Difference between revisions of "Part:BBa K4621153"

Revision as of 18:12, 10 October 2023

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 48h. 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(48h)

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.

Supplementary work in this module

After confirmation of their inducibility, we further conducted experiments to test if the induction of ectione operon could be achieved at different time points in a relative long-term fermentation, considering that the shrimp shell recycling fermentation typically last five-day long.

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

The fermentation experiments in this section were all conducted on the basis of using LB media, and there was no chitin added in the Ctrl group. In the fellow groups, 1% chitin powder was added at different time points, namely 0 d, 1 d, 2 d, 3 d, and 4 d. The test results indicated that the induction of ProP could be achieved from day 1 to day 3.

Sequence and Features