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A community of microbial gene expression destiny and its construction method and application

A construction method and a technology for recombining microorganisms, which are applied in the field of microbial gene expression community of destiny and its construction, can solve problems such as differences in gene expression levels, uneven gene expression, and group differences that are unfavorable for obtaining biological products, and achieve amplified production. , reliable and sustainable expression, reducing the effect of stress resistance

Active Publication Date: 2022-03-11
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the context of population diversity, bacterial colonies will have uneven gene expression, resulting in huge differences in the gene expression levels of product synthesis pathways, and there are also huge differences in the accumulation of target products by microorganisms (Guodong et al, 2013; Paolo et al, 2006); while large population differences are not conducive to obtaining maximum yields of biological products

Method used

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  • A community of microbial gene expression destiny and its construction method and application
  • A community of microbial gene expression destiny and its construction method and application
  • A community of microbial gene expression destiny and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, by in halophilic bacteria Halomonas bluephagenesis outer membrane protein gene wxya insert behind phaCAB Genetically obtained PHA-producing community strain

[0037] exist Halomonas bluephagenesis TD01 strain wxya Insertion behind the gene encoding an outer membrane protein, a strongly expressed essential gene phaC Gene, phaA Gene, phaB genes, achieved phaCAB The high-strength and stable expression of PHA has achieved the accumulation of high-concentration PHA, and the strain of the community of destiny for PHA production has been constructed.

[0038] The specific operation process is as follows:

[0039] Using CRISPR-Cas9 technology in Halomonas Halomonas bluephagenesis TD01 genome wxya post-gene insertion phaC Gene, phaA Gene, phaB Genes to obtain strains with reliable expression of genes related to PHA synthesis pathway. details as follows:

[0040] 1) Insert gene

[0041] The original expression vector used to express sgRN...

Embodiment 2

[0055] Embodiment 2, by in halophilic bacteria Halomonas campaniensis porin gene of LS21 porin front insert phaCAB Genetically obtained PHA-producing community strain

[0056] exist Halomonas campaniensis LS21 strain porin Insertion in front of the gene encoding a porin, a strongly expressed essential gene phaC Gene, phaA Gene, phaB genes, achieved phaCAB The high-strength and stable expression of PHA has achieved the accumulation of high-concentration PHA, and the strain of the community of destiny for PHA production has been constructed.

[0057] The specific operation process is as follows:

[0058] Using CRISPR-Cas9 technology in Halomonas Halomonas campaniensis LS21 Genome porin Gene front insertion phaC Gene, phaA Gene, phaB Genes to obtain strains with reliable expression of genes related to PHA synthesis pathway. details as follows:

[0059] 1) Insert gene

[0060] The original expression vector used to express sgRNA and donor DNA is pSEVA...

Embodiment 3

[0071] Example 3, inserting key genes in the ectoine synthesis pathway into Halomonas aydingkolgenesis In front of the essential genes of M1, the production genes and essential genes are co-expressed to form a community of destiny strains

[0072] in essential genes ftsZ (cell division related, moderately expressed), wxya (ribosome-associated gene, strongly expressed) and edd (the key gene in the ED pathway, weakly expressed) was respectively inserted into the key gene in the ectoine synthesis pathway ectB ( Figure 4 ), such that ectB The genes were expressed simultaneously with the three essential genes in a high-intensity and stable manner, realizing the stable production of ectoine, and constructing a community of destiny strain for ectoine production.

[0073] The specific operation process is as follows:

[0074] Using CRISPR-Cas9 technology in Halomonas Halomonas aydingkolgenesis in the M1 genome ftsZ Gene, wxya Gene, edd Gene front insertion ectB...

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Abstract

The invention discloses a microbial gene expression community of destiny and its construction method and application. The microbial gene expression community of destiny includes at least the essential genes of cells and genes related to the coding product synthesis pathway, and the essential genes are co-expressed with the genes related to the product synthesis pathway. It can realize the coexistence of life and death between biological cells and products, realize stable production of products, and improve production efficiency. By serially expressing the essential genes of microorganisms and the genes encoding the product synthesis pathway, the resistance of microorganisms to environmental changes is reduced, and more stable expression of target genes is achieved, which reduces the limitations and constraints of biomanufacturing, and is more conducive to Subsequent enlargement of production and improvement of product yield. The method will play an important role in biomanufacturing and bioindustrial technology, and greatly reduce the manufacturing cost of products.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular to the technical field of microbial genetic engineering, in particular to a microbial gene expression community of destiny and its construction method and application. Background technique [0002] Since modern times, the birth and development of bioengineering technology has had a huge impact on human production and life. As an emerging comprehensive application technology, bioengineering technology is based on biology and combines advanced engineering technologies such as chemical engineering, machinery and electronic computers to realize the manipulation and directional transformation of genetic material in cell factories. Reactors are cultivated on a large scale to achieve the production of large quantities of metabolites or their physiological functions (Muhammad et al, 2020; Otero et al, 2010). Due to its mild reaction conditions and environmentally friendly develop...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/75C12N15/81C12N15/80C12N15/82C12P7/625
CPCC12N15/70C12N15/75C12N15/81C12N15/80C12N15/82C12P7/625
Inventor 陈国强王子瑜纪梦珂王欢胡启跳赵翠环张李湛林艺娜马悦原黄悟哲郑陶然
Owner TSINGHUA UNIV
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