Gene recombination mediated by phiC312 and inheritance reformation for erythrocin producing bacterium
An erythromycin and bacteria-producing technology, applied in the field of genetic modification, can solve the problems such as the genetic modification of Saccharopolyspora rubrum that cannot be directly applied
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Embodiment 1
[0036] Example 1 Constructing the red Saccharopolyspora containing the bacterial attachment site attB target sequence in the chromosome
[0037] According to the method described in the literature ("Streptomyces Genetics Manual" Norwich, England: The John Innes Foundation; 2000), the polynucleotide containing the attB sequence of the bacterial attachment site was inserted into the red sugar polynucleotide by homologous recombination double exchange structure in the spore chromosome. In this example, we chose to insert the attB sequence into a NRPS gene cluster with a loss of function in the chromosome of Saccharopolyspora rubrum. In order to increase the integration efficiency mediated by ΦC31 integrase and carry out the second or multiple recombinations on the basis of the first site-specific recombination, we selected the attB sequence containing 8 copies as an insert to integrate into the red sugar in polyspora chromosomes. The insertion of this fragment will not have any...
Embodiment 2
[0056] Example 2 The 12-carbon hydroxylase EryK gene and the mycaminose methylase EryG gene of the macrocyclic skeleton in the biosynthesis of erythromycin were integrated into Saccharopolyspora red (S.erythraea E3-attB) attB target sequence
[0057] column
[0058] This example is a further modification based on the inventor's application for a Chinese patent titled 'A Strain, Construction Method and Use of an Optimized Erythromycin Fermentation Component' to construct a stable and enhanced expression of erythromycin During the biosynthesis process, the carbon 12 hydroxylase EryK and mycaminose methylase EryG production strains of erythromycin can improve the impurity components and yield of erythromycin.
[0059] Plasmid construction:
[0060] In order to construct an integrated plasmid based on ΦC31 integrase-mediated gene insertion, select the plasmid pSET152 ( Figure 5 ) as the plasmid parent. Digest containing ermE with EcoRI / SpeI * -eryK-eryG-eryK structure of the...
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