Chloroplast Engineering of Nitrogen Fixation

Through plastid genome engineering, we have achieved agronomic transformation of many important crops, such as herbicide resistance, drought tolerance, salt tolerance, insect resistance, pathogen resistance, nitrogen fixation, nutrition, and cytoplasmic male sterility. Here, Lifeasible is committed to providing reliable and economical chloroplast genetic engineering solutions for crops to enhance plant expression of nitrogenase.

Introduction

Nitrogen is a component of proteins, nucleic acids and other fundamental molecules in all living organisms and is extremely important in agriculture. Because nitrogen is unavailable to plants and animals, and nitrogen is harmful in the atmosphere, most nitrogen comes from the reduced or oxidized form of nitrogen in the soil during plant growth. Nitrogen availability is one of the main limiting factors for crop growth under most growing conditions. Farmers often use unsustainable levels of inorganic fertilizers to boost crop production. Finding alternatives to inorganic fertilizers is essential for sustainable and safe food production. Current biological solutions aim to fix N₂ into crops by establishing root symbiotic relationships with nitrogen-fixing bacteria found in legumes, by transferring nitrogenases to cereal crops, or directly by introducing nitrogenases into the plants themselves.

Fig. 1. Crop plants could be engineered to express nitrogenase. (Oldroyd G E D, et al., 2014)

Our Solutions

It is completely feasible to introduce nitrogenase into plant cells through the solution of chloroplast genetic engineering, and the nif gene integrated into the chloroplast genome can also produce functional products. This solution also faces the following two problems:

(1) The extreme sensitivity of nitrogenase and the irreversible inactivation of molecular oxygen contradict the main activity of the chloroplast—oxygenogenic photosynthesis.

(2) The assembly of active holoproteins is so complex that at least nine genes are required for expression in bacteria.

Faced with the above challenges, we used unicellular green algae as a platform to optimize plastid nitrogenase biogenesis, and then transferred this technology to plant models and ultimately to key crop species such as rice. Our goal is to engineer crop species for direct access to N₂. The strategy is as follows:

• Construction of recombinant plasmids and engineered strains.
• Reverse transcription-PCR (RT-PCR) and quantitative PCR (q-PCR).
• Measurement of nitrogen fixation activity.
• In vivo ¹⁵N₂ incorporation assay.
• Western blot analysis.

Attractive Advantages of Our Solutions

• Our solution avoids the need to import Nif proteins from the cytosol and also offers the potential for higher expression levels.
• We provide a simple, genetically tractable unicellular green algae platform with the advantages of rapid microbial culture, flexible metabolism, and well-established molecular tools for plastosome engineering.
• Our selected algae and gymnosperms express nitrogenase-like proteins encoded in the chloroplast genome.
• We used a simpler and more flexible photosynthesis platform as the SynBio testbed.

Lifeasible's goal is to provide customers around the world with fully customized chloroplast engineered solutions for enhancing plant expression of nitrogenase. Please contact us to discuss further details to ensure your next success.

References

1. Oldroyd G E D, Dixon R. (2014) Biotechnological solutions to the nitrogen problem[J]. Current opinion in biotechnology. 26: 19-24.
2. Larrea-Álvarez M, Purton S. (2021) The Chloroplast of Chlamydomonas reinhardtii as a Testbed for Engineering Nitrogen Fixation into Plants. Int J Mol Sci. 22(16):8806.