Part:BBa_K4621152
It consists of the chi19C hypothetical promoter and EctOperon.
Usage, Biology and Characterization
EctOperon contains ectA, ectB, ectC and ectD and can produce 5-Hydroxyectoine from lysine.
chi19C hypothetical promoter is a functional fragment in chitinase Chi19C 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 chitinase Chi19C was up-regulated when SCUT-3 was in chitin medium, and we speculated that its promoter chi19C hypothetical promoter may have properties that can be induced by chitin.
Testing and validation
We ligated the chi19C 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)
The performance test indicated that ectoine production showed almost no significant difference between induced condition and non-induced condition, but hydroxyectoine varies in production level and severity of inducibility. ProP, a nickname for promoter of LPMO, and ProC, a nickname for promoter of chi19C, presented the two most strict inducibility among four promoters tested. Besides, ProP and ProA, a nickname for promoter of chi18A, shows two greatest strength.
From our results, it’s easy to assume that ProP is the most applicable promoter to continue studying. At the same time, we gave the runner-up to ProC. ProA, with great leakage of expression, was insufficient for further engineering, considering that the chemical we aim to produce could be harmful for host cells’ growth, so we put severity over strength when assessing performance of promoters. In our later shrimp shell fermentation, it again proved our decision here to be reasonable and crucial, which would be shown later.
The production of ectoine and hydroxyectoine tested in this experiment was relative limited, up to about 60-70mg/L. Further optimization and scale-up attempts could be made to ensure the strains can effectively valorize shrimp shell in industrial processing.
By adding chitin at different days after fermentation, we also determined that the optimal time for chitin induction of chi19C 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-EctC at different induction times
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 3468
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 3468
Illegal NotI site found at 812
Illegal NotI site found at 1697
Illegal NotI site found at 2015 - 21INCOMPATIBLE WITH RFC[21]Unknown
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 3468
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 3468
Illegal NgoMIV site found at 1029
Illegal NgoMIV site found at 1435
Illegal NgoMIV site found at 1441
Illegal NgoMIV site found at 1732
Illegal NgoMIV site found at 2781
Illegal NgoMIV site found at 2973
Illegal NgoMIV site found at 3483 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 617
Illegal BsaI.rc site found at 797
Illegal BsaI.rc site found at 1579
Illegal BsaI.rc site found at 2107
Illegal BsaI.rc site found at 2362
Illegal BsaI.rc site found at 2452
Illegal BsaI.rc site found at 2527
Illegal SapI site found at 655
| None |