Part:BBa_K4891015

ptsGup-glk-glf-ptsGdown

It is the key part that is responsible for strengthening glucose utilization rate. The phosphotransferase system was replaced by heterogeneously introducing glucose facilitator in strain E. coli ptsG::glk-glf.

1 PCR verification

Colony PCR shows that glk and glf genes have been inserted into ptsG gene site of the genome in the host strain, and glk and glf genes are inserted into pBAD33 (Figures 1-2).

Figure 1 Insertion of glk and glf genes.

2 Shikimic acid biosynthesis

To determine the effect of ptsG::glk-glf on SA synthesis yield, we used YCY8 (MG1655 ΔldhA ΔadhE ΔpoxB ΔptA ΔaroK ΔaroL; pTrcHisA-aroG-aroB-aroD-aroE, pBAD33-tktA-talB) and YCY9 ( MG1655 ptsG::glf+glk ΔldhA ΔadhE ΔpoxB Δpta ΔaroK ΔaroL; pTrcHisA-aroG-aroB-aroD-aroE, pBAD33-tktA-talB) were compared. Theoretically, the yield of YCY9 should be higher than that of YCY8. However, Figure 2 shows that YCY9 has a 35% lower yield than YCY8. We speculate that the low expression level of the inserted heterologous gene glk-glf or the replacement of this glucose transport system may have affected the growth of the bacteria, and the real reason needs to be further investigated. In addition, we optimized the culture medium and inoculum amount for YCY9, hoping to increase SA yield.

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 and inoculum amount. Thus, we obtained the following test results (Tripathi et al., 2015).

3.1 Culture medium

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).

3.2 Inoculum levels

In addition, Wewe also investigated the effect of different inoculum levels of strain YCY9 on SA yield. For strain YCY9, SA yield increased with increasing inoculum. Among them, a yield of 1.875 g/L could be achieved at a 10% inoculum level, which significantly increased the SA yield compared to a 1% inoculum level (Fig 4). Thus, higher SA yields can be achieved with appropriately higher inoculum levels.

Figure 4 Influence of inoculum amount on SA synthesis in strain YCY9 (96 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.

Sequence and Features

Assembly Compatibility:

• 10
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• 12
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• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 630
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• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 630
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  Illegal AgeI site found at 927
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• 1000
  COMPATIBLE WITH RFC[1000]