Part:BBa_K4891008
ldhA
It is the key part that is responsible for optimizing metabolic network of the E. coli MG1655. Complicated metabolic flux usually leads to the inefficient production performance. With the disruption of the byproducts, intracellular PEP availability 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:
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Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Results
1 PCR verification
Colony PCR results show that ldhA, adhE, poxB and pta genes have been knocked out from the genome of the E. coli MG1655, and the recombinant strain is named as E. coli YCY1 (Figures 1-4).
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| Figure 1 Knock out of ldhA gene. |
The DNA fragments of knocked out and un-knocked out for ldhA gene are 1500 bp and 2490 bp, respectively. Besides, WT represents the wild-type strain E. coli MG1655.
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| Figure 2 Knock out of adhE gene. |
The DNA fragment of knocked out and un-knocked out for adhE gene are 1500 bp and 4176 bp, respectively.
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| Figure 3 Knock out of poxB gene. |
The DNA fragment of knocked out and un-knocked out for poxB gene are 2200 bp and 3919 bp, respectively.
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| Figure 4 Knock out of pta gene. |
The DNA fragment of knocked out and un-knocked out for pta gene are 1200 bp and 3345 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 5, 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.
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| Figure 5 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)
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