Periodic Reporting for period 1 - FRIDOM (Flux Race Investigation for Dissection Of Metabolic-bottlenecks: Leveraging the tremendous potential of algal metabolic diversity)
Reporting period: 2023-07-01 to 2025-12-31
FRIDOM aims to harness the high efficiency of algal metabolism for improving plant growth yield. The main underlying methodology used, which is unique to FRIDOM, is the ability to measure the actual metabolic rates of reactions in photosynthesis in live algal cells with very high-throuput (termed fluxome). By stable isotope labelling tools we developed for this project, we can quantify the rates of photosynthetic metabolism, in highly performing algal species, including the fastest alga on the plant, Chlorella ohadii, isolated from the Negev desert in Israel. In terms of experiments, this is extremely challenging as the half-times of many of these metabolic reactions is at the level of very few seconds. Once we are able to document these rates in several algae and under several growth conditions, we are able to identify reactions and associated enzymes which demonstrate superb rates in the highly-peroforming algae. This is the first step in raising targets from these algae for engineering into other, slower growing algae and plants. The other parts of FRIDOM involves our efforts to export the highly performing reactions from the fast algae, and introduce them into plants in order to test whether we can get them to grow faster or more efficiently. Once we succeed in finding such engineering targets, and find an improved growth phenotype, we use the same stable isotope labelling tools mentioned above to study the detailed mechanism of what happens inside the metabolic network of the engineered plants and algae. This is an informed approach to really understand what takes place inside their metabolic network, and not refer to the outcome as a 'black-box'. Cases where we can truly improve the growth yield of plants and algae, become immediately leads of projects aiming to reproduce the same engineering and effects in real crop plants, for improving their yield in the future, and supporting the increasing demand from agriculture.
During the last two years, we have made several major achievements in the progress of the FRIDOM project.
1. Establishment of massive fluxome analysis capacity - through the combination of (1) installing an automated stable isotope labelling sampler, (2) developing a new high-throughput analytical method for quantification of phosphorylated metabolites (key intermediates in photosynthetic metabolism), and (3) a simplified computational framework for genome-scale metabolic models, we have created the largest experimental pipelined for metabolic flux analysis in photosynthetic cultures, attracting interest of overwhelming amount of potential collaboration partners aiming to use our tools.
2. Generating a high-throughput algal metabolic engineering pipeline . thanks to modification of the modern tools like Modular Cloning (MoClo) to include metabolic-associated parts, we are now able to create 40-50 new engineered strains per year, providing a powerful capacity for testing our potential targets under several variations, and with minimal need for prioritization.
3. Engineering of highly performing crop lines - following introduction of algal metabolic architectures into Camlina sativa, a model seed crop plant, we were able to generate egineered plants with 20-30% increased biomass attracting the interests of seed companies.
1. Establishment of massive fluxome analysis capacity - through the combination of (1) installing an automated stable isotope labelling sampler, (2) developing a new high-throughput analytical method for quantification of phosphorylated metabolites (key intermediates in photosynthetic metabolism), and (3) a simplified computational framework for genome-scale metabolic models, we have created the largest experimental pipelined for metabolic flux analysis in photosynthetic cultures, attracting interest of overwhelming amount of potential collaboration partners aiming to use our tools.
2. Generating a high-throughput algal metabolic engineering pipeline . thanks to modification of the modern tools like Modular Cloning (MoClo) to include metabolic-associated parts, we are now able to create 40-50 new engineered strains per year, providing a powerful capacity for testing our potential targets under several variations, and with minimal need for prioritization.
3. Engineering of highly performing crop lines - following introduction of algal metabolic architectures into Camlina sativa, a model seed crop plant, we were able to generate egineered plants with 20-30% increased biomass attracting the interests of seed companies.
Sub-cellular localisation of key metabolites in algal cells - following discoveries on anomalies in metabolite levels across photosynthetic metabolism pathways in algae obtained within FRIDOM, we have harnessed super-resolution Mass-Spectrometry-imaging with the aim of identifying the sub-cellular distribution of these metabolites as the source of these anomalies. Specifically, we hypothesized that due to their unique cell structure, containing a well-developed pyrenoid, the algal part in the cells which is in charge of concentrating CO2, algae maintain a gradient of some photosynthetic metabolism intermediates. Despite the physical challenge, we were able to image key metabolites in a spatial resolution of 300-400 nm, for the first time in algae. Our findings regarding these metabolites challenge major existing paradigms in the field, and bear significant implications to the projects aiming to engineer algal pyrenoids into crop plants. We found that metabolic gradients must be balanced with the active pumping of CO2 in order to maintain an efficient activity of the carbon fixing pathways in photosynthesis.