Difference between revisions of "Part:BBa K4891007"
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With the disruption of shikimate catabolism, shikimate production could be improved.
 
With the disruption of shikimate catabolism, shikimate production could be improved.
  
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===Usage and Biology===
  
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4891007 SequenceAndFeatures</partinfo>
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===Results===
 
<h3>1 PCR verification</h3>
 
<h3>1 PCR verification</h3>
 
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.
 
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.
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| '''Figure 4 Shikimate biosynthesis in the engineered strains. (96 h)'''
 
| '''Figure 4 Shikimate biosynthesis in the engineered strains. (96 h)'''
 
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K4891007 SequenceAndFeatures</partinfo>
 
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 05:55, 9 October 2023


aroL

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

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 425
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 425
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 215
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 425
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 425
  • 1000
    COMPATIBLE WITH RFC[1000]

Results

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)