Adaptation to climate change in the rhizosphere across the millennia

Project Information

TOLERATE

Grant agreement ID: 101082049

DOI

10.3030/101082049

EC signature date 4 December 2023

Start date 1 December 2023

End date 30 November 2027

Funded under

• Food, Bioeconomy Natural Resources, Agriculture and Environment

Total cost

€ 3 909 870,00

EU contribution

€ 3 909 870,00

Coordinated by

DANMARKS TEKNISKE UNIVERSITET
Denmark

Periodic Reporting for period 1 - TOLERATE (Adaptation to climate change in the rhizosphere across the millennia)

Reporting period: 2023-12-01 to 2025-05-31

Summary of the context and overall objectives of the project

Climate change intensifies drought and temperature extremes, threatening agricultural productivity and the resilience of soil ecosystems. The rhizosphere—the narrow zone of soil influenced by plant roots—harbours microbial communities that play a critical role in plant adaptation to stress. However, most modern microbes have evolved under stable Holocene conditions and may lack the traits needed to cope with ongoing climatic pressures. The TOLERATE project addresses this challenge by exploring the genetic memory preserved in Arctic permafrost, where ancient DNA traces microbial responses to past warming events. By recovering and reactivating these ancient stress-resilience genes through synthetic biology, TOLERATE seeks to design microbial solutions for sustainable agriculture and bio-based manufacturing. The project’s overall objective is to identify, reconstruct, and validate genes conferring drought, osmotic, and thermal tolerance, introducing them into modern bacterial chassis to create bioinoculants and production strains. This work aligns with the EU Green Deal, Farm to Fork Strategy, and Circular Bioeconomy goals by providing tools to reduce irrigation, restore marginal soils, and replace fossil-derived industrial inputs with sustainable bio-based alternatives. The pathway to impact spans three dimensions: improved plant growth on degraded land, discovery of thermostable biomolecules for green manufacturing, and policy-relevant insights to update the regulatory framework for microbial biotechnology in climate adaptation.

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

During the first reporting period, TOLERATE established a functional paleogenomic and synthetic biology platform that bridges ancient genetic information with modern biotechnology. From 25 permafrost samples covering up to 600,000 years of Arctic history, 3.3 million genes and 61 high-quality metagenome-assembled genomes were reconstructed and authenticated. Among these, phasins and late embryogenesis abundant (LEA) proteins were identified as major contributors to drought and temperature adaptation. More than 30 ancient gene variants were synthesized, cloned, and tested in Pseudomonas putida, P. fluorescens, and Escherichia coli. Expression assays showed that ancient phasins increased solvent tolerance by up to 75% in E. coli and 25% in P. putida, while selected LEA proteins improved osmotic or solvent tolerance by 20-50%. Protein purification pipelines yielded gram-scale quantities for biophysical characterization, and constitutive expression systems were validated as most effective. Greenhouse trials with Pseudomonas protegens, P. capeferrum, and Mucilaginibacter gossypiicola demonstrated positive bioaugmentation effects, with barley biomass increasing by 67% under drought and enhanced chlorophyll and proline levels confirming physiological resilience. Microbiome analyses verified strain persistence and positive but transient modulation of native microbial communities. Together, these results advanced the project from TRL 1-2 to TRL 4-5 and provided the first experimental proof of concept for functional paleogenomics and climate-resilient bioinoculants.

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)

TOLERATE introduces the first validated workflow for transforming ancient DNA sequences into functional biomolecules. This innovation expands biotechnology into a temporal dimension, unlocking stress-adaptation traits lost through evolution. Scientifically, it delivers the first experimentally verified dataset of authentic ancient genes with measurable activity in living hosts. Technologically, it establishes a modular pipeline combining metagenomics, synthetic biology, and multi-stressor phenotyping, applicable to any bacterial platform. Practically, it demonstrates dual impact: (i) in agriculture, the engineered rhizobacteria enhance water retention and plant growth on marginal soils; and (ii) in industry, resurrected proteins function as stress-tolerant chaperones and biosurfactants. Preliminary life-cycle analyses suggest up to 20% water savings in bioaugmented crops and 40% lower greenhouse gas emissions from bio-based surfactant production. Remaining steps for full uptake include scaling fermentation processes, completing life-cycle sustainability assessments, and piloting field trials for long-term ecological validation. The project also identifies the need for harmonized regulatory guidance for microorganisms engineered with ancestral sequences, ensuring safe and sustainable market entry.