From secretion to synthetic microbial consortia: A modular approach to biomass deconstruction and sensing

Gupta, Mani (2025) From secretion to synthetic microbial consortia: A modular approach to biomass deconstruction and sensing. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Mani Gupta (20RS082)) 20RS082.pdf - Submitted Version Restricted to Repository staff only Download (15MB)

Abstract

The escalating global demand for energy and materials, coupled with the environmental imperative to decarbonize industrial processes, necessitates a fundamental shift from fossil-based feedstocks to renewable alternatives. Lignocellulosic biomass, an abundant and non-food resource, holds immense promise as a sustainable carbon source for the production of fuels and high-value chemicals. However, realizing this potential presents significant engineering challenges. Conventional biomanufacturing approaches, typically relying on single microbial strains, often suffer from metabolic burden, pathway interference, and high operational costs associated with biomass deconstruction and product purification. Furthermore, a lack of dynamic control and inter-strain coordination within complex biosynthetic pathways limits overall process efficiency and scalability. This thesis addresses these critical limitations by developing a modular and integrated biomanufacturing platform that employs rationally engineered microbial consortia, coupled with advanced sensing and separation technologies. The research provides a comprehensive blueprint for designing distributed biological systems capable of converting recalcitrant biomass into value-added products like tyrosine, with enhanced efficiency and sustainability. A primary focus of this work centered on overcoming the economic and environmental challenges of enzyme production and utilization. Chapter 2 details the engineering of an Escherichia coli strain for the extracellular secretion of a thermostable cellulase cocktail. We achieved high protein yields (up to 0.7 g/L) by leveraging the AnsB secretion tag, rigorously confirmed through immunoblotting and enzymatic assays. This innovative secretion strategy eliminates the need for costly and energy-intensive cell lysis and enzyme purification steps. A pivotal finding in this chapter is the demonstration that this engineered cellulase cocktail retains nearly 100% of its hydrolytic activity in unprocessed seawater. This groundbreaking observation offers a pathway to drastically reduce the freshwater footprint of industrial biomass saccharification, expanding the geographical viability and environmental sustainability of biorefineries to coastal regions. Building upon the success of extracellular enzyme production, Chapter 3 addresses the crucial challenge of enzyme reusability. It introduces a novel platform for magnetic nanoparticle-based enzyme immobilization and purification. We synthesized NTA-Ni@Fe₃O₄ magnetic nanoparticles (MNPs) capable of highly efficient, one-step purification of His-tagged enzymes directly from complex cell lysates and culture supernatants. Beyond their purification capabilities, these MNPs serve as a robust carrier for enzyme immobilization, enabling the magnetic recovery and subsequent reuse of the cellulase enzymes over multiple catalytic cycles. Experimental data confirmed that the MNP-immobilized enzymes maintained significant catalytic activity, validating their utility as a reusable, heterogeneous biocatalyst system. This innovation directly contributes to lowering operational costs and reducing waste in enzymatic bioprocesses, fostering a more circular economy in biomanufacturing. To enable dynamic monitoring and precise control within complex bioprocesses, Chapter 4 develops a sophisticated glucose-responsive whole-cell biosensor utilizing CRISPR interference (CRISPRi). This chapter addresses the inherent limitation of native glucose regulation, where high glucose concentrations typically repress gene expression. By employing a CRISPRi-based "logic inversion" circuit, we engineered an E. coli strain to produce a tunable, fluorescent output that directly correlates with glucose levels. The biosensor demonstrated high specificity for glucose and a robust linear response across a wide range of physiologically relevant concentrations. Its practical utility was rigorously validated by integrating it with a secreted β-glucosidase expression circuit, allowing for real-time, in situ monitoring of glucose production from cellobiose hydrolysis. This biosensor provides a critical feedback mechanism essential for optimizing metabolic fluxes and ensuring process stability in dynamic environments. Finally, Chapter 5 culminates the thesis by addressing the complex challenge of orchestrating multi-strain microbial consortia. Initial co-cultivation experiments of a cellulase-secreting strain and a tyrosine-producing strain revealed a significant lack of coordination, resulting in no detectable product. This underscored the critical need for a communication mechanism to ensure synchronous metabolic activity. To overcome this, we designed and systematically characterized a synthetic quorum sensing (QS) system based on the Vibrio fischeri LuxI/LuxR architecture. A comprehensive library of 81 sender-receiver promoter combinations was constructed and meticulously analysed to map inter-strain communication dynamics and identify optimal expression contexts. This extensive quantitative dataset informed the development of the "Promoter Recommender Tool for Lux QS Co-Culture." This computational tool provides a data-driven framework for the rational design and optimization of QS-regulated consortia, enabling precise temporal control over metabolic pathways and enhancing overall consortium performance.

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