Part:BBa_K2963039

Producing γ-PGA with different D/L glutamate monomer ratio(R).

Usage and Biology

The BCA genes from Bacillus sp. encode a γ-PGA synthetase located on the cell membrane which is capable of polymerizing glutamic acid to form poly-γ-glutamic acid. In Bacillus licheniformis, the BCA are called capBCA. We used the B* gene a mutant of B gene. The racE gene is derived from Bacillus subtilis and it encodes a racemase which can converts L-glutamate to D-glutamate.

We used this part to produce γ-PGA with different D/L monomer ratios. We assembled the capB*CA genes and the part containing racE gene (BBa_K2963032) together to construct this part using plasmid PZM1.

Characterization

We transferred this part into our chassis microorganism Corynebacterium glutamicum and used HPLC to detect the D/L monomer ratio of γ-PGA.The result shows as below.

Picture 1

Picture1 is the L-glutamate monomer ratio in γ-PGA we have produced using part BBa_K2963009 which do not link with racE gene and the result reaches about over 90%.

Picture 2

Picture2 is the result of BBa_K2963039. This part contains capBCA genes and racE gene which is under the control of tac promoter with one lacO. The L-glutamate monomer ratio reaches about 32%.

The results show that when we linked capB*CA genes with racE gene, the L-glutamate monomer ratio of γ-PGA is changing. This part is working. All the results show that we have achieved the goal of producing different D/L glutamate monomer ratio of γ-PGA preliminary.

References

1. Xu P, Vansiri A, Bhan N, et al. ePathBrick: a synthetic biology platform for engineering metabolic pathways in E. coli[J]. ACS Synthetic Biology, 2012, 1(7): 256-266.

2.Mutalik, Vivek K, et al. "Precise and reliable gene expression via standard transcription and translation initiation elements." Nature Methods 10.4(2013):354.