which are strong oxidants in cells, which can inhibit the synthesis of proteins, which may lead to the cleavage of nucleic acids and the peroxidation of unsaturated fatty acids. [0003] Synechocystis PCC6803 has a simple genetic background and complete disclosure of genetic information. It is regarded as a model organism for the study of new energy development and production of new raw materials. Studies have shown that Synechocystis is a new generation of biofuels such as ethanol and alkanes, as well as free fatty acids, industrial Quality producer of chemicals such as enzymes, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/65C12N15/74C12R1/01
CPCC07K14/195C12N15/65C12N15/74
Inventor 陈谷刘秤利许白雪林诗琪周健
Owner SOUTH CHINA UNIV OF TECH
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