Jean-Marc Daran

Flocculation is a key technological trait in lager brewing, governing fermentation performance, yeast recovery, and beer quality. In the allo-aneuploid hybrid yeast Saccharomyces pastorianus , the genetic basis of flocculation remains poorly resolved due to its complex dual sub-genome architecture. Here, we systematically re-annotated and functionally characterized the complete FLO gene repertoire of the Group II strain CBS 1483. Thirteen FLO genes were identified, including allelic variants and a previously uncharacterized adhesin, Flo12, containing a Hyphal_reg_CWP domain instead of the canonical PA14 lectin-binding domain. Structural modeling revealed strong conservation of Ca² ⁺ -binding residues in PA14 domains, alongside repeat-region diversification likely contributing to functional variability. Using optogenetic expression in a FLO -null background, we demonstrated that Spc I- FLO9 -1 and Spc I- FLO9-2 _1 are the strongest drivers of flocculation, exhibiting NewFlo-like sugar sensitivity. Transcriptomic analysis during 17°P wort fermentation showed dynamic induction of these genes coinciding with flocculation onset. Surprisingly, deletion of both loci in CBS 1483 did not abolish but only delayed sedimentation in wort, accompanied by improved maltose utilization and attenuation. These findings reveal functional redundancy and compensatory mechanisms within the FLO network of lager yeast, highlighting the genetic complexity underlying flocculation, and providing a molecular framework to inform yeast selection, strain development, and optimization of the lager fermentation processes. IMPORTANCE: Flocculation, the process by which yeast cells aggregate and settle, is essential for producing clear, high-quality lager beer, and for efficient yeast recovery during brewing. However, the genetic basis of this trait in lager yeast has remained poorly understood because these strains possess unusually complex hybrid genomes. In this study, we systematically identified and characterized the complete set of flocculation genes in the industrial lager yeast Saccharomyces pastorianus CBS 1483. We demonstrated that lager yeast flocculation is not controlled by a single dominant gene, but instead emerges from the combined action of several moderately active adhesion proteins that are expressed at low levels during fermentation. Surprisingly, deleting the two strongest candidate genes only delayed, rather than eliminated, sedimentation, revealing a robust compensatory network that preserves brewing performance. These findings refine the current understanding of yeast flocculation and provide a molecular framework for developing brewing strains with improved fermentation efficiency, product consistency, and flavor quality.

Microbial electrosynthesis (MES) is a promising technology for the valorization of CO ₂ into industrially relevant building blocks. The high-performing MES systems in terms of production rates of acetate (12.5–19.7 mmol L ⁻¹ _catholyte day ⁻¹ ), butyrate (1.9–12.2 mmol L ⁻¹ _catholyte day ⁻¹ ), and caproate (0.6–0.9 mmol L ⁻¹ _catholyte day ⁻¹ ) discussed in this study consist of mixed microbial communities. However, the microbial community members, metabolic pathways, and interactions driving product formation in MES communities remain poorly understood. To overcome these challenges, we conducted a comprehensive characterization of three high-performing MES communities, combining multi-omics with metagenome-scale metabolic modeling. Using a high-resolution metagenomic pipeline, we reconstructed high-quality genomes of 25 metagenome-assembled genomes present in our reactors, including six fully circular genomes. We report the presence of Clostridium aromativorans for the first time in a gas-fermenting system. In particular, our findings identified three acetogenic species, Eubacterium limosum , Sporomusa sphaeroides , and C. aromativorans , as key contributors to the production of acetate, butyrate, and caproate via the Wood-Ljungdahl and the reverse β-oxidation pathways. In addition, we found genes related to lactate and ethanol production from acetyl-CoA, along with proteomic evidence of lactate production. This paves the way for investigating the role of cross-fed metabolites such as lactate and ethanol as electron donors in chain elongation. Finally, meta-genome-scale metabolic modeling suggests that the communities might be sustained by the cross-feeding of specific cofactors such as pyridoxine, pantothenate, biotin, and thiamin. This study provides key insights into the structure and function of electrosynthetic communities, bringing us closer to the rational engineering of MES systems. IMPORTANCE: Microbial electrosynthesis (MES) offers a promising route to transform CO ₂ and renewable electricity into valuable platform chemicals. However, the microbial ecology governing the assembly and function of these systems remains poorly understood, limiting our ability to engineer them. By integrating high-resolution multi-omics with metabolic modeling, this study provides a systems-level framework to dissect the structure and function of electrosynthetic microbial communities. Understanding which organisms drive carbon fixation and chain elongation, how electrons and intermediates are transferred within the biofilm, and how metabolic dependencies structure these communities is essential for improving productivity and product specificity in MES. More broadly, this work highlights how integrated multi-omics approaches can resolve the functional organization of complex microbial ecosystems and provides methodological advances that are broadly applicable to the study and engineering of microbial communities in autotrophic, electricity-driven biotechnologies.

