Upscaling in vitro arbuscular mycorrhizal fungi inoculum production via combinatorial lipid metabolic engineering of host plants

Project Information

MycUpscaling

Grant agreement ID: 101018013

DOI

10.3030/101018013

Project closed

EC signature date 31 March 2021

Start date 1 September 2022

End date 31 January 2026

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 248 425,92

EU contribution

€ 248 425,92

Coordinated by

UNIVERSITE CATHOLIQUE DE LOUVAIN
Belgium

Periodic Reporting for period 1 - MycUpscaling (Upscaling in vitro arbuscular mycorrhizal fungi inoculum production via combinatorial lipid metabolic engineering of host plants)

Reporting period: 2022-09-01 to 2024-08-31

Summary of the context and overall objectives of the project

The MycUpscaling project addresses the major limitation in the large-scale, clean, and cost-efficient production of arbuscular mycorrhizal fungi (AMF) inoculum. Although AMF are critical symbiotic organisms supporting nutrient uptake in over 70% of land plants—particularly phosphorus—their industrial production remains constrained. Current large-scale methods mostly rely on in vivo soil-based systems, which are space-demanding and prone to microbial contamination. In contrast, in vitro systems, while cleaner, are technologically demanding and yield lower spore outputs, limiting their commercial application. This bottleneck is compounded by the fungi’s dependency on plant-derived lipids (especially TAGs) for spore formation and propagation, and the limited understanding of lipid transfer mechanisms at the plant–AMF interface.
The project’s outcomes are crucial for sustainable agriculture, aligning with the EU’s Green Deal and Farm-to-Fork strategies. By improving the mass production of biofortified AMF inoculum—rich in triacylglycerol and produced under sterile, controlled conditions—MycUpscaling offers a biotechnological alternative to synthetic fertilizers, reduces environmental impact, and improves crop resilience. This supports ecological intensification of farming practices, restores soil biodiversity, and enhances food security, particularly under the pressures of climate change and land degradation.
The overarching objective is to develop a novel platform for high-quality, high-yield in vitro AMF inoculum production by engineering host plant lipid metabolism. This goal is broken down into three specific and interlinked objectives:
Engineering plant lipid metabolism
– To identify optimal gene combinations that enhance TAG biosynthesis and transfer to AMF in Medicago truncatula roots, using a combinatorial “Push, Pull, Package” (PPP) metabolic engineering strategy.
Deciphering metabolic flux responses
– To trace how the plant’s lipid metabolic network is rewired in response to genetic modifications, by using isotopic labeling and lipidomics to monitor fatty acid flux toward AMF-compatible lipid species.
Assessing scalability and production cost
– To evaluate the engineered systems’ suitability for large-scale AMF spore production in Petri plates and bioreactors, and to perform cost-efficiency analyses to ensure industrial relevance.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Since the project start (01/12/2022), the MycUpscaling project has achieved significant progress across the outgoing phase (WSU) in Work Packages 1 and 2, with preparations underway for WP3 in the return phase (UCLouvain). The project pursued the overarching goal of engineering host plant lipid metabolism to enhance the production of triacylglycerol (TAG)-rich, in vitro–produced AMF spores.
The work unfolded in three major technical areas:
WP1 – Engineering Lipid Metabolism at the Symbiotic Interface
Over 100 combinatorial GoldenBraid 2.0 multigene constructs were assembled to overexpress lipid-related genes (WRI1, RAM1, RAM2, FatM, STR1, STR2).
Promoter diversification strategies were implemented to mitigate transformation inefficiencies.
Transgenic Medicago truncatula hairy roots (R108 genotype) were successfully generated and shown to accumulate higher TAG levels.
Several engineered lines, such as R2WFDS1S2O1 and R1R2FDS1S2O1, significantly increased AMF spore production in vitro (up to 2500 spores/plate).
WP2 – Characterization of Lipid Flux and Functional Validation
Selected high-performing transgenic lines were subjected to [14C]-acetate metabolic flux analysis.
Results confirmed redirection of acyl flux from membrane lipid biosynthesis to storage lipid pathways, especially in lines expressing RAM1, RAM2, and FatM.
TLC and GC analyses validated increased incorporation of labeled fatty acids into TAGs and fungal-specific lipid markers (e.g. 16:1ω5).
Enhanced lipid accumulation correlated with increased fungal colonization and sporulation efficiency.
Main Results Achieved So Far
Creation of a library of engineered M. truncatula lines optimized for lipid transfer to AMF.
Demonstration that combinatorial engineering (PPP strategy) significantly improves both TAG accumulation and AMF spore production.
Proof-of-concept for lipid flux redirection and enhanced symbiotic output in engineered systems.
Preparation of protocols and materials for scale-up testing in bioreactors at UCLouvain.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The MycUpscaling project has made pioneering advancements in plant–microbe synthetic biology, pushing well beyond the current state of the art in several key areas:
It is the first project to engineer the host plant specifically to biofortify its microbial symbiont (AMF) by enhancing triacylglycerol (TAG) production and transfer.
It applied, for the first time in AMF systems, a combinatorial “Push–Pull–Package” (PPP) lipid metabolic engineering strategy, which had previously only been used in seed oil enhancement.
The project developed a library of multigene transgenic Medicago truncatula lines with optimized root expression of lipid biosynthesis and transport genes.
It demonstrated through radioisotopic flux tracing and lipidomic profiling that engineered lines redirect acyl flux away from membrane lipids toward storage lipids beneficial for AMF spore formation—evidence never before shown in this symbiotic system.
These scientific advancements bridge plant lipid metabolism, fungal physiology, and synthetic biology, setting a new benchmark for future research on microbial bio-inoculants.

As the project enters its return phase (at UCLouvain), the following milestones are expected:
Scale-up validation of selected engineered lines in Petri dish and bioreactor systems, measuring spore production efficiency, root colonization, and hyphal development.
Cost-efficiency analysis of producing biofortified AMF spores compared to conventional inocula.
Optimization of bioreactor culture conditions (e.g. aeration, nutrient flow) for large-scale, sterile AMF production.
Completion of microscopy imaging and targeted lipidomics to spatially resolve lipid flux in the symbiotic interface.
Submission of a peer-reviewed publication and final dissemination actions including public events and industrial outreach.
Socio-Economic and Societal Impact
The project directly addresses two major societal needs:
Sustainable Agriculture and Fertilizer Reduction
By enabling cost-effective, clean AMF inoculum production, MycUpscaling supports the shift away from synthetic phosphorus fertilizers—mitigating environmental pollution and reducing dependence on non-renewable resources.
Industrial Competitiveness and Bioeconomy
The project lays the groundwork for commercial-scale AMF production technologies with potential applications in crop biostimulants, horticulture, and ecological restoration. The biofortified spores can improve plant nutrition, stress resilience, and yield—factors highly relevant to Europe’s agrotech industry.
Scientific Capacity and Innovation
The fellow has gained unique training in mycorrhiza in vitro culture, synthetic biology, metabolic flux analysis, fostering EU leadership in the emerging field of microbiome-based agricultural inputs.
Wider Societal Benefits
By contributing to agroecological transitions, improved soil health, and climate-resilient agriculture, MycUpscaling is aligned with the EU Green Deal, Farm to Fork strategy, and the UN Sustainable Development Goals (SDGs 2, 12, 13, and 15).

Combinatorial lipid engineering strategy of mycorrhized host plants for mass-production of bio-forti