Difference between revisions of "Part:BBa K4891005"

Revision as of 07:47, 8 October 2023

aroGfbr-aroB-aroD-aroE

It is the key gene element for constructing shikimate biosynthetic pathway. Using this recombinant plasmid, shikimate could be generated from phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) by multi-enzyme cascade reaction.

Usage and Biology

1 Plasmid Construction

Colony PCR results show that aroGopt, aroB, aroD, and aroE genes have been inserted into plasmid pTrcHisA (Fig 1).

Fig 1. Construction of pTrcHisA-aroGfbr-aroB-aroD-aroE.

2 Protein expression analysis

SDS-PAGE analysis shows that aroGopt, aroB, aroD and aroE genes have been expressed and belong to water-soluble, indicating successful construction of pTrcHisA-aroGfbr-aroB-aroD-aroE (Fig 2).

Fig 2. SDS-PAGE assay in E. coli YCY6.

YCY6 (MG1655 ΔldhA ΔadhE ΔpoxB Δpta ΔaroK ΔaroL; pTrcHisA-aroG-aroB-aroD-aroE) Note: The protein molecular size of AroG, AroB, AroD, and AroE is 38.0 kDa, 38.9 kDa, 27.5 kDa, and 29.4 kDa, respectively.

3 Shikimic acid biosynthesis

To determine the effect of this composite component on the synthesis yield of SA, we used YCY7 (MG1655 ΔldhA ΔadhE ΔpoxB Δpta ΔaroK ΔaroL; pBAD33-tktA-talB) and YCY8 (MG1655 ΔldhA ΔadhE ΔpoxB ΔptA ΔaroK ΔaroL; pTrcHisA-aroG-aroB-aroD-aroE, pBAD33-tktA-talB) for comparison. Due to the incorporation of aroG, aroB, aroD, and aroE genes, YCY8 increased its yield by nearly tripled compared to YCY7, reaching 1.06g/L. Overall, the composite component has the function of increasing the yield of SA. We also conducted experiments such as media optimization for YCY8 in hopes of increasing SA production.

Fig 3 Shikimate biosynthesis in the engineered strains. (96 h)

Sequence and Features