Gene Knockout for Lycopene Overproduction

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Researchers Hal Alper, Kohei Miyaoku and Greg Stephanopoulos have recently shown the effectiveness of using directed evolution for optimizing a synthetic construct. Previously, the researchers had engineered a strain of E. coli capable of synthesizing lycopene, a carotenoid which naturally occurs in tomatoes but has more recently been incorporated in vitamin tablets for its antioxidant capabilities. The group was interested in further optimizing the lycopene output of this strain. Using computer modeling, they identified a series of gene knockouts sites which were predicted to increase the lycopene production. However, strains engineered with 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.”

The researchers used directed evolution to find gene knockouts which 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 (LINK). This random knockout library was then tested through plating, which revealed increases in lycopene production efficiency in the E. coli as increases in red colony color. The three best knockout E. coli selected by this screening to determine the beneficial gene knockout sites. All three knockout sites were different from the seven predicted by their models. Two of these knockouts interrupted genes which had been previously undescribed.

The team was interested in determining how effective the three gene knockouts were 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 and defined 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 and when compared to the completely systematically-predicted knockout strain in batch fed culture (Fig. 1b).


(Alper, 2005 - Permission Pending)

Figure 1: The two measurements lycopene production in knockout strains of lycopene producing bacteria. (a) A landscape displaying the 64 strains resulting for all possible combinations of gene knockouts selected through systematic modeling and directed evolution (combinatorial knockouts). Lycopene production for each strain was measured at the end of a 48-h shake-flask fermentation and amount of lycopene produced was quantified through extraction form the cell pellet with acetone and supernatant absorbance at 475 nm. Of interest is global maximum strain ΔgdhA ΔaceE ΔPyjiD, which contains a knockout of the ΔPyjiD gene selected through directed evolution testing. (b) Lycopene production of the best knockout strains in batch-fed culture. From left to right, the K12 strain from which combinatorial mutants were derived, the preengineered parental strain from which the systematically-selected knockout strains were derived, global maximum strain ΔgdhA ΔaceE ΔfdhF, global maximum strain ΔgdhA ΔaceE ΔPyjiD, and the global minimum strain and two local maximum strains from landscape 1a. Of interest is knockout strain ΔgdhA ΔaceE ΔPyjiD (fourth from the left). This strain, which has a gene knockout selected through directed evolution testing, shows the same maximum in lycopene productivity as the entirely systematically-predicted strain ΔgdhA ΔaceE ΔfdhF (third from left), but also shows an earlier peak in this productivity and more sustained lycopene production.