Gluconobacter oxidans shuttle vector for gene expression

A technology for oxidizing glucose and shuttle carriers, which is applied in the field of genetic engineering, can solve the problems of limited application potential, poor stability and transformation efficiency of Gluconobacter oxydans, lack of multiple plasmid vectors, etc., and achieve the effect of improving transformation efficiency and improving efficiency

Active Publication Date: 2022-03-04
JIANGNAN UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the expression vectors used in Gluconobacter oxidans are mainly pBBRI-MCS series plasmids or their derivative plasmids. This series of plasmids are widely host plasmids, and their stability and transformation efficiency in Gluconobacter oxidans are relatively poor.
Due to the lack of efficient transformation plasmids in Gluconobacter oxidans and the lack of co-expression of multiple plasmid vectors in Gluconobacter oxidans, this severely limits its application potential in industrial production

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  • Gluconobacter oxidans shuttle vector for gene expression
  • Gluconobacter oxidans shuttle vector for gene expression
  • Gluconobacter oxidans shuttle vector for gene expression

Examples

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Embodiment 1

[0044] Embodiment 1 Construction of Gluconobacter oxidans shuttle plasmid

[0045] According to the results of the genome sequencing of Gluconobacter oxydans WSH-003, Gluconobacter oxidans WSH-004, Gluconobacter oxidans 1.049, and Gluconobacter oxydans 621H genome, through the analysis of the endogenous plasmid gene of Gluconobacter oxydans, such as SEQ ID NO. 1 to 15 gene sequences encoding replication proteins shown in SEQ ID NO.15.

[0046] Primer pairs (shown in Table 1) were designed to amplify the replicating protein sequence. Using the total genomic DNA of Gluconobacter oxydans as a template, PCR amplification was carried out with this primer pair, and Primer Star MasterMix (Takara Company) high-fidelity enzyme was selected for PCR amplification. 95°C, 15s, 56°C, 15s, 72°C, 1min-3min (determined according to the size of different replicating proteins); extension 72°C, 5min. The PCR product was subjected to product purification. The specific steps are:

[0047] Using...

Embodiment 2

[0067] Example 2 Construction of Fluorescent Protein Expression Vectors Using Shuttle Vectors

[0068] Design the primer pair (as shown in Table 2) for amplifying the fluorescent sequence, use the shuttle vector constructed in Example 1 as a template, carry out PCR amplification with the primer pair in Table 3, and select Primer Star MasterMix (Takara Company) high-fidelity enzyme Carried out, the conditions were pre-denaturation at 95°C, 3min; amplification stage 30 cycles, according to 95°C, 15s, 56°C, 15s, 72°C, 1min; extension at 72°C, 5min. The specific steps are:

[0069] Using the p1-Kana constructed in Example 1 as a template, and using T-F / p1-R as primers, amplify the p1-Kana vector fragment; pass the p1-Kana vector fragment and the fluorescent protein fragment shown in SEQ ID NO.16 through Gibson The assembly method is constructed as a fluorescent protein expression vector p1-mcherry;

[0070] Using the p2-Kana constructed in Example 1 as a template, and using T-F / ...

Embodiment 3

[0093] Example 3 Construction of shuttle vectors resistant to different antibiotics

[0094]The antibiotic resistance gene sequences shown in SEQ ID NO.21-SEQ ID NO.25 were synthesized respectively. A primer pair (as shown in Table 5) for amplifying the fluorescent sequence was designed, and the expression vector containing the fluorescent protein was amplified with the primers shown in Table 5. Choose Primer Star MasterMix (Takara Company) high-fidelity enzyme, the conditions are pre-denaturation at 95°C, 3min; the amplification stage is 30 cycles, according to 95°C, 15s, 56°C, 15s, 72°C, 1min; extension at 72°C, 5min .

[0095] Using the p5-k-EGFP constructed in Example 2 as a template, and using T-Res-F / T-Res-R as amplification primers, the T carrier fragment with fluorescent protein was amplified; the synthesis shown in SEQ ID NO.22 The DNA fragment encoding antibiotic resistance was connected with the T carrier fragment with fluorescent protein and the DNA fragment of a...

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Abstract

The invention discloses a gluconic acid bacteria shuttle carrier used for high-efficiency gene expression, and belongs to the field of genetic engineering. The present invention screens the possible replication protein of the endogenous plasmid of Gluconobacter oxydans, constructs a series of shuttle vectors by using the endogenous replication protein of this type, transfers it into Gluconobacter oxidans for verification, and finds that its transformation efficiency and wide host Compared with the plasmid pBBR-MCS, it has been greatly improved; and the shuttle vector is used for protein expression, so that the L-sorbose production of Gluconobacter oxidans is increased by 7.8% compared with the expression of the broad host vector, and the substrate conversion time Shortened by 27.2%. The invention greatly improves the efficiency of the genetic modification of the gluconobacterium oxydans, and realizes different requirements for the genetic modification of the gluconobacterium oxydans.

Description

technical field [0001] The invention relates to a gluconic acid bacteria shuttle vector used for high-efficiency gene expression, and belongs to the field of genetic engineering. Background technique [0002] Gluconobacter oxidans is a strictly aerobic Gram-negative bacteria belonging to the genus Glucobacterium, Acetobacteriaceae, which mainly inhabit high-sugar environments, such as fruits, flowers and honey. Gluconobacter has the following characteristics that make it widely used in industry: (1) can quickly and incompletely oxidize sugar and alcohol substrates to generate corresponding aldehydes, ketones and acids; (2) membrane-bound dehydrogenase catalytic activity The site is generally located in the periplasmic space, and the substrate does not need to be transported across the membrane during the catalytic reaction, while the corresponding product is directly secreted into the medium; (3) The catalytic reaction is highly regioselective and stereoselective. The chara...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74C12N15/70C12N1/21C12R1/01C12R1/19
CPCC12N15/74C07K14/195
Inventor 周景文陈坚刘立曾伟主堵国成李江华
Owner JIANGNAN UNIV
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