The high cost of cultivation media remains one of the barriers to the commercialization of cultivated meat. Current serum‐free media reduce reliance on fetal bovine serum but only achieve modest cost savings. Especially at larger scales (> 20 m ³ ), pharmaceutical‐grade amino acids and recombinant proteins remain major cost drivers. Techno‐economic analyses suggest that replacing purified amino acids is essential to achieve price parity with conventional meat. Microbial hydrolysates, also referred to as extracts, offer a promising alternative, as these can supply complex mixtures of amino acids and peptides at lower cost and with greater scalability than chemically defined formulations. In particular, yeast and bacterial extracts combine high protein (and therefore high amino acid) content, rapid growth, and established industrial‐scale production. Comparative analyses indicate that microbial amino acid profiles broadly overlap with those of animal cells and traditional meat, but this similarity does not translate into meeting cellular amino acid demand. Consumption data indicate that key amino acids, such as glutamine, cysteine, serine and arginine, would be supplied insufficiently by microbial extracts. This mismatch highlights the need for engineering of microbial biomass and optimization of extraction methods. Extraction methods such as autolysis, enzymatic lysis, or physical disruption strongly influence nutrient release and composition, underscoring the need for standardizing microbial extract preparation. In addition, challenges remain in ensuring consistency, safety, and bioavailability of nutrients, as microbial extracts also contain nucleic acids and potential toxins. This review summarizes current knowledge on microbial hydrolysates for cultivated meat media, including biomass composition, extraction methods, and analytical tools to assess quality and performance. We identify key knowledge gaps, particularly in quantitative amino acid consumption data for relevant cell lines and performance testing in scalable cultures. Addressing these gaps could enable cost‐effective media development and accelerate research toward sustainable, commercially viable cultivated meat.

Isoprene, a highly volatile hydrocarbon with numerous industrial applications, has traditionally been produced from petrochemical sources through processes associated with significant environmental impacts. Microbial production of isoprene has emerged as a promising and more sustainable alternative. In this study, the potential of the non-conventional yeast Kluyveromyces marxianus to produce isoprene from a renewable feedstock was explored, contributing to the development of a more sustainable process. K. marxianus was engineered to produce isoprene from glucose through the expression of an isoprene synthase gene, and the gene copy number of this enzyme was found to significantly influence isoprene production. Furthermore, enhancing the supply of the isoprene precursors acetyl-CoA and dimethylallyl diphosphate (DMAPP) via engineering of the mevalonate pathway led to increased production. A higher headspace-to-culture ratio in sealed serum bottles also facilitated isoprene accumulation. Importantly, isoprene production was achieved from the cellulosic fraction of pretreated Eucalyptus globulus wood. To our knowledge, this is the first report of isoprene production in K. marxianus using a lignocellulosic feedstock, providing proof of concept for its potential in integrated processes based on sustainable substrates under stressful conditions.

Lager beer, made by fermentation of barley malt using the hybrid yeast Saccharomyces pastorianus, is extremely popular worldwide. The low genetic variation of S. pastorianus strains limits the scope of development for novel lager beer types. The wild ancestor Saccharomyces eubayanus has potential for use in lager brewing, but its application is constrained by formation of phenolic off-flavours (POF+ phenotype) and limited uptake of the wort α-oligoglucosides maltose and maltotriose. Here, we addressed both limitations, using screening and evolutionary engineering of the Himalayan S. eubayanus strain ABFM5L.1, one of the closest known relatives of S. eubayanus subgenome of modern hybrid lager yeasts. Prior to improving α-oligoglucoside utilisation, 4-vinylguaiacol formation was abolished by mutagenesis and screening for POF⁻ mutants. Subsequently, evolution for improved sugar utilisation and reverse engineering identified SeMALR1 (the S. eubayanus ortholog of S. cerevisiae MAL63) as a key regulatory control point. Single-nucleotide substitutions in this maltose regulon activator gene enabled rapid maltose uptake and partial maltotriose utilisation in a previously maltose-negative background. The evolved non-genetically modified POF⁻ strains showed near-complete maltose consumption (>90%–95%) and partial maltotriose consumption (∼25%–32%) in industrial wort. These results reveal substantial regulatory flexibility in Holarctic S. eubayanus and support its further development as a novel lager brewing yeast.

