Huerta Cepas, J. | bioRxiv Science

bioRxiv2026

Coupled enzyme discovery, evolution and synthetic yeast chassis adaptation for microbial biopolymer valorisation

The valorisation of biological polymers requires microbial systems that can both access recalcitrant substrates and convert the resulting carbon into useful products. Although microbial genome and metagenome resources provide an expanding reservoir of candidate depolymerizing and modifying enzymes, most discovery workflows remain disconnected from enzyme optimisation and host adaptation. Here we present a coupled sequence-based enzyme discovery, enzyme evolution and synthetic yeast chassis adaptation strategy for microbial biopolymer valorisation. Focusing on laccases for the depolymerisation of lignin as a proof of concept, we combine sequence data mining for enzyme discovery, modular yeast surface display for functional screening, directed evolution for enzyme optimisation and synthetic yeast genome diversification for chassis improvement. In our study, surface display enabled functional benchmarking and recovery of improved laccase variants and synthetic-genome-enabled diversification provided a route to explore host configurations that influence display and enzyme performance. By integrating enzyme-level and chassis-level optimisation, this framework addresses a central bottleneck in converting microbial biodiversity by computational sequence repository mining into deployable biomanufacturing systems. Our results establish laccases as tractable entry points for oxidative biopolymer conversion and provide a generalizable platform for engineering yeast systems for sustainable carbon valorisation.

synthetic biology↗

bioRxiv2026

Metabolic commitment and nitrogen control of diazotrophy in the diazoplast-containing diatom Epithemia adnata

Earths nitrogen cycle is central to sustaining ecosystem productivity and global biogeochemical balance. Although biological N2-fixation is well characterized in prokaryotes and plant symbioses, in other eukaryotic lineages it remains poorly understood. Diatoms of the family Rhopalodiacea harbor diazoplasts, endosymbiotic spheroid bodies specialized for N2-fixation. This makes these diatoms genuine N2-fixing eukaryotes that represent a unique model for organelle evolution, parallel but distinct from haptophyte nitroplasts. Here, we report the isolation and stable cultivation of an Epithemia adnata strain, the sequencing of its diazoplast genome and its proteomic profile when growing diazotrophically in the light or darkness, or upon exposure to ammonium. Our analyses reveal that ammonium induced broad down-regulation of diazoplast proteins, particularly those linked to N2-fixation, ATP synthesis, and central carbon metabolism underscoring a general regulatory commitment toward diazotrophic metabolism tightly coupled to host carbon and nitrogen status. The pentose phosphate pathway and ferredoxin-NADP oxidoreductase appear as likely source of reductant to nitrogenase. A striking enrichment of chaperones, peroxiredoxins, bacterioferritin-like proteins, and DpsA might stabilize nitrogenase and buffer against oxidative stress during light-driven diazotrophy. Importantly, we identified a plasmid-encoded GlpF as a putative glycerol transporter, pointing to glycerol-mediated host-symbiont metabolic integration in the extant symbiosis and possibly a crucial innovation during the early evolutionary stages of its establishment. Thus, diazoplast activity is not autonomous but requires integration with host carbon and nitrogen status, establishing glycerol transport, reductant supply, stress mitigation, and nutrient-responsive regulation as pivotal mechanisms of nitrogenase activity and host integration. These findings have broad implications for biogeochemical cycling, organellogenesis, and synthetic biology strategies aimed at engineering N2-fixation in crop plants. SignificanceN2-fixing eukaryotes are increasingly recognized as abundant algae containing bacterial-derived diazotrophic endosymbionts, representing an underappreciated component of global N cycling. Diazoplasts in rhopalodiacean diatoms represent a compelling example of such endosymbionts specialized for N2-fixation. By combining genomic sequencing and proteomic analysis, we demonstrate their metabolic specialization, host integration, and regulatory commitment to diazotrophy. These findings reinforce the emerging view that diazoplasts function as organelle-like entities dedicated to N2-fixation, dependent on host-supplied carbon while contributing fixed N in return. Beyond giving evolutionary insights into organellogenesis, this work establishes a framework for translational applications, such as engineering N2-fixation into agricultural plants. Such advances could reduce reliance on synthetic fertilizers, influence biogeochemical cycles, and promote sustainable food production.

plant biology↗