Engineering

Abstract:

Shikimic acid (SA) is an important natural biochemical metabolite in plants and microorganisms that is known for antimicrobial and anti-inflammatory activities. Our project completed the metabolic engineering modification of Escherichia coli (E. coli) MG1655 to achieve SA accumulation. Among them, the YCY9 strain was able to produce 1875 mg/L of shikimic acid by continuous cultivation for 96 h at 10% inoculum, which greatly improved the target yield and provided the possibility of achieving mass production.

1. Overview of our design

We have identified strategies for metabolic engineering modifications E. coli based on genomic metabolic network modeling, which are divided into the following main points (See Design for more details):

1. Enhancement of PEP levels:
(1) blocking the PTS by knocking out the ptsG gene;
(2) blocking PEP branching pathways, including knocking out of ldhA, adhE, poxB, and pta genes;

2. Increase the E4P content: overexpression of tktA and talB genes;

3. Optimize the shikimic acid synthesis pathway: (1) overexpression of aroG, aroB, aroD, and aroE genes, enhance shikimic acid synthesis metabolic flux; (2) knockout of aroK and aroL genes to block the shikimic acid catabolic pathway;

4. In addition, we introduced glk and glf genes from Zymomonas mobilis (mainly by glk-glf integration into the ptsG locus)

Note: ptsG, glucose-specific EIICB protein components; glk, glucokinase; glf, glucose-promoting diffusion protein; tktA, transketonase; talB, transaldolase; ldhA, lactate dehydrogenase; adhE, alcohol dehydrogenase; poxB, pyruvate dehydrogenase; pta, phosphate transacetylase; aroG, DAHP synthase; aroB, 3-dehydroquinic acid synthetase; aroD, 3-dehydroquinic acid dehydratase; aroE, shikimic dehydrogenase; aroK/aroL, shikimic acid kinase.

2. Metabolic engineering modification - construction of chassis strains

Due to a lack of time during the summer, we turned to our lab instructors to knock out ldhA, adhE, poxB, pta, aroK, and aroL. In this study, the target gene was amplified by PCR and then ligated into pTrcHisA plasmid vector using T4 DNA ligase, which was transfected into DH5α receptor cells and transferred to solid medium for overnight culture, and the following results were verified by colony PCR:

2.1

As shown by the PCR of this colony, colony #1 was able to amplify the aroG and aroB genes with specific primers of about 2,000 bp, indicating that these two genes were transformed into the bacteria.

2.2

As shown by the PCR of this colony, colonies such as #1 and #2 were able to amplify the aroD and aroE genes with specific primers with a size of about 1600 bp, indicating that these two genes have been transformed into the bacteria.

2.3 To overexpress tktA and talB, we used PCR to amplify the target gene and then ligated it to pBAD33 plasmid vector using T4 DNA ligase, which was transfected into DH5α cells and transferred to a solid medium for overnight cultivation, and the following are the results of colony PCR verification:

As shown by the PCR of this colony, colonies such as #1 and #2 were able to amplify the tktA and talB genes with specific primers with a size of about 3000 bp, indicating that these two genes have been transformed into the bacteria.

2.4

As shown by the PCR of this colony, colonies #1 and #2 were able to amplify the glk and glf genes with specific primers of about 3000 bp, indicating that these two genes have been integrated into the bacterial genome.

So far, we've made all the metabolic modifications in E. coli MG1655.

3. Function Test

3.1 Strain growth testing

To test the growth of engineered strains after cutting off the PEP branching pathway and the SA catabolic pathway. We found that knocking out the PEP branch pathway had no significant effect on the growth of the bacteria in NBS medium with or without supplemental amino acids. In contrast, after further knockdown of aroK and aroL genes and disruption of the SA catabolic pathway, the growth of the bacterium was significantly inhibited, which might be due to the reduction of intracellular L-tyr, L-phe, and L-try amino acids. The inhibition of bacterial growth was alleviated to some extent by additional amino acid supplementation. Therefore, the medium used in subsequent experiments must be supplemented with a certain amount of amino acids to ensure normal growth and propagation of the engineered strains.

3.2 SA yield of different strains

Based on the disruption of the PEP branching pathway and the SA catabolic pathway, we enhanced the expression of genes for enzymes related to the SA synthesis pathway, aroG, aroB, aroD, and aroE, i.e., YCY6; further, we overexpressed the expression of enzymes related to the E4P synthesis pathway, tktA and talB, i.e., YCY8; and finally, on the basis of which we replaced the glucose transport system with glf-glk to reduce the consumption of PEP, the precursor of SA synthesis, i.e., YCY9. We tested the SA yield of the above different strains and found that the yield of YCY8 was able to reach 1.06 g/L within 96 h, indicating that cutting off the PEP branching pathway and the SA catabolic pathway and overexpressing the SA synthesis pathway and the E4P synthesis pathway were able to effectively increase the SA production. However, the modification of the glucose transport system did not contribute to further increase the SA production, and we speculate that this may be due to the low expression level of glf-glk, which makes it difficult to achieve a synergistic effect.

4. Optimization of culture conditions to improve SA production

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

4.1 Inoculum levels

We 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 8). Thus, higher SA yields can be achieved with appropriately higher inoculum levels.

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

Summary：

In this project, we measured the growth of the engineered E. coli MG1655 and found that additional amino acid supplementation could rescue the growth defects of the engineered bacteria to a certain extent, which was favorable to the production of SA. By further enhancing the SA synthesis pathway and E4P synthesis pathway based on cutting off the PEP branching pathway and SA catabolism pathway, the production of SA in the YCY8 strain was able to reach 1.06 g/L within 96 h. However, the production of YCY9 was not as good as that of YCY8, probably due to the low level of glf-glk expression. Furthermore, we carried out the optimization of inoculum amount and medium and found that inoculation with 10% inoculum amount into the optimized medium could bring out the advantages of YCY9, with a yield of 1.87 g/L. In addition, we found that the optimized medium also increased the production of SA by 13-16 times compared to the basal medium within 48 hours. These optimized conditions that have been tested for SA yield enhancement will be integrated for synergistic effects in the future. The increased production of shikimic acid will help improve the immunity of livestock and poultry, reduce the use of antibiotics and potentially huge economic losses, and achieve the goal of sustainability.