Part:BBa_K4621153

It consists of the LPMO hypothetical promoter and EctOperon.

Usage, Biology and Characterization

EctOperon contains ectA, ectB, ectC and ectD and can produce 5-Hydroxyectoine from lysine.

LPMO hypothetical promoter is a whole functional sequence in front of the Lytic polysaccharide monooxygenase LPMO gene, which was cloned from SCUT-3 gene. In our project, through transcriptome analysis, qPCR and other means, we found that the gene expression of monooxygenase LPMO was up-regulated when SCUT-3 was in chitin medium, and we speculated that its promoter Pro lpmo may have properties that can be induced by chitin.

Testing and validation

We ligated the LPMO hypothetical promoter with EctOperon and overexpressed it in Streptomyces SCUT-3 using an integrated plasmid. The modified strain was inoculated in 40 mL LB medium and fermented at 40℃ and 220 rpm for 120h. The production of Ectoine and 5-Hydroxyectoin in the presence or without chitin (the first day of fermentation) was detected to evaluate the chitin inducability of the promoter. The results are shown in FIG. 1.

Fig.1 Fermentation of products with or without the addition of an inducer（chitin) (a).Ectoine (b).5-Hydroxyectoin(72h)

By adding chitin at different days after fermentation, we also determined that the optimal time for chitin induction of LPMO hypothetical promoter was the second day of fermentation, and the specific experimental data are shown in FIG. 2:

Fig.2 Fermentation of the product of SCUT-3-EctP at different induction times

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 3437
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 3437
  Illegal NotI site found at 781
  Illegal NotI site found at 1666
  Illegal NotI site found at 1984
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 963
  Illegal XhoI site found at 1137
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 3437
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 3437
  Illegal NgoMIV site found at 998
  Illegal NgoMIV site found at 1404
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  Illegal NgoMIV site found at 1701
  Illegal NgoMIV site found at 2750
  Illegal NgoMIV site found at 2942
  Illegal NgoMIV site found at 3452
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 586
  Illegal BsaI.rc site found at 766
  Illegal BsaI.rc site found at 1548
  Illegal BsaI.rc site found at 2076
  Illegal BsaI.rc site found at 2331
  Illegal BsaI.rc site found at 2421
  Illegal BsaI.rc site found at 2496
  Illegal SapI site found at 624