Controlling cellular differentiation of industrial enzyme production in Bacillus subtilis

Projektinformationen

BIOSTAB-DIFF

ID Finanzhilfevereinbarung: 101106245

DOI

10.3030/101106245

Projekt abgeschlossen

EK-Unterschriftsdatum 17 April 2023

Startdatum 1 September 2023

Enddatum 31 August 2025

Finanziert unter

• Marie Skłodowska-Curie Actions (MSCA)

Gesamtkosten

Keine Daten

EU-Beitrag

€ 203 464,32

Koordiniert durch

UNIVERSITEIT VAN AMSTERDAM
Netherlands

Periodic Reporting for period 1 - BIOSTAB-DIFF (Controlling cellular differentiation of industrial enzyme production in Bacillus subtilis)

Berichtszeitraum: 2023-09-01 bis 2025-08-31

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

The project BIOSTAB-DIFF (“Controlling cellular differentiation of industrial enzyme production in Bacillus subtilis”) aimed to improve microbial enzyme productivity and stability by addressing bistable sporulation, a process that generates heterogeneous populations and reduces yield consistency in industrial fermentations. The central hypothesis was that blocking sporulation at defined checkpoints, together with nutrient optimization, would stabilize enzyme secretion and enhance productivity.

The overall objective was to investigate and control cellular heterogeneity in B. subtilis for biotechnological applications. Specific goals were: (1) to construct and characterize sporulation-deficient strains (ΔspoIIE and ΔsigF), (2) assess the impact of these genetic modifications on growth and fitness, (3) evaluate enzyme production under controlled fermentation conditions, (4) optimize nutrient formulations for maximizing yield and secretion stability, and (5) disseminate findings through publications, presentations, and training.

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

Two sporulation-deficient B. subtilis strains (ΔspoIIE and ΔsigF) were successfully engineered using molecular cloning, PCR, and sequencing. These strains were transformed with an amylase-expressing plasmid and tested under 27 different culture conditions varying in glucose, raffinose, and yeast extract composition. Growth kinetics and enzyme secretion were monitored through optical density, amylase activity, and nutrient utilization assays.

Key findings include:

ΔspoIIE supported higher and more stable enzyme production than ΔsigF, validating early sporulation blockade as an effective strategy.

Optimal nutrient conditions (1% glucose + 2% yeast extract) balanced growth with secretion efficiency.

Raffinose acted as a secretion stabilizer rather than a primary carbon source, contributing to consistent enzyme titers.

Combined genetic and nutrient strategies provided a clear proof-of-concept for controlling bistability to enhance industrial enzyme production.

Deliverables achieved included construction and validation of mutant strains, comprehensive growth and secretion profiling, and identification of high-yield conditions. A manuscript is in preparation for submission to Microbial Cell Factories. Dissemination included a presentation at the Swammerdam Institute for Life Sciences seminar, and preliminary findings were integrated into other research lines within the host group.

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

This project provides the first systematic demonstration that disrupting early sporulation checkpoints in B. subtilis can be used to stabilize and enhance enzyme production. Unlike existing approaches focusing mainly on plasmid stability or promoter engineering, this strategy establishes sporulation control as a novel route to improve microbial performance.

The discovery that raffinose functions as a secretion stabilizer is also innovative, offering new opportunities for fermentation design. Together, these results advance microbial biotechnology, synthetic biology, and industrial fermentation by showing how cellular differentiation can be managed to achieve stable high-yield enzyme production.

Applications include:

Bioprocess optimization: reduced sporulation-related variability, lower costs, and improved efficiency.

Food and pharmaceutical industries: more reliable enzyme supply for processing applications.

Sustainable biomanufacturing: alignment with the European Green Deal through resource efficiency and reduced environmental footprint.

The work also strengthened the researcher’s expertise in microbial genetics, fermentation, and dissemination, supporting career development toward independent leadership in enzyme biotechnology.