Synthetic biology approach for designing of photosynthetic escherichia coli

Abstract

Increasing carbon dioxide concentration is a very serious problem. To solve this problem, various approaches have been done. Between of these approaches, photosynthesis is the most important approach because this is a key step for the carbon cycle of the Earth. An increased amount of carbon dioxide production must be fixed by photosynthetic carbon fixation for carbon circulation.

In this study, converting a chemoheterotroph organism to a photoautotroph was studied using synthetic biology approach. First, introducing a photosystem in Escherichia coli was done to generate energy. For this result, light converted to chemical energy, which formed ATP. The energy conversion amount was 8.28 mmol ATP/gDCW·h. This result was done by introducing the proteorhodopsin, which absorbs specific wavelength. In this study, confirming co-expression of the different proteorhodopsin was done. At this result, The co-expressed proteorhodopsin absorbs wider wavelength of light, so the light usage efficiency can improve.

Next, the dark reaction was studied. To circulate carbon fixation, Calvin cycle was introduced. Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC.7942 Calvin cycle and Synechocystis sp. PCC 6803 carboxysome was introduced in Escherichia coli. The carboxysome functions to concentrate carbon dioxide and block oxygen, so Rubisco can fix carbon dioxide more efficiently. The forming $\beta$-carboxysome was shown in situ and in vitro images. The Rubisco activity was shown in vitro and in vivo. In this result, confirmed the Calvin cycle activity in Escherichia coli.

Inside of the carboxysome, there are many enzymes, such as Rubisco and carbonic anhydrase. For encapsulation, selective specific enzymes replace with Rubisco was done in this study. The carboxysome without Rubisco gene was expressed and the empty carboxysome was confirmed with TEM image. The fumarase was encapsulated in the carboxysome to confirm the encapsulation. The fumarase encapsulated carboxysome was purified and an activity assay was done. As expected the result showed fumarase was indeed encapsulated in carboxysome. Through this system, we can express enzyme encapsulate in the carboxysome and this system expect to generate make anaerobic enzymes expression actively in an aerobic cell growth condition.