Part:BBa_K4891006

aroK

With the disruption of shikimate catabolism, shikimate production could be improved.

Usage and Biology

We engineered the shikimate pathway of E. coli MG1655 to efficiently accumulate SA. We knocked out ptsG, ldhA, adhE, poxB, and pta genes to achieve the production of precursor substance PEP. To increase intracellular E4P content, we overexpressed tktA and talB genes. To enhance product accumulation, we overexpressed aroG, aroB, aroD, and aroE genes, while knocking down aroK and aroL genes to cut off the metabolic flux, thus accomplishing the accumulation of shikimic acid. Besides, a non-phosphorylated pathway, that is, glk and glf genes (mainly by glk-glf integration into the ptsG locus) is introduced to enhance glucose utilization. To achieve the goal above-mentioned, we totally constructed 26 parts this year. See table below:

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 200
  

1 PCR verification

Colony PCR results show that aroK and aroL genes have been knocked out from the genome of the host strain (Figures 1-2), and the recombinant strains are named as strains E. coli YCY2-E. coli YCY3, respectively.

Figure 1 Knock out of aroK gene.

The DNA fragment of knocked out and un-knocked out for aroK gene are 1800 bp and 2322 bp, respectively.

Figure 2 Knock out of aroL gene.

The DNA fragment of knocked out and un-knocked out for aroL gene are 1800 bp and 2325 bp, respectively.

2 Growth assay

To evaluate whether the elimination of shikimate catabolism resulted in an auxotroph for amino acids, the growth curve in strains E. coli YCY1-YCY3 is examined. As seen in Figure 3, the growth rate and glucose consumption of strain E. coli YCY2-3 are significantly slowed down without the addition of L-tyrosine, L-phenylalanine, and L-tryptophan. In contrast, the growth status of these strains is improved after supplementing aromatic amino acids.

Figure 3 Growth curve in the engineered strains without (left) or with amino acid (right).

WT (Escherichia coli MG1655), YCY1 (MG1655 ΔldhA ΔadhE ΔpoxB Δpta), YCY2 (MG1655 ΔldhA ΔadhE ΔpoxB Δpta ΔaroK), YCY3 (MG1655 ΔldhA ΔadhE ΔpoxB Δpta ΔaroK ΔaroL)

3 Shikimic acid biosynthesis

To determine the effect of this composite component on the synthesis yield of SA, we used YCY4 (MG1655 ΔldhA ΔadhE ΔpoxB Δpta; pTrcHisA-aroG-aroB-aroD-aroE) and YCY6 (MG1655 ΔldhA ΔadhE ΔpoxB Δpta ΔaroK ΔaroL; pTrcHisA-aroG-aroB-aroD-aroE) for comparison. Due to the knockout of aroG and aroL, YCY6 increased SA yield by nearly 35 times compared to YCY4, reaching 0.75g/L. Overall, the composite component has the function of increasing the yield of SA.

Figure 4 Shikimate biosynthesis in the engineered strains. (96 h)