Engineered Bacteria Platform Unlocks Industrial Production of Plant Protein-Boosting Enzyme

A team of bioengineers in Barcelona has built a standardized genetic toolkit that turns the harmless soil bacterium Bacillus subtilis into a miniature factory for protein-glutamine glutaminase, an enzyme with the potential to transform the texture and functionality of plant-based foods. The work, published in Applied Microbiology and Biotechnology, tackles a problem that has long frustrated food technologists: although the enzyme is highly promising, its natural producer, the bacterium Chryseobacterium proteolyticum, makes it in quantities far too small for industry, and the protein only becomes active after a complicated proteolytic activation step. By combining modular DNA assembly with careful bioreactor engineering, the researchers have demonstrated a scalable route to producing the enzyme outside the cell, where it can be harvested easily and safely.

Protein-glutamine glutaminase, often abbreviated PGG, belongs to a family of enzymes that modify glutamine residues within proteins. When applied to plant proteins such as those from soy, pea or wheat, it can convert glutamine side chains into glutamic acid, changing the charge profile of the protein and, in turn, its solubility, emulsifying capacity and gel-forming behavior. That matters enormously for the fast-growing plant-based food sector, where manufacturers struggle to replicate the mouthfeel and stability that animal proteins deliver naturally. An enzyme that can be sprinkled into a formulation to improve functionality without chemical additives is an attractive proposition, but only if it can be manufactured at scale, consistently and affordably.

The Barcelona group, led by Clàudia Lliso-Pascual, Sergi Abad, Marc Carnicer and Antoni Planas, working between the Institut Químic de Sarrià of Universitat Ramon Llull and the biotechnology company CYGYC BIOCON, chose Bacillus subtilis as their production host for good reason. This Gram-positive bacterium is famous for secreting proteins directly into the culture medium, a trait exploited for decades in the industrial production of enzymes for detergents and food processing. Secretion simplifies purification dramatically: instead of cracking open cells and separating a soup of intracellular contaminants, manufacturers can simply collect the culture supernatant, in which the target protein is already enriched. Yet B. subtilis does not secrete every protein equally well, and finding the right combination of genetic controls is largely a matter of trial and error.

To bring order to that trial and error, the researchers constructed a modular secretion platform based on Golden Gate cloning, a DNA assembly method that allows genetic parts to be swapped in and out of a common backbone with standardized overhangs. In their architecture, a promoter, which controls how strongly the gene is transcribed, and a signal peptide, which directs the growing protein through the secretion machinery, could be combined systematically in a single construct design. This meant that dozens of promoter-signal peptide pairings could be screened under identical conditions, with the gene of interest held constant. Such standardization is the essence of modern synthetic biology: rather than redesigning each construct from scratch, engineers assemble variants like interchangeable modules and let the data decide which configuration wins.

The screening combined a reporter assay with direct measurement of enzyme activity, allowing the team to identify which combinations genuinely delivered functional protein to the medium rather than merely producing transcripts. The clear winner was a configuration pairing the Pgrac100 promoter with the amyQ signal peptide, a signal sequence derived from the alpha-amylase gene that has a long track record in Bacillus secretion systems. In the laboratory strain B. subtilis 168, this construct supported robust secretion of the PGG enzyme from Chryseobacterium proteolyticum, designated CpPGG. Intriguingly, the secreted protein emerged as an inactive proenzyme, and the host bacterium itself carried out the maturation step, processing the proenzyme into its active form through its own extracellular proteases.

That host-mediated activation initially looked like a convenient bonus, but it became a liability at scale. When the researchers moved their best construct into 3-liter batch bioreactors running on rich medium, the culture reached 2.9 units per milliliter of enzyme activity in the supernatant, a respectable starting point for a difficult-to-secrete enzyme. However, when they shifted to fed-batch cultivation in a chemically defined medium, a strategy used industrially to push cell densities and product titers higher, a new problem surfaced. The very proteases that B. subtilis uses to mature the proenzyme began to degrade the accumulated protein, and extracellular proteolysis emerged as a major limitation under high-cell-density conditions. In other words, the same biology that activated the enzyme also destroyed it.

The solution was conceptually elegant: decouple secretion from activation. The team transferred their construct into B. subtilis KO7-S, a protease-deficient strain that has been engineered to lack many of the extracellular degradative enzymes that plague protein production in this organism. The strain was kindly provided by Dr. Jordi Ferrando and Prof. Pere Picart of the University of Barcelona, whose expertise in Bacillus biology and transformation supported the work. In KO7-S, the non-processed proenzyme accumulated stably in the culture supernatant, protected from the proteolytic buzz-saw that had undermined production in strain 168. The trade-off was that the accumulated protein was inactive, but that turned out to be a manageable problem rather than a fatal one.

With the proenzyme safely stockpiled outside the cells, the researchers activated it on their own terms. They treated the supernatant with a controlled in vitro activation step using a food-grade neutral protease, an enzyme acceptable for use in food processing, to cleave the pro-peptide and unleash the catalytic activity. This controlled approach yielded 12.8 plus or minus 0.8 units per milliliter of supernatant, a more than fourfold improvement over the batch bioreactor result in the protease-rich host. Just as importantly, the decoupling strategy gives manufacturers precise control over when and how the enzyme becomes active, which simplifies quality control and avoids the unpredictable mixture of intact and degraded protein that plagued the earlier system.

The broader significance of the study lies less in any single number than in the engineering logic it demonstrates. Efficient PGG production, the authors conclude, requires coordinated control of three distinct processes: secretion, extracellular stability and proenzyme activation. Each of these can be optimized independently, and the modular platform makes that optimization systematic rather than ad hoc. The same Golden Gate architecture could, in principle, be applied to other industrially interesting enzymes that suffer from poor secretion or proteolytic degradation in Bacillus hosts, turning the platform into a general-purpose chassis for extracellular protein production. The work also highlights the value of food-grade solutions throughout the process, from the neutral protease used for activation to the generally recognized safety profile of B. subtilis itself.

For the plant-based food industry, the prospect of a reliable, scalable supply of protein-glutamine glutaminase could accelerate the development of next-generation meat and dairy alternatives with improved texture, solubility and nutritional performance. For bioprocess engineers, the study offers a textbook example of how modular cloning, host strain selection and bioreactor strategy must advance together: a brilliant genetic construct is worthless if the fermentation destroys its product, and a high-titer fermentation is worthless if the protein it yields is inactive. The Barcelona team’s platform, funded in part by an industrial doctorate grant from the Catalan government’s AGAUR agency, bridges those worlds, and its open-access publication means that other laboratories can immediately adopt and extend the modular design. As demand for functional plant proteins continues to climb, engineered microbial factories like this one are likely to play an increasingly central role in feeding the world’s appetite for sustainable protein.

Subject of Research: Modular secretion engineering in Bacillus subtilis for scalable production of protein-glutamine glutaminase

Article Title: A modular secretion platform in Bacillus subtilis enables scalable production of protein-glutamine glutaminase

Article References: Lliso-Pascual, C., Abad, S., Carnicer, M., & Planas, A. (2026). A modular secretion platform in Bacillus subtilis enables scalable production of protein-glutamine glutaminase. Applied Microbiology and Biotechnology. https://doi.org/10.1007/s00253-026-14008-4

Image Credits: AI Generated

DOI: 10.1007/s00253-026-14008-4

Keywords: Bacillus subtilis, protein-glutamine glutaminase, protein secretion, Golden Gate cloning, modular cloning, proenzyme activation, bioreactor cultivation, plant proteins, food-grade enzymes, protease-deficient strains, synthetic biology, industrial biotechnology