Recombinant streptomyces diastatochromogenes with reinforced toyF expression, construction method and uses thereof

[0029] Example 1: Amplification of target gene and construction of recombinant amylase-producing Streptomyces chromosome

[0030] To S. diastatochromogenes 1628 chromosome genome as template, using PtoyF F Nde I and PtoyF R Not I is the primer, PCR amplification obtained contains Nde I and Not I The toyF genes at the two restriction sites are connected with pMD18-T Vector to construct the cloning vector pMD18-T-toyF ( Nde I + Not I), the cloning vector pMD18-T-toyF ( Nde I + Not I) Transform into recipient Escherichia coli, spread it on an ampicillin-resistant LB agar plate, culture overnight at 37°C, randomly select positive transformants for identification and send them to Shanghai Shenggong for sequencing and sequence analysis. Nde I and Not I double digestion to get Nde I and Not I. The toyF genes at the two restriction sites are connected with the same double-cut Streptomyces integrative shuttle expression vector pIB139 to obtain the recombinant shuttle expression vector pIB139-toyF. After being verified by restriction enzyme digestion, it is transferred into E.coli ET12567 (pUZ8002), screen positive transformants on kana-resistant and apramycin-resistant LB plates E.coli ET12567 (pUZ8002, pIB139-toyF), with E.coli ET12567 (pUZ8002, pIB139-toyF) is the donor, the amylase Streptomyces chromogenes is the acceptor, and pIB139-toyF is integrated into the amylase Streptomyces chromogenes using the conjugative transfer method S. diastatochromogenes On chromosome 1628, positive transformants were screened on apramycin-resistant MS plates to obtain recombinant amylase Streptomyces chromosome 1628-TOYF.

[0031] To S. diastatochromogenes Chromosome 1628 is used as a template. Two primers are designed to amplify toyF by PCR. The primers are designed as follows:

[0032] toyF F Nde I: 5’-CGC CATATG GTGACTGCTG CGTCTGC-3’

[0033] toyF R Not I: 5’-CGC GCGGCCGC TCAGAGAATCGCTCCGGG-3’

[0034] Recombinant plasmid pMD18-T-toyF with restriction endonuclease Nde I and Not I double enzyme digestion verification such as figure 2 As shown, the recombinant plasmid pMD18-T-toyF double-enzyme digestion to obtain DNA fragments of about 1.4 kb and 2.7 kb, which are the same size as the toyF gene fragment and plasmid pMD18-T, respectively, indicating that the recombinant cloning vector is connected correctly. The recombinant plasmid pMD18-T-toyF was sequenced, and the sequence analysis showed that the inserted fragment was a 1443 bp sequence, encoding 480 amino acids, and the relative molecular weight was about 53 kDa, which was cleaved with adenosuccinates of many Streptomyces that have been reported. Enzymes have high homology, of which the highest is 89%. The GenBank accession number of the amplified toyF gene is: JQ267374.1. Connect the toyF gene into the pET28a vector to construct the pET28a-toyF recombinant vector, and transfer the recombinant vector into E. coli BL21, pick the positive transformants in 10 mL of LB medium, culture overnight at 37°C with shaking, transfer the next day, IPTG Induce expression, such as image 3 As shown, the recombinant bacteria had obvious characteristic bands after induction, and the adenosine succinate lyase enzyme activity was measured to be 24 U/mg total protein, indicating that the toyF gene was successfully expressed in E. coli.

[0035] The results of restriction digestion verification of integrated recombinant shuttle expression plasmid pIB139-toyF are as follows Figure 4 As shown, the recombinant plasmid pIB139-toyF is Nde I and Not The 1.4 kb fragment released by I double enzyme digestion is consistent with the size of the toyF gene, indicating that the plasmid pIB139-toyF was constructed correctly and integrated into the amylase Streptomyces chromogenes ( Streptomyces diastatochromogenes ) On the chromosome of 1628, a few single colonies were randomly picked on the apramycin-resistant plate and cultured in CP medium for many times, and then the chromosomes were extracted. PCR experiments can amplify apramycin resistance gene apr ( Figure 5 ), which proved that the recombinant amylase Streptomyces chromogenes 1628-TOYF was successfully constructed and genetically stable.

[0036] Example 2: Verification of fermentation performance of amylase-producing Streptomyces chromogenes original and recombinant bacteria

[0037] Compared with the original strain, the adenosine succinate lyase enzyme activity of the recombinant bacteria is increased by 0.9 times, which is better for the recombinant strain 1628-TOYF and the original strain. S. diastatochromogenes In 1628, a 250 mL shake flask fermentation experiment was conducted to verify the effect of enhancing the adenosuccinate lyase activity of the amylase-producing Streptomyces chromophore on the production of toyotomycin from the perspective of fermentation. The speed of the reciprocating shaker was 200 r/min, 28°C, and the fermentation was 96h. The control group was the starting strain of Streptomyces chromogenes. As shown in Table 1, the yield of the recombinant strain Fengjiamycin was higher than that of the original strain. The final production of the recombinant strain Fengjiamycin reached 162.64 mg/L, which was about 20.5% higher than the original strain, and the repeatability was good. Description of producing strains in Fengjiamycin S. diastatochromogenes In 1628, the expression of adenosuccinate lyase—ToyF, a key enzyme that enhances the biosynthetic pathway, helps to increase the production of Toyogamycin during the fermentation process.

[0038] Bacteria Toyoungamycin production (mg/L) 1628 134.97 1628-TOYF 162.64