Biology subjects

Garcia-Ruiz, E.

Publications and source records attributed to Garcia-Ruiz, E..

3 recordsLinked to original sources

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↗

bioRxiv2022

Design, Construction, and Functional Characterization of a tRNA Neochromosome in Yeast

Here we report the design, construction and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the [~]190 kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporated orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enable an orthogonal SCRaMbLE system capable of adjusting tRNA abundance. Following construction, we obtained evidence of a potent selective force once the neochromosome was introduced into yeast cells, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up new opportunities to directly test hypotheses surrounding these essential non-coding RNAs. HighlightsO_LIDe novo design, construction and functional characterization of a neochromosome containing all 275 nuclear tRNA genes of Saccharomyces cerevisiae. C_LIO_LIIncreasing the copy number of the 275 highly expressed tRNA genes causes cellular burden, which the host cell likely buffers either by selecting for partial tRNA neochromosome deletions or by increasing its ploidy. C_LIO_LIThe tRNA neochromosome can be chemically extracted and transformed into new strain backgrounds, enabling its transplantation into multi-synthetic chromosome strains to finalize the Sc2.0 strain. C_LIO_LIComprehensive functional characterization does not pinpoint a singular cause for the cellular burden caused by the tRNA neochromosome, but does reveal novel insights into its tRNA and structural chromosome biology. C_LI

synthetic biology↗

bioRxiv2021

Stable and functionally diverse versatile peroxidases by computational design directly from sequence

White-rot fungi secrete a repertoire of high-redox potential oxidoreductases to efficiently decompose lignin. Of these enzymes, versatile peroxidases (VPs) are the most promiscuous biocatalysts. VPs are attractive enzymes for research and industrial use, but their recombinant production is extremely challenging. To date, only a single VP has been structurally characterized and optimized for recombinant functional expression, stability and activity. Computational enzyme optimization methods can be applied to many enzymes in parallel, but they require accurate structures. Here, we demonstrate that model structures computed by deep-learning based ab initio structure prediction methods are reliable starting points for one-shot PROSS stability-design calculations. Four designed VPs encoding as many as 43 mutations relative to the wild type enzymes are functionally expressed in yeast whereas their wild type parents are not. Three of these designs exhibit substantial and useful diversity in reactivity profile and tolerance to environmental conditions. The reliability of the new generation of structure predictors and design methods increases the scale and scope of computational enzyme optimization, enabling efficient discovery and exploitation of the functional diversity in natural enzyme families.

biochemistry↗