Random Gene Knockout to Maximize Product Yield
Contents
The Experiment
The Problem
Researchers Hal Alper, Kohei Miyaoku and Greg Stephanopoulos have recently demonstrated the effectiveness of using directed evolution to optimize a synthetic construct. Previously, the researchers engineered a strain of E. coli to synthesize lycopene, a carotenoid that naturally occurs in tomatoes. Lycopene is an antioxidant that has recently been incorporated into vitamin tablets, creating a commercial pressure to increase the efficiency and yield of lycopene synthesis.
Computer Modeling to Increase Product Yield
To further optimize lycopene production by the e. coli strain, the authors used computer modling to identify a series of gene knockouts sites that were predicted to increase lycopene production. However, the strains engineered to contain these knockouts were still unable to produced lycopene at the predicted “stoichiometric maximum.” This finding led the researchers to hypothesize that lycopene production was “limited by unknown kinetic or regulatory factors unaccounted for in the stoichiometric models.”
Directed Evolution: Random Gene Knockout
The researchers used directed evolution to find other gene knockouts that might affect these unexplored regions of the cell. A library of lycopene-producing E. coli with random gene knockout was achieved by introducing genome-wide integrating transposons to the cells in vivo. This random knockout library was then plated and tested for lycopene production. In this assay, lycopene production was proportional to the red color of bacterial colonies. The three best knockouts selected by this screening were there molecularly characterized. All three knockout sites were differed that had been predicted to be beneficial using their models. Two of these knockouts interrupted genes which had been previously undescribed. Thus, by using a random strategy, they discovered new genes whose expression affected the production of the desired product.
Computer Modeling Versus Random Gene Knockout
The team went on to determine the relative effectiveness of the three gene knockouts at increasing lycopene production when compared to the knockouts they had predicting with computer modeling. To answer this question, the researchers created 64 unique strains of the lycopene-producing E. coli representing all possible combinations of the three knockouts selected by directed evolution, the seven model-predicted knockouts, and the two parental strains from which the “evolved” and model-predicted strains were derived. Lycopene production of the 64 strains was measured by extracting the lycopene from the each colony after a defined interval of time and quantifying lycopene level by absorbance spectroscopy at 475. Of the two global maxima of this experiment, one had a knockout selected through directed evolution testing (Fig. 1a). Furthermore, this particular knockout strain also showed an earlier peak in lycopene production when compared to the completely systematically-predicted knockout strain in batch fed culture (Fig. 1b).
