Part:BBa_K4891025

tktA-talB

PEP is derived from the pentose phosphate pathway, and the overexpression of tktA and talB genes is beneficial for strengthening metabolic flow, thereby improving the synthesis efficiency of shikimic acid.

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
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2872
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 232
  Illegal AgeI site found at 337
  Illegal AgeI site found at 1669
  
• 1000
  COMPATIBLE WITH RFC[1000]

1 PCR verification

Colony PCR shows that tktA and talB genes have been inserted into pBAD33 (Figure 1).

Figure 1 Construction of pBAD33-tktA-talB

2 Shikimic acid biosynthesis

To determine the effect of this complex composition on SA synthesis yield, we used YCY6 (MG1655 ΔldhA ΔadhE ΔpoxB Δpta ΔaroK ΔaroL; pTrcHisA-aroG-aroB-aroD-aroE) and YCY8 (MG1655 ΔldhA ΔadhE ΔpoxB ΔptA ΔaroK ΔaroL; pTrcHisA-aroG-aroB-aroD-aroE, pBAD33-tktA-talB) were compared. YCY8 achieved a 40% yield increase over YCY6, reaching to 1.06g/L. In conclusion, the composite components worked to increase SA yield. We also optimized the culture medium for YCY8 to increase SA production.

Figure 2 Shikimate biosynthesis in the engineered strains. (96 h)

3 Fermentation Profiles

After analyzing the above results and reviewing the paper, we concluded that the yield of SA could be further improved by optimizing the culture conditions, such as medium composition. Thus, we obtained the following test results (Tripathi et al., 2015). From the growth curve measurement experiments, we learned that the growth of nutrient-deficient strains needs to be rescued by additional supplementation with the appropriate amino acids, and based on this, we optimized the fermentation medium. We found that the culture medium may greatly influence SA synthesis of YCY8 and YCY9 strains. We incubated YCY8 and YCY9 with NBS medium and optimized medium (the recipe of optimized medium is in the protocol) for 48 h, respectively, and measured the SA production. In general, YCY8 and YCY9 synthesized low yields of SA in the NBS medium, but were able to accumulate SA in the optimized medium: YCY8 yielded 1.31 g/L and YCY9 yielded 1.05 g/L (Fig 3). Detailed, we added L-tyr, L-phe, L-try, yeast extract, citric acid, and other substances to supplement the bacterial nutritional deficiencies.

Figure 3 Shikimate biosynthesis in the optimized fermentation conditions (48 h).

Reference

Tripathi P, Rawat G, Yadav S, Saxena RK. Shikimic acid, a base compound for the formulation of swine/avian flu drug: statistical optimization, fed-batch and scale-up studies along with its application as an antibacterial agent. Antonie Van Leeuwenhoek. 2015;107(2):419-431.