Overview
Our project is going to modify E. Coli MG1655 to achieve the shikimic acid production. In brief, the metabolic engineering modification is divided into the following aspects:1. Enhancement of PEP levels: (1) blocking the PTS by knocking out the ptsG gene; (2) Blocking PEP branching pathways, including lactate, acetate, and ethanol synthesis pathways (knockout of ldhA, adhE, poxB, and pta genes); 2. Increase the E4P content: overexpression of tktA and talB genes;
3. Construct and 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. Enhancing glucose utilization by heterologously introducing a non-phosphorylated pathway, that is, inserting glk and glf genes (mainly by glk-glf integration into the ptsG locus);
In E. coli, glucose is translocated intracellularly via the phosphotransferase system (PTS) and phosphorylated to form glucose-6-phosphate catalyzed by either hexokinase or glucokinase. Glucose 6-phosphate is then converted to phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) via the glycolysis (EMP) and pentose phosphate (PPP) pathways, respectively. A portion of PEP and E4P together enter the shikimate acid pathway to produce aromatic amino acids, and another portion enters the tricarboxylic acid (TCA) cycle to produce energy. In addition, PEP is converted to substances such as lactic acid, acetic acid, and ethanol.
Genome-scale metabolic model (GSMM) of Escherichia coli (iML1515)
To identify the key gene targets for improving the shikimate biosynthesis in the metabolic network, the iML1515 was used to analyze the flux variation of reactions in the network while increasing the shikimate output rate. When glucose was a substrate, 88 reactions were screened from all reactions in the metabolic network (Fig.1A). The normalized flux of these reactions showed a monotonous increasing trend, suggesting these reactions should be potential targets directly affecting the biosynthesis of shikimate. Based on metabolic pathways (Fig.1D), the reactions involved in the shikimate biosynthesis pathway (including aroE, aroD, aroG and aroB) showed a flux-increased trend with shikimate output rate, and the reactions involving precursor PEP accumulation (including tktA and talB) also have slightly increased in flux (Fig.1B), demonstrating that these targets should be enhanced in project of improving shikimate biosynthesis. Whereas, the reaction involved in the glucose phosphotransferase system (including ptsG, ptsH, ptsI), pyruvate dehydrogenase (coding by poxB gene) and shikimate kinase (coding by aroK and aroL) showed a deceased trend in flux variation (Fig1.C), which weakens the precursor PEP consumption pathway and shikimate consumption pathway.
The specific steps are as follows:
1.Step 1: Blocking the PEP branching pathway
Fig 2. PEP branching pathway
The shikimic acid synthesis pathway starts with PEP and E4P, and increasing intracellular PEP levels theoretically increases shikimic acid production. The branching pathways of PEP include the lactate synthesis pathway, acetate synthesis pathway, and ethanol synthesis pathway. In order to accumulate more PEP intracellularly, we need to reduce unnecessary consumption of PEP as well as energy.
The conversion of phosphoenolpyruvate to pyruvate is reversible in dynamic equilibrium. To accumulate more phosphoenolpyruvate, the flow of pyruvate to the lactate, acetate, and ethanol synthesis pathways was blocked by knocking out the genes for adhE, pta, poxB, and ldhA enzymes. Since the conversion of pyruvate to acetyl coenzyme A in the tricarboxylic acid cycle is very important for the normal growth and metabolism of E. coli, this pathway cannot be blocked.
Fig 3. Partial plasmid for blocking the PEP branching pathway
2.Shikimic acid pathway The shikimic acid pathway is a common metabolic pathway in the synthesis of aromatic amino acids. Glucose through glycolysis and pentose phosphate pathway to produce PEP and E4P, respectively, the two in the aroG catalyzed into the shikimic acid pathway, after a series of enzymatic reactions (in the order of aroB, aroD, aroE, respectively) to synthesize shikimic acid, and ultimately produce tyrosine, tryptophan, phenylalanine, three of the aromatic amino acids. In the wild-type strain, shikimic acid is an intermediate metabolite in the aromatic amino acid synthesis pathway and does not accumulate in the cell.
Fig 4. Shikimic acid pathway
(1) Step 2: Cutting off the shikimic acid decomposition pathwayThe downstream catabolic pathway also exists in the shikimic acid synthesis pathway in E. coli. In order to maximize the synthesis and secretion of shikimic acid, we will block the downstream catabolic pathway of shikimic acid by knocking out the aroK and aroL genes with gene knockout technology.
Fig 5. Plasmid for knocking out aroK and aroL
(2) Step 3: Enhancement of the shikimic acid synthesis pathway As shown in Fig 4, the shikimic acid synthesis pathway was systematically improved by metabolic engineering. Briefly, the shikimic acid synthesis pathway starts with the synthesis of DAHP (3-deoxy-D-arabino-heptulosonate-7-phosphate) by PEP and E4P under the catalytic action of aroG, and then shikimic acid is synthesized under the catalytic action of a series of enzymes such as aroB, aroD, and aroE. To increase the yield of shikimic acid, we overexpressed these enzymes.
Fig 6. Plasmid for enhancement of the shikimic acid synthesis pathway
3.Step 4: Increase intracellular E4P synthesis
Fig 7. Pentose phosphate pathway
The pentose phosphate pathway is a glucose catabolic pathway that is prevalent in animals, plants, and microorganisms but is involved in different proportions in different species. In addition to providing energy, the pentose phosphate pathway in organisms primarily provides a variety of raw materials for anabolism. For example, it provides NADPH for fatty acid biosynthesis, ribose 5-phosphate for nucleotide coenzymes and nucleotide synthesis, and E4P for aromatic amino acid synthesis.
As mentioned above, the shikimic acid synthesis pathway starts with PEP and E4P, so increasing the amounts of precursors of the shikimic acid synthesis pathway could theoretically increase shikimic acid production. In addition to enhancing the accumulation of intracellular PEP, we also enhanced the expression of two enzyme genes, tktA and talB, in the pentose phosphate pathway to increase the level of intracellular E4P. PEP and E4P are two important precursors for the synthesis of shikimic acid, and the accumulation of both is beneficial for the improvement of shikimic acid production.
Fig 8. Plasmid for increasing intracellular E4P synthesis
Step 5: Optimize the glucose transport system
Fig 9. Glucose transport system
The glucose facilitator-glucose kinase (Glf-Glk) pathway from the motile fermentative bacterium Zymomonas mobilis promotes the bacterial uptake of glucose. According to the papers, the transport of glucose, the substrate of glycolysis, i.e. the transport of glucose from the culture medium across the cell membrane into the cell, can take two forms: the PTS transport system and the non-phosphorylated Glf-Glk transport system. However, the PTS transport system requires a large amount of PEP during transport, thus reducing the amount of substrate (PEP) in the shikimic acid synthesis pathway and decreasing shikimic acid production.
Glucose has both of the above modes of transport. The PTS transport system requires PEP consumption for glucose transport, which is not conducive to subsequent PEP accumulation. Therefore, we chose to eliminate the PTS transporter from the two transporter systems and introduced the glf-glk transporter system into the ptsG locus by recombinant technology to meet the energy requirements for bacterial growth. The introduction of this pathway into microorganisms can alleviate the side effect of slow microbial growth caused by the knockout of the PTS system, as well as improve the efficiency of glucose utilization, and at the same time, it can allow more carbon metabolism flow into the subsequent relevant synthetic pathway, which is expected to increase the production of shikimic acid
Fig 10. Plasmid for optimizing the glucose transport system
