The Chloroplast of Chlamydomonas reinhardtii as a Testbed for Engineering Nitrogen Fixation into Plants

Abstract

Eukaryotic organisms such as plants are unable to utilise nitrogen gas (N2) directly as a source of this essential element and are dependent either on its biological conversion to ammonium by diazotrophic prokaryotes, or its supply as chemically synthesised nitrate fertiliser. The idea of genetically engineering crops with the capacity to fix N2 by introduction of the bacterial nitrogenase enzyme has long been discussed. However, the expression of an active nitrogenase must overcome several major challenges: the coordinated expression of multiple genes to assemble an enzyme complex containing several different metal cluster co-factors; the supply of sufficient ATP and reductant to the enzyme; the enzyme’s sensitivity to oxygen; and the intracellular accumulation of ammonium. The chloroplast of plant cells represents an attractive location for nitrogenase expression, but engineering the organelle’s genome is not yet feasible in most crop species. However, the unicellular green alga Chlamydomonas reinhardtii represents a simple model for photosynthetic eukaryotes with a genetically tractable chloroplast. In this review, we discuss the main advantages, and limitations, of this microalga as a testbed for producing such a complex multi-subunit enzyme. Furthermore, we suggest that a minimal set of six transgenes are necessary for chloroplast-localised synthesis of an ‘Fe-only’ nitrogenase, and from this set we demonstrate the stable expression and accumulation of the homocitrate synthase, NifV, under aerobic conditions. Arguably, further studies in C. reinhardtii aimed at testing expression and function of the full gene set would provide the groundwork for a concerted future effort to create nitrogen-fixing crops.

Chlamydomonas: Fast tracking from genomics

“…Chlamydomonas is also a model for understanding cilia/flagella and basal body biogenesis and their functions in motility and sensing the environment (Dutcher, 2020; Dutcher & O'Toole, 2016; Hayer‐Hartl, 2017; Ostrowski et al, 2011; Silflow & Lefebvre, 2001; Wostrikoff & Mackinder, 2023). It is increasingly used to dissect the cell cycle (Cross & Umen, 2015; Strenkert et al, 2019), nutrient limitation (Blaby‐Haas & Merchant, 2013; Calatrava et al, 2023; Sanz‐Luque & Grossman, 2023; Schmollinger et al, 2014), optogenetics (Berndt et al, 2016; Deisseroth & Hegemann, 2017), and interactions with other organisms (Calatrava et al, 2018; Torres et al, 2022) and in the production of recombinant proteins and high‐value chemicals, potentially in chloroplasts (Cutolo et al, 2022; Larrea‐Alvarez & Purton, 2021).…”

A novel phenylpropanoid methyl esterase enables catabolism of aromatic compounds that inhibit biological nitrification

“…Another approach is to limit the application of chemical fertilizers. For this, researchers and several companies (e.g., Pivot Bio) are developing engineered microbial and plant 9 systems that can be deployed in agricultural soils to enhance fixation of elemental nitrogen (N 2 ) into biologically available nitrogen (NH 3 ) [9][10][11][12] . Supplementing soils with these engineered N 2 -fixing bacteria can substantially reduce the amount of chemical N-fertilizers required for robust crop yields.…”

Multi-Sample Detection of Soil Nitrate Nitrogen Using a Digital Microfluidic Platform

“…Soil nitrate nitrogen serves as a primary reservoir of vital nitrogen for the flourishing of crops [1][2][3]. Inadequate levels of soil nitrate nitrogen could have a significant detrimental effect on grain productivity, whereas excessive application of nitrogen fertilizers may result in resource inefficiency and environmental contamination [4][5][6][7].…”