Part:BBa_K2963033

Ptac(two lacOs)-racE- Producing γ-PGA with different D/L monomer ratio

Our project used this part to produce γ-PGA with different D/L glutamate monomer ratios.This part contains the racE gene, which was under the control of a modified tac promoter with another additional operator gene (lacO). We used racemase activity data and real-time PCR data to characterize and compare the different expression intensities of racE gene using different structures of tac promoters.

Usage and Biology

The racE gene is derived from Bacillus subtilis. This gene encodes a racemase which can converts L-glutamic acid to D-glutamate.

Characterization

We used real-time quantitative PCR and enzyme activity assay to compare the different expressions of racE gene under the control of tac promoter containing different numbers of operator genes in Corynebacterium glutamicum. The results were shown in Figure 1. The expression level of the racE gene at 8 h, 16 h, 24 h decreased by 29.99%, 58.86% and 62.34% respectively. The results showed that the expression intensity of racE was effectively reduced at each transcriptional stage by the tandem of two lacOs compared to one lacO. At the same time, we tested the activity of the racemase in cells. The enzyme activity under the control of one lacO is higher than that with two lacOs. The data is shown in Figure 2.

Figure1

Figure 1. The expression level of the racemase gene racE at 8 h, 16 h, 24 h decreased by 29.99%, 58.86% and 62.34% respectively. The results show that the expression intensity of racE was effectively reduced at each transcriptional stage by the tandem of two lacOs compared to one lacO.

Figure2

Figure 2. we tested the enzyme activity of the racemase in cells. The enzyme activity under the control of one lacO is higher than that with two lacOs.

References

1.Keitarou K. Roles and regulation of the glutamate racemase isogenes, racE and yrpC, in Bacillus subtilis[J]. Microbiology, 2004, 9(150): 2911-2920.

2. Feng Jiang, Gaofu Qi, Zhixia Ji. Expression of glr gene encoding glutamate racemase in Bacillus licheniformis WX-02 and its regulatory effects on synthesis of poly-γ-glutamic acid[J]. Biotechnology Letters, 2011, 33: 1837-1840.