A Simple Method for Highly Evolved Enzymes
Researchers Neunschwander et al. had been working with the enzyme chorismate mutase (EcCM), which is responsible for converting the metabolic intermediate chorismate to prephanate. The researchers wanted to introduce a “five-amino-acid hinge loop” in one of the enzyme’s helixes. However, this insertion drastically affected the enzyme’s capacity to catalyze the chorismate to prephanate reaction (Fig. 1).
The team hoped to utilize directed evolution to restore enzymatic activity to the new form of the enzyme, hEcCM.
Problems with Directed Evolution
A mutant library of the hEcCM gene was generated using error-prone PCR and DNA shuffling and selection was run in vivo in E. coli auxotrophic for chorismate mutase. However, the hEcCM enzyme produced using this protocol, tEcCM, did not have an increase in enzymatic activity (Fig. 1).
(Neunschwander et al., 2007 - Permission Pending)
Figure 1 - Quantification of the catalytic activity of the wild-type chorismate mutase enzyme (EcCM), chorismate with the engineered helix-loop (hEcCM), the evolved enzyme after two rounds of directed evolution (tEcCM), and the evolved enzyme after directed evolution with "selectio" expression vector (EcCM-200/4 - see Fig. 2). Catalytic activity was determined by an in vitro enzyme specific assay.
Finding a Solution
The authors hypothesized that if they were able increase selective pressure for catalytic activity during the selection process, directed evolution would be much more effective at restoring the enzymatic activity of hEcCM.
To achieve their goal, the authors came up with the following solution: transform an auxtrophic strain of E. coli with an expression vector containing the hEcCM gene and devise a way to keep the enzyme at very low concentrations. Inefficient catalyst activity would result in lethality for the cell (a common selection scheme). Since the levels of the mutated hEcCM would be kept low, only the most efficient enzymes would survive through the selection scheme.
The researchers changed the expression of hEcCM in two ways to keep the enzyme at low concentrations. First, they inserted the hEcCM gene behind a Ptet promoter cassette, which allowed the team to control the cellular levels of the enzyme as a function of tetracycline concentration (Fig. 2). Secondly, they inserted an ssrA tag behind the enzyme, making the enzyme susceptible to degradation by the protease ClpXP (Fig. 2).
(Neuenschwander et al., 2007 - permission pending)
Figure 2 - The constructed expression vector used to evovle the hEcCM gene. The combination of tetracycline-induced expression and constant degradation by the protease ClpXP meant the hEcCM enzyme was kept at constant low levels during selection, which increased selective pressure for higher rates of activity in the enzyme.
To test the effectiveness of this new method, they created a mutated library of the tEcCM gene using error-prone PCR and DNA shuffling and inserted these genes into this new expression vector. These plasmids were then transformed into the auxotrophic strain of E. coli and the organisms were subjected to a single round of directed evolution. The most efficient mutant of this experiment, EcCM-200/4, displayed a level of enzymatic activity comparable to the original wild-type chorismate mutase gene (Fig. 1).
Advantages of the Method
- The method Neuenschwander et al. proved useful and restoring enzymatic activity to their synthetically-engineered catalyst.
- The "selection vector" used in this experiment is modular: when selective pressure is too low to improve proteins in other experiments with directed evolution, the "selection vector" can be used to increase selective pressure on the protein for phenotype improvement.
Disadvantages of the Method
- The technique requires the use of auxotropihc organisms to produce the selected pressure required to drive the improvement in the protein of interest.
- The protein of interest must be essential to the life of the organism which may not be the case with completely novel proteins engineered through synthetic biology.
- The method requires the knowledge of gene function, which often is not the case.
- This method generates a large number false positives due to some E. coli receiving two or more copies of the expression plasmid.