Difference between revisions of "Part:BBa K4621133"

Latest revision as of 11:53, 11 October 2023

The composite Parts of SCUT-3-GABAi9 that achieve high GABA production functiont.

Usage, Biology and Characterization

gadB is the coding sequence for glutamate decarboxylase, GAD is a pyridoxal 5'phosphate (PLP)-dependent decarboxylase widespread in bacteria yeast and filamentous fungi. The carboxyl group (-COOH) on the catalytic glutamate molecule is released to CO2, and 4-Aminobutanoate is generated at the same time, which participates in the utilization of α-Ketoglutaricacid in the TCA cycle.[1] The specific synthesis route is shown in FIG. 1.

Fig.1 Biosynthetic pathway of GABA

This CRISPRi fragment contains the dCas9 and sgRNA necessary for the CRISPRi system. The CRISPRi system is suitable for a variety of Streptomyces.[2] In this study, it was used to inhibit the expression of gabT gene in SCUT-3 to produce more GABA.

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-gadB-i9 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 and shrimp shells.

Fig.2 Fermentation using common LB

From the result we can observe significant improvements of GABAi9 compared to GABA and WT.

Then we conducted shrimp shell fermentation to these 3 strains.

Fig.3 Fermentation using shrimp shells

From the illustration, GABAi9 still produce the highest level of GABA, with also the highest level of FAA in the fermentation broth.

Reference

[1] Guo, X. F., Hagiwara, T., Masuda, K., and Watabe, S. (2011) Biosci. Biotechnol. Biochem. 75, 1867-1871. [2] 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