Presentation Information
[2ASBA-10]Opto-engineering biology with rhodopsins: Developing optoenergetic platform cell factories with light-driven proton pump systems
○Kiyotaka Hara1 (1. University of Shizuoka (Japan))
Keywords:
ATP,Rhodopsin,Escherichia coli,Saccharomyces cerevisiae
Research and development aimed at enhancing the fundamental and versatile functions of microorganisms and creating common platform cells for producing various useful chemicals has been actively pursued in recent years. By installing the gene clusters constituting the synthesis pathway for the target chemicals into the platform cell, intracellular biosynthesis activity of the target chemicals can be enhanced. For the microbial base of platform cell factories, aerobic fermentation using chemoheterotrophic microorganisms, such as Escherichia coli and budding yeast Saccharomyces cerevisiae, is often employed due to reasons including their rapid growth speeds and well-established synthetic bioengineering and metabolic engineering tools.
A fundamental subject of microbial platform cell factories cultivated aerobically is that the ATP supply becomes a limiting factor for cellular metabolism due to the limitation of dissolving oxygen into the culture medium. In microbial fermentative production, ATP regeneration, while crucial for cellular processes, conflicts with efficient target chemical production because ATP regeneration exhausts essential carbon sources also required for target chemical biosynthesis. To wrestle with this dilemma, we harnessed the power of microbial rhodopsins with light-driven proton pumping activity to supplement with ATP, thereby facilitating the bioproduction of various chemicals. As a result, the proton concentration gradient formed across the membrane is used as a proton motive force, enabling the ATP synthase enzyme present in the membrane to regenerate ATP.
We demonstrated a light-driven ATP supply and redistribution of metabolic carbon flows to target chemical synthesis by installing delta rhodopsin (dR), a microbial light-driven proton-pumping rhodopsin from Haloterrigena turkmenica, in Escherichia coli. In addition, we identified novel rhodopsins with higher proton pumping activities than dR, and created an engineered cell for in vivo self-supply of the chromophore, all-trans-retinal. In S. cerevisiae, maintaining acidification inside the vacuoles consumes large amounts of ATP to pump protons from the cytosol to the vacuolar lumen by the vacuolar ATPase (V-ATPase), which is localized in vacuolar membrane. In our recent study, dR was also expressed and localized in the vacuolar membrane of S. cerevisiae. The light-driven proton pumping activity of the purified vacuole with dR was observed as the pH change outside the vacuole. A light-induced increase in the intracellular ATP content was shown in the yeast harboring vacuoles with dR. These optoenergetic bacteria and yeast can potentially solve the fundamental energy limitation problem of platform cell factories for various bioproduction applications. Our concept exploiting the light-powering ATP supplier offers a potential increase in carbon use efficiency for microbial productions through metabolic reprogramming.
A fundamental subject of microbial platform cell factories cultivated aerobically is that the ATP supply becomes a limiting factor for cellular metabolism due to the limitation of dissolving oxygen into the culture medium. In microbial fermentative production, ATP regeneration, while crucial for cellular processes, conflicts with efficient target chemical production because ATP regeneration exhausts essential carbon sources also required for target chemical biosynthesis. To wrestle with this dilemma, we harnessed the power of microbial rhodopsins with light-driven proton pumping activity to supplement with ATP, thereby facilitating the bioproduction of various chemicals. As a result, the proton concentration gradient formed across the membrane is used as a proton motive force, enabling the ATP synthase enzyme present in the membrane to regenerate ATP.
We demonstrated a light-driven ATP supply and redistribution of metabolic carbon flows to target chemical synthesis by installing delta rhodopsin (dR), a microbial light-driven proton-pumping rhodopsin from Haloterrigena turkmenica, in Escherichia coli. In addition, we identified novel rhodopsins with higher proton pumping activities than dR, and created an engineered cell for in vivo self-supply of the chromophore, all-trans-retinal. In S. cerevisiae, maintaining acidification inside the vacuoles consumes large amounts of ATP to pump protons from the cytosol to the vacuolar lumen by the vacuolar ATPase (V-ATPase), which is localized in vacuolar membrane. In our recent study, dR was also expressed and localized in the vacuolar membrane of S. cerevisiae. The light-driven proton pumping activity of the purified vacuole with dR was observed as the pH change outside the vacuole. A light-induced increase in the intracellular ATP content was shown in the yeast harboring vacuoles with dR. These optoenergetic bacteria and yeast can potentially solve the fundamental energy limitation problem of platform cell factories for various bioproduction applications. Our concept exploiting the light-powering ATP supplier offers a potential increase in carbon use efficiency for microbial productions through metabolic reprogramming.
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