Part:BBa_K4891012

poxB

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.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 651
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 651
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 651
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 651
  
• 1000
  COMPATIBLE WITH RFC[1000]

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).

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.

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.

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.

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.

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)