Biofilm-based microbial electrosynthesis (MES) is a promising technology that converts CO2 into industrially relevant organic compounds using renewable energy sources. The highest performing MES systems reported to date consist of biofilm-driven microbial communities. However, for a successful deployment of this technology, we still need to overcome key challenges, including the long colonization time of the biocathode and the difficulty of controlling the product profile. To address these challenges, it is crucial to better understand the key microbial components responsible for the desired metabolic products, and how they assemble and function as a community. In this study, we conducted an in-depth characterisation of three high-performing mixed MES communities using metagenomics, metaproteomics and advanced analysis of metagenome-derived metabolic networks. Our findings identified Eubacterium limosum, Sporomusa sphaeroides and Clostridium aromativorans as key contributors to the production of acetate, butyrate and caproate via the Wood-Ljungdahl and the reverse β-oxidation pathways. A higher production of butyrate and caproate was observed in reactors with higher abundance of C. aromativorans, an organism only recently discovered and never reported in gas-fermenting systems before. The recovered genome of C. aromativorans, reconstructed in a single circular fragment, provides a more comprehensive genomic representation than the current reference. In addition, we found genes related to lactate and ethanol production from acetyl-CoA in the metagenomes, including proteomic evidence for lactate production. This study provides key insights into the microbial players, metabolic processes and microbial community features driving product formation in biofilm-based MES, bringing this technology closer to industrial application.

Hanseniaspora species gained attention due to the ability of these species to ferment simple sugars and to actively contribute to the development of bouquet aromas in wine and cider fermentations. We present a chromosome-level assembly of an isolate of Hanseniaspora mollemarum that would enhance its potential applications.

Flocculation in Saccharomyces cerevisiae is a critical phenotype with ecological and industrial significance. This study aimed to functionally dissect the contributions of individual FLO genes (FLO1, FLO5, FLO9, FLO10, FLO11) to flocculation by employing an optogenetic circuit (OptoQ-AMP5) for precise, light-inducible control of gene expression. A FLO-null platform yeast strain was engineered allowing the expression of individual FLO genes without native background interference. Each FLO gene was reintroduced into the FLO-null background under the control of OptoQ-AMP5. Upon light induction, strains expressing FLO1, FLO5, or FLO10 demonstrated strong flocculation, with FLO1 and FLO5 forming large and structurally distinct aggregates. FLO9 induced a weaker phenotype. Sugar inhibition assays revealed distinct sensitivities among flocculins, notably FLO9’s novel sensitivity to fructose and maltotriose. Additionally, FLO-induced changes in cell surface hydrophobicity were quantified, revealing that FLO10 and FLO1 conferred the greatest hydrophobicity, correlating with their aggregation strength. This work establishes a robust platform for investigating flocculation mechanisms in yeast with temporal precision. It highlights the phenotypic diversity encoded within the FLO gene family and their differential responses to environmental cues. The optogenetic system provides a valuable tool for both fundamental studies and the rational engineering of yeast strains for industrial fermentation processes requiring controlled flocculation.

Reductive amination is one of the most synthetically direct routes to access chiral amines. Several Imine Reductases (IREDs) have been discovered to catalyze reductive amination (Reductive Aminases or RedAms), yet they are dependent on the expensive phosphorylated nicotinamide adenine dinucleotide cofactor NADPH and usually more active at basic pH. Here, we describe the discovery and synthetic potential of an IRED from Rhodococcus erythropolis (RytRedAm) that catalyzes reductive amination between a series of medium to large carbonyl and amine compounds with conversions of up to >99% and 99% enantiomeric excess at neutral pH. RytRedAm catalyzes the formation of a substituted γ-lactam and N-methyl-1-phenylethanamine with stereochemistry opposite to that of fungal RedAms, giving the (S)-enantiomer. This enzyme remarkably uses both NADPH and NADH cofactors with K _M values of 15 and 247 μM and turnover numbers k _cat of 3.6 and 9.0 s^-1, respectively, for the reductive amination of hexanal with allylamine. The crystal structure obtained provides insights into the flexibility to also accept NADH, with residues R35 and I69 diverging from that of other IREDs/RedAms in the otherwise conserved Rossmann fold. RytRedAm thus represents a subfamily of enzymes that enable synthetic applications using NADH-dependent reductive amination to access complementary chiral amine products.