Novel integrase and application thereof to efficiently modifying saccharopolyspora spinosa

A technology of Saccharopolyspora spinosa and integrase, applied in applications, enzymes, fungi, etc., can solve problems such as lack of attB, unstable genetics, and difficult genetic operations

Active Publication Date: 2015-11-25
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Both Saccharopolyspora spinosa and the erythromycin-producing bacterium Saccharopolyspora rubrum belong to the genus Saccharopolyspora, but the genetic manipulation of the former is very difficult, and the efficiency of introducing plasmids by conjugative transfer or protoplast transformation is very low; Saccharopolyspora spinosa The bacterial chromosome does not have attB required for complete phiC31 integration. By identifying the possible pseudo-attB recognition site, the general-purpose plasmid pSET152 containing phiC31 integration-related elements can be introduced at a very low efficiency (lower than 10-7 conjugative transfer efficiency); Homologous fragment recombination can introduce exogenous genes or increase the copy number of endogenous genes, but most of them are limited to single exchange, which will directly affect the functions of upstream and downstream genes, so it is usually necessary to select possible neutral sites, and stable inheritance may not be possible

Method used

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  • Novel integrase and application thereof to efficiently modifying saccharopolyspora spinosa
  • Novel integrase and application thereof to efficiently modifying saccharopolyspora spinosa
  • Novel integrase and application thereof to efficiently modifying saccharopolyspora spinosa

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0191] Embodiment 1: Introduction and stability test of plasmid

[0192] The constructed plasmids were respectively transformed into Escherichia coli S17-1, and transformed into S. spinosa by E. coli-Saccharopolyspora indirect conjugative transfer. The purified resistant colonies were uploaded on the non-resistant plate for the first and second generations, and the spores were collected and diluted to obtain a single colony. Pick 100-200 single colonies on the resistant plate and non-resistant plate respectively, culture at 30°C, and detect the proportion of non-resistant colonies (colonies with plasmid loss). The results are shown in Table 3.

[0193] Table 3 Plasmid Stability Test

[0194]

[0195]

[0196] The results show that the stability of autonomously replicating plasmids in S. spinosa is different. After non-resistance selection, the loss rate of plasmids is high, which can be used to selectively lose plasmids in the shuffling screening of circulating genomes...

Embodiment 2

[0197] Example 2: Spinosyn production by fermentation of Saccharopolyspora spinosa.

[0198] The Saccharopolyspora spinosa spores on the mature slant were inoculated in the seed shaker flask with a sterile inoculation shovel, the amount of the seed shaker flask was 25ml / 250ml, and cultivated at 28°C and 220r / min for 64-72h. Insert the fermentation medium with 10% inoculum amount, the volume of the fermentation shaker flask is 25ml / 250ml shaker flask, cultivate at 28°C 220r / min for 8-10d,

[0199] Sampling was performed to detect spinosyn production.

[0200] HPLC analysis conditions for determination of spinosyn yield: chromatographic column: Agilent Zorbax Eclipse XDB-C8 4.6×12.5 (5 μm); detection wavelength: 246nm; mobile phase: methanol: acetonitrile: water = 45:45:10, plus 0.005% ammonium acetate ; Flow rate: 1.0ml / min.

[0201] The formula of Saccharopolyspora spinosa slant medium is: whole milk powder 2%, glucose 0.5%, yeast extract powder 0.3%

[0202] The formula of...

Embodiment 3

[0204] Example 3: Application of resistance plasmid markers in genome shuffling screening of Saccharopolyspora spinosa

[0205] The bacterial strain SN304 containing the hygromycin resistance gene plasmid pSN11 marker and the bacterial strain SN0207 containing the apramycin resistance gene plasmid pWT295 marker were respectively prepared into protoplasts, and the protoplast fusion test was carried out according to the principle of patent ZL200710043687.7 ( Refer to literature 12 for the specific method, randomly pick 100 fusion sons for fermentation detection, and the fermentation titer increases by 39%, and the highest increase reaches 41% ( Figure 4 ). The fusion son can further lose one of the resistances, combined with physical or chemical mutagenesis, to obtain a compound parent, and perform the next round of genome shuffling, and repeat multiple rounds of fusion to screen for high-yielding strains.

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Abstract

The invention provides a novel integrase and application thereof to genetically modifying saccharopolyspora spinosa. The integrase particularly comes from saccharopolyspora endophytica CCTCC AA208003, and exogenous DNA (deoxyribonucleic acid) can be specifically locally integrated on saccharopolyspora endophytica chromosome by the integrase via specific cis-form components. In addition, independent free replication regions of different strain sources are discovered in the saccharopolyspora spinosa by an inventor via research on genetic factors of different strains of the saccharopolyspora spinosa. The novel integrase and the application have the advantages that vectors constructed on the basis of the integrase and sequences of the replication regions can be used for genetically modifying the saccharopolyspora spinosa, accordingly, novel spinosad analogues can be possibly discovered, and the spinosad fermentation yields can be possibly increased; different resistance molecular markers can be carried by plasmids, accordingly, the novel integrase can be used for inter-species or intergenetic genome shuffling operation on the saccharopolyspora spinosa, and efficient genome shuffling can be implemented.

Description

technical field [0001] The invention relates to the field of genetic engineering or molecular biology, in particular to an integrase that can be used for the genetic transformation of Saccharopolyspora spinosa, which identifies a series of functional genes by means of a plasmid vector, maintains the stable inheritance of the plasmid vector in Saccharopolyspora spinosa, and provides The genetic engineering of Saccharopolyspora (nucleic acid sequence functional analysis, homologous expression, heterologous expression or overexpression, molecular markers, etc.) and genome optimization provide effective molecular genetic manipulation tools to quickly realize the improvement of Saccharopolyspora spinosa and Increase spinosad production. Background technique [0002] Saccharopolyspora spinosa (Saccharopolysporaspinosa) belongs to the genus Saccharopolyspora, which can produce a variety of novel macrolide compounds A83543 (1). At present, at least 30 structural analogs have been i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/80C12N1/15C12N15/31
Inventor 覃重军夏海洋党福军陈建
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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