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Recombinant production of enzymes for the paper industry

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Universitat de Barcelona. Departament de Biologia, Sanitat i Medi Ambient

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[eng] Enzymes have become indispensable tools in modern bioprocessing, offering sustainable and high-specificity alternatives to traditional chemical catalysts. In the pulp and paper industry, cellulases and amylases are increasingly used for reducing energy consumption, water use, and reliance on harsh chemicals. However, industrial implementation is often constrained by the need for catalytic activity under extreme conditions, such as elevated temperatures and alkaline pH. Successful industrial use thus requires both robust enzyme candidates and production systems capable of achieving high titers at low cost. Bacillus subtilis is a preferred microbial host in industrial platforms due to its GRAS (generally regarded as safe) or QPS (qualified presumption of safety) status and secretion capacity, yet its utility is often limited by bottlenecks in heterologous protein expression and secretion pathway, often leading to failed projects in both academic and industrial settings. Within this context, this thesis presents a cohesive set of four publications that address these challenges through enzyme discovery and host strain engineering for developing advanced tools to enhance heterologous protein production in B. subtilis. The first paper reports on the identification and characterization of StachCel5, a novel GH5 endoglucanase mined from the genome of Stachybotrys chartarum, a well-known fungus producing thermostable and alkali-tolerant cellulases. Expressed in Komagataella phaffii, StachCel5 exhibited excellent thermostability at 50 °C, broad pH activity (pH 4 9), and strong specificity for -1,4-glucans. Application in pulp refining demonstrated a 25% reduction in mechanical energy demand while improving fiber bonding and paper strength without compromising fiber integrity. These findings underline its potential for industrial use beyond paper and pulp industry, including in the biofuel, food, and detergent sectors. Nonetheless, for industrial application, enzymes must be produced efficiently and at scale to be economically viable. The second paper of this thesis focuses on developing a modular, high-titer expression platform in B. subtilis. By employing the CRISPR-Cas9 system alongside promoter optimization, gene copy number modulation, and secretion enhancement, a stable and scalable AmyQ -amylase production platform was established. A multi-copy strain was designed with up to six chromosomal amyQ copies under the dual promoter PamyQ-Pcry3A, combined with overexpression of the chaperone PrsA and signal peptidase SppA. The engineered strain reached capacity. To overcome the time-intensive process of iterative genome editing, the third and fourth papers introduce a novel CRISPR-Cas9-based multiplex genome engineering platform for B. subtilis, featuring a colorimetric screening system based on the crtMN operon from Staphylococcus aureus. This operon confers a bright yellow pigmentation on the host, which is visibly lost upon successful gene replacement, enabling rapid, PCR-free selection of successful edited clones. Three open-access, patent-free B. subtilis KO7-S strains were constructed with one to three chromosomal crtMN insertions, along with a CRISPR-Cas9 single-plasmid toolkit (Paper 3). This system enables simultaneous, scarless integration of up to three gene copies in a single step, thus dramatically accelerating strain construction timelines. Building on this chassis, the fourth study demonstrates its use in carotenoid biosynthesis. By co-integrating multiple crtMN copies, enhancing precursor supply through fpps overexpression, and deleting yisP, a stable B. subtilis strain was created that pr -copy plasmid systems, without the drawbacks of plasmid instability or antibiotic selection. Together, these works define a robust pipeline for industrial biocatalyst development: from the discovery of robust enzymes suitable for process-relevant conditions, to the construction of stable and high-expression microbial platforms. This thesis contributes practical tools and strategies towards more sustainable, energy-efficient, and economically viable bioprocesses.

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[spa] Las enzimas se utilizan cada vez más en la industria como alternativas sostenibles a los catalizadores químicos. En el sector papelero, celulasas y amilasas contribuyen a reducir el consumo de energía, agua y productos químicos, aunque su aplicación a escala industrial requiere de biocatalizadores estables en condiciones extremas y sistemas de producción eficientes. Bacillus subtilis se emplea ampliamente en la producción de enzimas gracias a su estatus GRAS y a su elevada capacidad secretora, si bien la expresión y secreción de proteínas heterólogas a menudo requiere optimización. Esta tesis aborda estos retos a través de cuatro estudios complementarios. El primero describe la identificación de StachCel5, una nueva endoglucanasa de Stachybotrys chartarum, estable a 50 °C y activa en un amplio intervalo de pH, capaz de reducir hasta un 25% el consumo energético en el refinado de pasta papelera y con un gran potencial de aplicación en biocombustibles, alimentación y detergentes. El segundo presenta una estrategia racional y secuencial para optimizar la expresión de enzimas en B. subtilis mediante CRISPR-Cas9. Combinando la optimización de promotores, el incremento del número de copias génicas y la reducción de los cuellos de botella en la vía secretora, se desarrolló una cepa recombinante capaz de secretar elevadas cantidades de α-amilasa AmyQ en biorreactor, potencialmente escalable a nivel industrial. Los dos últimos estudios introducen una herramienta de ingeniería genómica basada en CRISPR-Cas9 junto con un sistema de cribado colorimétrico fundamentado en el operón crtMN, que permite la selección rápida de integraciones múltiples. Mediante esta tecnología también se obtuvieron cepas de B. subtilis aplicadas a la biosíntesis de carotenoides C₃₀, alcanzando niveles de producción comparables a los de sistemas plasmídicos, pero sin los inconvenientes asociados a la inestabilidad y a la dependencia de antibióticos. En conjunto, esta tesis aporta nuevas herramientas para el descubrimiento de enzimas robustas y para el desarrollo de plataformas microbianas de alto rendimiento, contribuyendo al avance hacia bioprocesos más sostenibles, eficientes y económicamente viables.

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Tesis Doctorals - Departament - Biologia, Sanitat i Medi Ambient

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Recombinant production of enzymes for the paper industry