# Toward food-grade production of the Bacteroides helcogenes protein-glutamine glutaminase with an optimized Bacillus subtilis strain

## TL;DR

Researchers optimized a Bacillus subtilis strain to produce a food-grade enzyme from Bacteroides helcogenes, achieving high yields without antibiotics.

## Contribution

A novel antibiotic-free, high-yield production method for a food-grade enzyme using CRISPR/Cas9 in Bacillus subtilis.

## Key findings

- Site-specific integration of the PGB gene into B. subtilis 007 increased enzyme activity to 9.5 µkat/Lculture.
- Deleting the sfp locus reduced foaming and improved production efficiency.
- Multiple genomic integrations enabled efficient PGB secretion without antibiotics.

## Abstract

Protein-glutamine glutaminases (PGs; EC 3.5.1.44) have gained attention in the food industry due to their application in plant protein products. The recently discovered PG from Bacteroides helcogenes (PGB) has especially been shown to provide promising characteristics for improving the techno-functional properties of plant proteins. A prerequisite for food enzymes, such as the PG, is their production with an expression host that meets food safety and yield requirements. The antibiotic-free and secretory production of the PGB was targeted in this study using the undomesticated Bacillus subtilis 007. The CRISPR/Cas9-mediated approach enabled specific genomic PGB integrations, while simultaneously deleting unwanted B. subtilis traits. Firstly, the PGB expression cassette was integrated into the sigF gene, leading to an asporogenic strain and extracellular activity of 4.1 µkat/Lculture in bioreactor cultivations. However, excessive foaming hampered the production process tremendously. Consequently, a second PGB copy was integrated into the sfp locus, which is involved in the production of lipopeptides, such as surfactin. As a result, the PGB activity was increased to 5.4 µkat/Lculture, and foaming during cultivation was reduced significantly. The introduction of a third PGB copy for preventing cell motility did not increase production; however, the integration into the well-established amyE locus improved the PGB yield during reactor cultivations. A final extracellular activity of 9.5 µkat/Lculture was reached. The multiple genomic integrations of the PGB gene enabled the efficient PGB secretion in an optimized B. subtilis host without the need for antibiotics.

• Site-specific PGB integration enabled by genome sequencing of B. subtilis 007.

• Antibiotic-free and secretory PGB production with an optimized B. subtilis host.

• Increased PGB production reaching 9.5 µkat/Lculture.

The online version contains supplementary material available at 10.1007/s00253-025-13681-1.