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Engineering a Rhodopsin-Based Photo-Electrosynthetic System in Bacteria for CO₂ Fixation.

ABSTRACT: A key goal of synthetic biology is to engineer organisms that can use solar energy to convert CO₂ to biomass, chemicals, and fuels. We engineered a light-dependent electron transfer chain by integrating rhodopsin and an electron donor to form a closed redox loop, which drives rhodopsin-dependent CO₂ fixation. A light-driven proton pump comprising Gloeobacter rhodopsin (GR) and its cofactor retinal have been assembled in Ralstonia eutropha (Cupriavidus necator) H16. In the presence of light, this strain fixed inorganic carbon (or bicarbonate) leading to 20% growth enhancement, when formate was used as an electron donor. We found that an electrode from a solar panel can replace organic compounds to serve as the electron donor, mediated by the electron shuttle molecule riboflavin. In this new autotrophic and photo-electrosynthetic system, GR is augmented by an external photocell for reductive CO₂ fixation. We demonstrated that this hybrid photo-electrosynthetic pathway can drive the engineered R. eutropha strain to grow using CO₂ as the sole carbon source. In this system, a bioreactor with only two inputs, light and CO₂, enables the R. eutropha strain to perform a rhodopsin-dependent autotrophic growth. Light energy alone, supplied by a solar panel, can drive the conversion of CO₂ into biomass with a maximum electron transfer efficiency of 20%.

SUBMITTER: Davison PA

PROVIDER: S-EPMC9680020 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Engineering a Rhodopsin-Based Photo-Electrosynthetic System in Bacteria for CO2 Fixation.

Davison Paul A PA Tu Weiming W Xu Jiabao J Della Valle Simona S Thompson Ian P IP Hunter C Neil CN Huang Wei E WE

ACS synthetic biology 20221020 11

A key goal of synthetic biology is to engineer organisms that can use solar energy to convert CO2 to biomass, chemicals, and fuels. We engineered a light-dependent electron transfer chain by integrating rhodopsin and an electron donor to form a closed redox loop, which drives rhodopsin-dependent CO2 fixation. A light-driven proton pump comprising Gloeobacter rhodopsin (GR) and its cofactor retinal have been assembled in Ralstonia eutropha (Cupriavidus necat ...[more]

PMID: 36264158

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Project description:A grand challenge for the next century is in facing a changing climate through bioengineering solutions. Biological nitrogen fixation, the globally consequential, nitrogenase-catalyzed reduction of atmospheric nitrogen to bioavailable ammonia, is a vital area of focus. Nitrogen fixation engineering relies upon extensive understanding of underlying genetics in microbial models, including the broadly utilized gammaproteobacterium, Azotobacter vinelandii (A. vinelandii). Here, we report the first CRISPR interference (CRISPRi) system for targeted gene silencing in A. vinelandii that integrates genomically via site-specific transposon insertion. We demonstrate that CRISPRi can repress transcription of an essential nitrogen fixation gene by ~60%. Further, we show that nitrogenase genes are suitably expressed from the transposon insertion site, indicating that CRISPRi and engineered nitrogen fixation genes can be co-integrated for combinatorial studies of gene expression and engineering. Our established CRISPRi system fills an important gap for engineering microbial nitrogen fixation for desired purposes.IMPORTANCEAll life on Earth requires nitrogen to survive. About 78% of the atmosphere alone is nitrogen, yet humans cannot use it directly. Instead, we obtain the nitrogen we need for our survival through the food we eat. For more than 100 years, a substantial portion of agricultural productivity has relied on industrial methods for nitrogen fertilizer synthesis, which consumes significant amounts of nonrenewable energy resources and exacerbates environmental degradation and human-induced climate change. Promising alternatives to these industrial methods rely on engineering the only biological pathway for generating bioaccessible nitrogen: microbial nitrogen fixation. Bioengineering strategies require an extensive understanding of underlying genetics in nitrogen-fixing microbes, but genetic tools for this critical goal remain lacking. The CRISPRi gene silencing system that we report, developed in the broadly utilized nitrogen-fixing bacterial model, Azotobacter vinelandii, is an important step toward elucidating the complexity of nitrogen fixation genetics and enabling their manipulation.

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Engineering a Rhodopsin-Based Photo-Electrosynthetic System in Bacteria for CO₂ Fixation.

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Engineering a Rhodopsin-Based Photo-Electrosynthetic System in Bacteria for CO<sub>2</sub> Fixation.

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Engineering a Rhodopsin-Based Photo-Electrosynthetic System in Bacteria for CO2 Fixation.

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Engineering a Rhodopsin-Based Photo-Electrosynthetic System in Bacteria for CO&lt;sub&gt;2&lt;/sub&gt; Fixation.

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Engineering a Rhodopsin-Based Photo-Electrosynthetic System in Bacteria for CO₂ Fixation.

Engineering a Rhodopsin-Based Photo-Electrosynthetic System in Bacteria for CO<sub>2</sub> Fixation.