Pseudomonas stutzeri mutant strain as well as construction method and application thereof

A technology of Pseudomonas stutzeri and mutant strains, which is applied in the field of microorganisms, can solve the problems of unsatisfactory commercialization and low nitrogenase activity of DSM4166, and achieve the improvement of gene expression level of nitrogenase synthesis and broad commercial use. value effect

Active Publication Date: 2020-06-16
SHANDONG UNIV +1
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Problems solved by technology

[0003] Due to the fact that the nitrogenase activity of Pseudomonas stutzeri DSM4166 is not high at present and has not reached a satisfactory degree of commercialization, the genetic engineering method is used to further artificially design the nitrogen-fixing gene cluster by streamlining the nitrogen-fixing gene cluster and replacing the high-strength promoter. And reconstruct and optimize the biological nitrogen fixation pathway, greatly improve its nitrogen fixation capacity, strive to break through the limit of natural nitrogen fixation, and achieve a satisfactory degree of commercialization

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  • Pseudomonas stutzeri mutant strain as well as construction method and application thereof
  • Pseudomonas stutzeri mutant strain as well as construction method and application thereof
  • Pseudomonas stutzeri mutant strain as well as construction method and application thereof

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Embodiment 2

[0040] The construction and screening of embodiment 2 engineering strain DSM4166-TP (nifR) comprises the following steps:

[0041] 1. Screening of strong constitutive promoters in Pseudomonas stutzeri DSM4166

[0042] Pseudomonas stutzeri DSM4166 were cultured under four different culture conditions, LB medium, 30℃, 37℃; PMM medium, 30℃, 37℃.

[0043] The growth curves of P. stutzeri DSM4166 under four culture conditions were measured, as shown in Figure 1 .

[0044] Under each culture condition, three time points were selected for transcriptome sequencing, specifically:

[0045] Table 1 Selection of time points for transcriptome sequencing under different culture conditions

[0046]

[0047] The genes whose expression level is in the top 5% cut off were selected from the transcriptome data, as shown in Table 2.

[0048] Table 2 Genes with higher transcription levels in P. stutzeri DSM4166 under different culture conditions

[0049]

[0050]

[0051]

[0052] ...

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Abstract

The invention belongs to the technical field of microorganisms, and relates to a mutant strain as well as a construction method and application thereof. The pseudomonas stutzeri mutant strain is pseudomonas stutzeri DSM4166-TP (nifR), is preserved in China General Microbiological Culture Collection Center on December 20, 2019, and has a preservation number of CGMCC No.19081. The pseudomonas stutzeri mutant strain has a preservation number of CGMCC No.19081. The pseudomonas stutzeri mutant strain has the beneficial effects that compared with the existing wild type DSM4166 strain, the pseudomonas stutzeri mutant strain has the advantages that the nitrogenase synthesis gene expression level is greatly improved, and the activity of the produced nitrogenase is also greatly higher than that of the wild type DSM4166 strain. The pseudomonas stutzeri mutant strain disclosed by the invention can be industrially produced on a large scale and has a wide commercial value.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and relates to a mutant strain and its construction method and application. Background technique [0002] Pseudomonas stutzeri DSM4166 (Pseudomonas stutzeri DSM4166, formerly known as CMT.9.A) is a functional strain with nitrogen fixation ability, which was isolated from the rhizosphere of German sorghum (Sorghum nutans) in 1897. The bacterium has been preserved in the German Microorganism Collection Center (Deutsche SammLung von Mikroorganismen und Zellkulturen GmbH) at present, and the preservation number is DSM4166. The bacteria can convert nitrogen in the air into ammonium that can be directly used by plants under the conditions of no ammonia and microaerobic conditions. At present, the genome sequencing of this strain has been completed, and it is found that its genome contains a Nitrogen-fixing gene island (Nif), which is located between the cobs (PSTAA1334) and gshp (PSTAA1391) gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12Q1/66C12Q1/04C05F11/08C12R1/38
CPCC07K14/21C12Q1/66C12Q1/04C05F11/08
Inventor 于广乐王海龙涂强张友明
Owner SHANDONG UNIV
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