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Engineering bacterium for high-yield production of spinosad J/L as well as construction method and application of engineering bacterium

A spinosyn and construction method technology, applied in microorganism-based methods, applications, genetic engineering and other directions, can solve the problems of easy off-target, RNAi can not gene transcription or translation, false positives, etc., to achieve the effect of increasing yield

Pending Publication Date: 2022-02-01
QILU PHARMA INNER MONGOLIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods all have various defects: inactivating spnK through homologous recombination requires two-step screening, and the efficiency is very low; screening spnK gene mutants through mutagenesis requires larger-scale screening, and specialized Poor consistency; RNAi cannot completely inhibit gene transcription or translation, and it is easy to off-target and lead to false positive or false negative results
Therefore, whether the CRISPR / ddCpf1 system can be applied to a specific host environment needs to be tested and explored. At present, there is no application of this technology in the prior art

Method used

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  • Engineering bacterium for high-yield production of spinosad J/L as well as construction method and application of engineering bacterium
  • Engineering bacterium for high-yield production of spinosad J/L as well as construction method and application of engineering bacterium
  • Engineering bacterium for high-yield production of spinosad J/L as well as construction method and application of engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Construction of recombinant CRISPR / ddCpf1-crRNA plasmid

[0051] Such as figure 2 As shown, the spnK gene is used as the target gene (sequence shown in SEQ ID NO.1), the PAM recognition sequence TTTN or TTN of the Cpf1 enzyme is searched in the ORF of the spnK gene, and the 23 nucleotides after the PAM sequence are selected as protospacer. The off-target risk of protospacers was assessed by manual design, and all protospacers in the spnK gene ORF were listed, and a total of 63 protospacer sequences were obtained for screening and evaluation. These 63 protospacer sequences were named crRNA-1 to crRNA-63, and were verified later It is known that crRNA-1, crRNA-2 and crRNA-3 have the best inhibitory effect on the spnK gene, and their sequences are shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively; crRNA-4 , crRNA-5 is used as the control group of subsequent embodiment, and its sequence is shown in SEQ ID NO.5 and SEQ ID NO.6 respectively, and othe...

Embodiment 2

[0065] The construction of embodiment 2 transformants and genetically engineered bacteria

[0066] The recombinant plasmid pQL-spnK-crRNA-1 is used to construct a genetically engineered bacterium with high yield of spinosyn J / L, the specific method is as follows:

[0067] (1) Transformation of Escherichia coli

[0068] The recombinant plasmid pQL-spnK-crRNA-1 prepared in Example 1 was transformed into competent Escherichia coli S17-1 (purchased from Takara Company), and then the transformant was picked into a small test tube of 4ml LB medium (Apr 100 μg / ml) After shaking at 37°C for 12 hours, transfer to a 250ml Erlenmeyer flask with 50ml of LB at 2% inoculum size, and shake at 37°C for about 2 hours, so that the OD value of the bacterial solution is between 0.4 and 0.6, and transfer the bacterial solution into a 50ml flask Centrifuge in a sterile plastic centrifuge tube (4000rpm, 10min, 4°C), pour off the supernatant, wash the bacteria twice with 20ml LB (4000rpm, 10min, 4°C...

Embodiment 3

[0077] Example 3 Preparation of Spinosyn J / L by Genetic Engineering Bacteria QL-1 / pQL-spnK-crRNA

[0078] Taking genetically engineered bacteria QL-1 / pQL-spnK-crRNA-1 as an example to prepare spinosyn J / L, the specific steps are as follows:

[0079] Pick the genetically engineered bacteria QL-1 / pQL-spnK-crRNA-1 in Example 2 and inoculate them in the same slant medium, and after cultivating them at 28°C for 8 days, inoculate them in the same seed culture solution, and inoculate them at 28°C After culturing for 3 days, it was transferred to a fermentation medium, cultured at 28° C. and 240 r / min for 9 days, and then bottled to obtain a fermentation broth.

[0080] The seed medium components are as follows: 3.0% glucose, 1.0% soluble starch, 2.0% cottonseed cake powder, 0.2% soybean cake powder, 0.2% yeast powder, 1.0% corn steep liquor, 0.5% calcium carbonate, pH 7.0, all mass percentage.

[0081] The components of the fermentation medium are as follows: 6.0% glucose, 3.0% sol...

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Abstract

The invention relates to an engineering bacterium for high-yield production of spinosad J / L as well as a construction method and application of the engineering bacterium. Saccharopolyspora spinosa QL-1 is used as an original strain, crRNA is inserted to inhibit the spnK gene expression of the Saccharopolyspora spinosa QL-1 until the spnK gene is inactivated, and the QL-1 / pQL-spnK-crRNA genetically engineering bacterium with high-yield production of spinosad J / L is constructed. The CRISPRi technology is introduced into the saccharopolyspora spinosa for the first time, and the spnK gene is inhibited and inactivated by a CRISPR / ddcpf1 method, so that the high-level expression of the spinosad J / L is realized, and the purpose of high yield of the spinosad J / L is achieved. The constructed QL-1 / pQL-spnK-crRNA engineering bacterium can be stably passaged, and the yield of spinosad J / L of the saccharopolyspora spinosa can be obviously improved and is 8-9 times that of wild saccharopolyspora spinosa.

Description

technical field [0001] The invention relates to a high-yielding spinosyn J / L engineering bacterium and its construction method and application, belonging to the field of biotechnology. Background technique [0002] Spinosad is a macrolide biopesticide extracted from the fermentation broth of Saccharopolyspora spinosa. It has the characteristics of pollution-free, high efficiency, low toxicity and easy degradation. It is not harmful to non-target animals and is not carcinogenic to mammals. Spinosyn is a mixture in which spinosyns A and D are the major components and have the highest activity against key insect targets. Spinosyn J and L (two minor components in the spinosyn mixture) are precursors to ethyl spinosyn, a second-generation spinosyn insecticide. Ethyl spinosyn is a mixture of 5,6-dihydro-3'-ethoxy spinosyn J (main component) and 3'-ethoxy spinosyn L (see the accompanying drawings figure 1 ), which is obtained from the two-step chemical synthesis of ethylation an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/31C12N1/21C12N15/70C12P19/62C12N9/22C12N15/113C12R1/01C12R1/19
CPCC12N15/74C07K14/195C12N15/70C12P19/62C12N1/20C12N9/22C12N15/113C12N2310/20
Inventor 黄科学何天景翟晓云刘家亨
Owner QILU PHARMA INNER MONGOLIA
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