Part:BBa_K4621133
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
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 5966
Illegal PstI site found at 3860
Illegal PstI site found at 4094
Illegal PstI site found at 5306 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 5943
Illegal SpeI site found at 5966
Illegal PstI site found at 3860
Illegal PstI site found at 4094
Illegal PstI site found at 5306 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1899
Illegal BglII site found at 2694
Illegal BglII site found at 5463
Illegal BamHI site found at 1639
Illegal BamHI site found at 2988
Illegal BamHI site found at 5931
Illegal BamHI site found at 6136
Illegal XhoI site found at 2312
Illegal XhoI site found at 4658 - 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 5966
Illegal PstI site found at 3860
Illegal PstI site found at 4094
Illegal PstI site found at 5306 - 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 5966
Illegal PstI site found at 3860
Illegal PstI site found at 4094
Illegal PstI site found at 5306
Illegal NgoMIV site found at 53
Illegal NgoMIV site found at 1242
Illegal NgoMIV site found at 3256
Illegal NgoMIV site found at 3627
Illegal NgoMIV site found at 3830
Illegal AgeI site found at 470
Illegal AgeI site found at 690 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 4838
Illegal BsaI.rc site found at 5717
Illegal SapI site found at 2230
Illegal SapI site found at 3520
Illegal SapI.rc site found at 4717
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