Method for producing prodigiosin based on PNTs
A technology for producing prodigiosin and prodigiosin, which is applied in the biological field and can solve the problems of low yield and hindering the industrialization process of microbial fermentation
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Embodiment 1
[0032] Example 1: Serratia marcescens engineered bacteria JNB5-1 / pigF PNT , JNB5-1 / pigN PNT Build
[0033] Specific steps are as follows:
[0034] (1) Construction of pETC-28a plasmid
[0035] Using the pET-28a plasmid as a template, amplify the pET-28a plasmid backbone by reverse PCR (excluding the kanamycin resistance gene); use the pKK 232-8 plasmid as a template to amplify the chloramphenicol resistance gene by PCR; Use homologous recombination kit to homologously recombine the plasmid backbone and the chloramphenicol resistance gene to obtain the recombined product; transform the recombined product into E. coli BL21 to obtain the transformed product; spread the transformed product on LB solid medium (containing 12.5 μg) ·ML -1 Chloramphenicol), inverted culture in a constant temperature incubator at 37°C for 8-12 hours to obtain transformants; pick the transformants to inoculate into LB liquid medium, and shake flask culture at 37°C and 180rpm for 8-12 hours The plasmid is ext...
Embodiment 2
[0095] Example 2: Production of Prodigiosin
[0096] Specific steps are as follows:
[0097] Taking Serratia marcescens JNB5-1 as a control, the Serratia marcescens engineering bacteria JNB5-1 / pigF obtained in Example 1 were selected respectively PNT , JNB5-1 / pigN PNT Inoculate single colony of LB liquid medium (containing 50μg·mL -1 Apramycin and 50μg·mL -1 Clindamycin), shaking culture at 37°C and 200 rpm for 12 hours to obtain seed liquid; the seed solution was inoculated into the fermentation medium at 6% (v / v) inoculum at 30°C and 200 rpm Ferment for 96h to obtain fermentation broth.
[0098] Detect the content of prodigiosin in the fermentation broth (see results Figure 5 ), the results showed that: when fermented for 96h, the production of prodigalin in the fermentation broth obtained by Serratia marcescens JNB5-1 was 5.36g / L, and the Serratia marcescens engineered bacteria JNB5-1 / pigF PNT , JNB5-1 / pigN PNT The production of prodigalin in the fermentation broth obtained by fe...
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