Abstract. Some biological particles and macromolecules are particularly efficient ice nuclei (IN), triggering ice formation at temperatures close to 0 ∘C. The impact of biological particles on cloud glaciation and the formation of precipitation is still poorly understood and constitutes a large gap in the scientific understanding of the interactions and coevolution of life and climate. Ice nucleation activity in fungi was first discovered in the cosmopolitan genus Fusarium, which is widespread in soil and plants, has been found in atmospheric aerosol and cloud water samples, and can be regarded as the best studied ice-nucleation-active (IN-active) fungus. The frequency and distribution of ice nucleation activity within Fusarium, however, remains elusive. Here, we tested more than 100 strains from 65 different Fusarium species for ice nucleation activity. In total, ∼11 % of all tested species included IN-active strains, and ∼16 % of all tested strains showed ice nucleation activity above −12 ∘C. Besides Fusarium species with known ice nucleation activity, F. armeniacum, F. begoniae, F. concentricum, and F. langsethiae were newly identified as IN-active. The cumulative number of IN per gram of mycelium for all tested Fusarium species was comparable to other biological IN like Sarocladium implicatum, Mortierella alpina, and Snomax®. Filtration experiments indicate that cell-free ice-nucleating macromolecules (INMs) from Fusarium are smaller than 100 kDa and that molecular aggregates can be formed in solution. Long-term storage and freeze–thaw cycle experiments revealed that the fungal IN in aqueous solution remain active over several months and in the course of repeated freezing and thawing. Exposure to ozone and nitrogen dioxide at atmospherically relevant concentration levels also did not affect the ice nucleation activity. Heat treatments at 40 to 98 ∘C, however, strongly reduced the observed IN concentrations, confirming earlier hypotheses that the INM in Fusarium largely consists of a proteinaceous compound. The frequency and the wide distribution of ice nucleation activity within the genus Fusarium, combined with the stability of the IN under atmospherically relevant conditions, suggest a larger implication of fungal IN on Earth’s water cycle and climate than previously assumed.

Background Non-ribosomal peptide synthetase-like (NRPS-like) enzymes are highly enriched in fungal genomes and can be discriminated into reducing and non-reducing enzymes. Non-reducing NRPS-like enzymes possess a C-terminal thioesterase domain that catalyses the condensation of two identical aromatic α-keto acids under the formation of enzyme-specific substrate-interconnecting core structures such as terphenylquinones, furanones, butyrolactones or dioxolanones. Ascocoryne sarcoides produces large quantities of ascocorynin, which structurally resembles a terphenylquinone produced from the condensation of p-hydroxyphenylpyruvate and phenylpyruvate. Since the parallel use of two different substrates by a non-reducing NRPS-like enzyme appeared as highly unusual, we investigated the biosynthesis of ascocorynin in A. sarcoides. Results Here, we searched the genome of A. sarcoides for genes coding for non-reducing NRPS-like enzymes. A single candidate gene was identified that was termed acyN. Heterologous gene expression confirmed that AcyN is involved in ascocorynin production but only produces the non-hydroxylated precursor polyporic acid. Although acyN is embedded in an ascocorynin biosynthesis gene cluster, a gene encoding a monooxygenase required for the hydroxylation of polyporic acid was not present. Expression analyses of all monooxygenase-encoding genes from A. sarcoides identified a single candidate that showed the same expression pattern as acyN. Accordingly, heterologous co-expression of acyN and the monooxygenase gene resulted in the production of ascocorynin. Structural modelling of the monooxygenase suggests that the hydrophobic substrate polyporic acid enters the monooxygenase from a membrane facing entry site and is converted into the more hydrophilic product ascocorynin, which prevents its re-entry for a second round of hydroxylation. Conclusion This study characterises the first naturally occurring polyporic acid synthetase from an ascomycete. It confirms the high substrate and product specificity of this non-reducing NRPS-like enzyme and highlights the requirement of a monooxygenase to produce the terphenylquinone ascocorynin.

Some biological particles and macromolecules are particularly efficient ice nuclei (IN), triggering ice formation at temperatures close to 0 • C. The impact of biological particles on cloud glaciation and the formation of precipitation is still poorly understood and constitutes a large gap in the scientific understanding of the interactions and co-evolution of life and climate. To investigate the frequency and distribution of IN activity within the fungal genus Fusarium, more than 100 strains from 65 different Fusarium species were screened. In total, ∼11 % of all tested species included ice nucleation-active (IN-5 active) strains, and ∼16 % of all tested strains showed IN activity above -14 • C. Besides Fusarium species with known IN activity, F. armeniacum, F. begoniae, F. concentricum, and F. langsethiae were newly identified as IN-active. The cumulative number of IN per gram of mycelium for all tested Fusarium species was comparable to other biological IN like Sarocladium implicatum, Mortierella alpina, and Snomax ® . Filtration experiments suggest that the single cell-free Fusarium IN is smaller than 100 kDa, and that aggregates can be formed in solution. Long-term storage and freeze-thaw cycle experiments revealed 10 that the Fusarium IN remain active in solution for several months and after repeated freezing and thawing. Oxidation and nitration reactions, as occurring during atmospheric aging, did not affect the activity of the Fusarium IN. The high frequency of Fusarium and the wide distribution of IN activity within the genus, combined with the high stability of the IN, suggest a significant impact of fungal IN on the Earth's water cycle and climate.

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