Difference between revisions of "Part:BBa K4800015"

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GabT

GabT, 4-aminobutyrate-2-oxoglutarate transaminase from Escherichia coli K-12 is the initial enzyme of one of two 4-aminobutyrate (GABA) degradation pathways. It belongs to the aminotransferase subgroup II of pyridoxal 5'-phosphate (PLP)-dependent enzymes, which requires pyridoxal phosphate to function. GabT also functions as a 5-aminovalerate aminotransferase during degradation of L-lysine.

Characterization

The gene of GabT was amplified from the genome of E. coli MG1655, and integrated into pTrc99a vector to obtain the plasmid pTrc99a-GabT (BBa_K4800046). We performed colony PCR on bacteria containing the existing pTrc99a-GabT plasmid. Based on the results of colony PCR, the gene fragment of GabT was between 1000 and 2000 bp (Figure 1), which was consistent with our expectation of the fragment size of GabT.

Plasmid pTrc99a-GabT were transferred into strain E. coli BL21 to determine its express. The cells were inoculated and cultured in the LB medium at 37℃. 0.05 mM of IPTG was added into the culture to induce protein expression when cells grew into an OD₆₀₀ of 0.6-0.8. After overnight induction and cultivation, the cells were harvested and resuspended in 50 mM Tris-HCl buffer (pH 8.0). The suspended cells were then lysed by ultrasonication to release the intracellular proteins. SDS-PAGE results confirmed that the molecular weight of GabT protein was correct, which was consistent with the expected molecular weight of 43.0 kDa (Figure 2).

Determining the activity and usage for 1, 5-pentanediol production

GabT catalyzes 5-aminovalerate to glutarate semialdehyde. As the instability and detection difficulty of glutarate semialdehyde, we determined the activity of GabT in a cascade reaction with aldehyde reductase of Yahk by detecting the 5-hydroxyvalerate production. The cell that contained empty plasmid was used as the control. As the results shown in Figure 3, GabT from E. coli exhibited the ammonia transfer activity towards 5-aminovalerate, and the product of 5-hydroxyvalerate was successfully detected.

Next, we test the activity GabT for the 1,5-pentanediol (1,5-PDO) production when it was co-expressed with carboxylate reductase MmCAR and aldehyde reductase of Yahk by a whole cell process. We used 5-AVA as the starting substrate for whole-cell catalysis. As shown in Figure 4, the production of 1,5-PDO by a cascade reaction containing GabT was successfully detected, indicating that GabT could be employed as a component in the synthetic 1,5-PDO pathway for its production.

After obtaining the results of whole-cell catalysis, we chose to use GabT as the transaminase to construct 1,5-PDO fermentation strains for further verification. Two plasmids including pTrc99a-davB-davA-GabT (BBa_K4800054) and pACYCDuet-sfp-MmCAR-YahK (BBa_K4800053) were constructed and transformed into E. coli NT1003 strain to obtain the engineered strain of E. coli NT1003-P1. After the fermentation of 72 h, the fermentation broth contained 2.82 mM (0.293 g/L) of 1,5-PDO (Figure 5).

We further expressed the plasmid pTrc99a-davB-davA-GabT containing GabT together with the plasmid of pRSFDuet-sfp-MmCAR-YahK (BBa_K4800055) to obtain the engineered strain E. coli NT1003-P2. Similarly, 8.09 mM (0.843 g/L) 1,5-PDO could be successfully produced by the engineered strain E. coli NT1003-P2 (Figure 6).

These results confirmed that GabT enabled the synthesis of 1,5-PDO in E. coli through an artificial synthetic pathway, which support us to do the following strain optimization design and provided a good basis for the subsequent construction of 1,5-PDO producing strains.

Sequence and Features