Controlling cellular differentiation of industrial enzyme production in Bacillus subtilis

Project Information

BIOSTAB-DIFF

Grant agreement ID: 101106245

DOI

10.3030/101106245

Project closed

EC signature date 17 April 2023

Start date 1 September 2023

End date 31 August 2025

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 203 464,32

Coordinated by

UNIVERSITEIT VAN AMSTERDAM
Netherlands

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

Reporting period: 2023-09-01 to 2025-08-31

Summary of the context and overall objectives of the project

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.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

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.