Difference between revisions of "Part:BBa K4621132"

Revision as of 18:32, 10 October 2023

Complex functional parts of SCUT-3-EctPi8 that induce high Hydroxyectoine production.

Usage, Biology and Characterization

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.

This CRISPRi fragment contains four parts : BBa _ K4621070, BBa _ K3875019, BBa _ K4621031, and BBa _ K3081104, covering the dCas9 and sgRNA necessary for the CRISPRi system. The CRISPRi system is suitable for a variety of Streptomyces.[1] In this study, it was used to inhibit the expression of GQS52 _ 08040 gene in SCUT-3 to produce more ectoine and hydroxyectoine.

Fig.1Mechanism of CRISPRi

Testing and validation

Due to the limited types of plasmids available for SCUT-3, we inserted the CRISPRi fragment into the previously constructed plasmid PZ-ProP-Ect-i8 by homologous recombination to achieve simultaneous stable expression of multiple target genes. Subsequently, we verified the effectiveness of the CRISPRi system in the fermentation of ordinary LB, high-salt LB and shrimp shells.

Fig.2 Fermentation under the high salt environment

Under 1% NaCl, CRISPRi-introduced strains show decrease on the total production when compared to ProP, and for Pi4 and Pi8, they nearly offset the positive effect of ProP overexpressing. This result did not align with our expectation. To further invest about which factor influence the performance of CRISPRi, we conducted another experience which replace the common LB medium to LB medium with 5% NaCl. We wished to test if the performance was affected by insufficient metabolic flux.

As shown in Figure 2B, under 5% NaCl, CRISPRi indroduced strains Pi4 showed significant increase among all strains tested.

Fig.3 Fermentation using shrimp shells

From Figure 3A we can see that Pi4 creates the highest yield of ectoine and hydroxyecyoine. In amino acid analysis of Figure 3B, we can see that the shrimp shell degradation level in modified bacteria is even higher than wild type.

Reference

[1] Zhao Y, Li L, Zheng G, Jiang W, Deng Z, Wang Z, Lu Y. CRISPR/dCas9-Mediated Multiplex Gene Repression in Streptomyces. Biotechnol J. 2018 Sep;13(9):e1800121. doi: 10.1002/biot.201800121. Epub 2018 Jul 4. PMID: 29862648.

Sequence and Features