Part:BBa_K4891005

aroGfbr-aroB-aroD-aroE

It is the key gene element for constructing shikimate biosynthetic pathway. Using this recombinant plasmid, shikimate could be generated from phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) by multi-enzyme cascade reaction.

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 BglII site found at 2175
  Illegal XhoI site found at 934
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1965
  Illegal AgeI site found at 2509
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2362
  Illegal SapI.rc site found at 241
  

1 Plasmid Construction

Colony PCR results show that aroGopt, aroB, aroD, and aroE genes have been inserted into plasmid pTrcHisA (Fig 1).

Fig 1. Construction of pTrcHisA-aroGfbr-aroB-aroD-aroE.

2 Protein expression analysis

SDS-PAGE analysis shows that aroGopt, aroB, aroD and aroE genes have been expressed and belong to water-soluble, indicating successful construction of pTrcHisA-aroGfbr-aroB-aroD-aroE (Fig 2).

Fig 2. SDS-PAGE assay in E. coli YCY6.

YCY6 (MG1655 ΔldhA ΔadhE ΔpoxB Δpta ΔaroK ΔaroL; pTrcHisA-aroG-aroB-aroD-aroE) Note: The protein molecular size of AroG, AroB, AroD, and AroE is 38.0 kDa, 38.9 kDa, 27.5 kDa, and 29.4 kDa, respectively.

3 Shikimic acid biosynthesis

To determine the effect of this composite component on the synthesis yield of SA, we used YCY7 (MG1655 ΔldhA ΔadhE ΔpoxB Δpta ΔaroK ΔaroL; pBAD33-tktA-talB) and YCY8 (MG1655 ΔldhA ΔadhE ΔpoxB ΔptA ΔaroK ΔaroL; pTrcHisA-aroG-aroB-aroD-aroE, pBAD33-tktA-talB) for comparison. Due to the incorporation of aroG, aroB, aroD, and aroE genes, YCY8 increased its yield by nearly tripled compared to YCY7, reaching 1.06g/L. Overall, the composite component has the function of increasing the yield of SA. We also conducted experiments such as media optimization for YCY8 in hopes of increasing SA production.

Fig 3. Shikimate biosynthesis in the engineered strains. (96 h)

4 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 4). Detailed, we added L-tyr, L-phe, L-try, yeast extract, citric acid, and other substances to supplement the bacterial nutritional deficiencies.

Fig 4 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.