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Methods and compositions for improving engineered microbes

A composition and genetic engineering technology, applied in biochemical equipment and methods, botanical equipment and methods, fertilization methods, etc., can solve problems such as unclear pathways

Inactive Publication Date: 2020-08-14
皮沃特生物股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although tremendous progress has been made in understanding the nitrogen-fixing symbiosis between rhizobacteria and legumes, the pathways by which this knowledge can be used to induce nitrogen-fixing nodules on non-legume crops remain unclear
At the same time, the challenge of providing adequate supplemental sources of nitrogen, such as fertilizers, will continue to increase with the growing need to increase food production

Method used

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  • Methods and compositions for improving engineered microbes
  • Methods and compositions for improving engineered microbes
  • Methods and compositions for improving engineered microbes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0315] Example 1: Transcriptome profiling of candidate microorganisms

[0316] Transcriptome profiling of strain CI010 was performed to identify promoters active in the presence of ambient nitrogen. Strain CI010 was grown in defined nitrogen-free medium supplemented with 10 mM glutamine. Total RNA was extracted from these cultures (QIAGEN RNeasy kit) and subjected to RNAseq sequencing by Illumina HiSeq (SeqMatic, Fairmont, CA). Geneious was used to map sequencing reads to CI010 genomic data and identify highly expressed genes under the control of proximal transcriptional promoters.

[0317] Tables 3 and 4 list the genes and their relative expression levels measured by RNASeq sequencing of total RNA. Sequences of proximal promoters are recorded for mutagenesis of nif pathways, nitrogen utilization-related pathways, colonization-related pathways, or genes with desired expression levels.

[0318] table 3

[0319]

[0320]

[0321] Table 4

[0322]

Embodiment 2

[0323] Example 2: Mutagenesis of Candidate Microorganisms

[0324] λ-Red-mediated knockout

[0325] Several mutants of candidate microorganisms were generated using plasmid pKD46 or derivatives containing a kanamycin resistance marker (Datsenko et al. 2000; PNAS 97(12):6640-6645). A knockout cassette was designed with 250 bp of homology flanking the target gene and generated via overlap extension PCR. Candidate microorganisms were transformed with pKD46, grown in the presence of arabinose to induce λ-Red mechanical expression, ready for electroporation, and transformed with the knockout cassette to generate candidate mutagenized strains. As shown in Table 5, thirteen candidate mutants of the nitrogen fixation regulatory genes nifL, glnB and amtB were generated using four candidate microorganisms and one laboratory strain, Klebsiella oxytoca M5A1.

[0326]

[0327] Table 5: List of individual knockout mutants generated by λ-Red mutagenesis

[0328] Oligonucleotide-directe...

Embodiment 3

[0332] Example 3: In-Plant Phenotypes of Candidate Microorganisms

[0333] Colonization of plants by candidate microorganisms

[0334]Colonization of desired host plants by candidate microorganisms is quantified by short-term plant growth experiments. Maize plants were inoculated with strains expressing RFP from a plasmid or from a Tn5 integrated RFP expression cassette. Plants were grown in sterile sand and non-sterile peat media and inoculated by pipetting 1 mL of cell culture directly onto the coleoptiles of germinating plants three days after germination. The plasmid is maintained by watering the plants with a solution containing the appropriate antibiotic. After three weeks, plant roots were collected, rinsed three times in sterile water to remove visible soil, and divided into two samples. A root sample was analyzed by fluorescence microscopy to identify localization patterns of candidate microorganisms. Microscopic examination was performed on the finest intact plan...

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Abstract

The present disclosure provides a bacterial composition, comprising: at least one genetically engineered bacterial strain that fixes atmospheric nitrogen in an agricultural system, wherein the bacterial strain comprises a modification in or one or more genes selected from the group consisting of bcsll, bcslll, yjbE, fhaB, pehA, glgA, otsB, treZ, and cysZ. The present disclosure further provides abacterial composition and method for increasing the colonization of a plant growth promoting bacterial strain on a plant, wherein the plant growth promoting bacterial strain has been remodeled to increase colonization of said plant, In a further aspect, the present disclosure provides methods of increasing nitrogen or nitrogen fixation available to a plant.

Description

[0001] cross reference [0002] This application claims the benefit of U.S. Provisional Application 62 / 543,288, filed August 9, 2017, which is incorporated herein by reference. Background technique [0003] By 2050, the Food and Agriculture Organization of the United Nations predicts that total food production must increase by 70% to meet the needs of a growing population, a challenge exacerbated by a number of factors, including dwindling freshwater resources, increased competition for arable land, and rising energy prices , input costs increase and crops may need to adapt to the stress of drought, heat and a more extreme global climate. [0004] One area of ​​interest is improved nitrogen fixation. Nitrogen (N 2 ) is the main component of the Earth's atmosphere. Additionally, the element nitrogen (N) is an important component of many chemical compounds that make up living organisms. However, many organisms cannot directly use N 2 to synthesize chemicals used in physiolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C05F11/08C07K14/195C12N1/20C12N1/21C12N15/52C12N15/63
CPCC05F11/08C07K14/195C12N15/63C12Y302/01015C12Y302/01141C12N9/00C12N9/1051C12N9/16C12N9/2402C12Y204/01021C12Y301/03012C12N15/52C12N15/03A01N63/20A01C21/00C07K14/235C12N15/74
Inventor S·布洛克K·特米A·坦瑟
Owner 皮沃特生物股份有限公司
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