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

Nannochloropsis oceanica occupies a special place among photosynthetic chassis organisms. It is a haploid alga whose nuclear genome has been fully sequenced and extensively annotated, and it naturally produces high levels of triacylglycerol rich in eicosapentaenoic acid, a high-value omega-3 long-chain polyunsaturated fatty acid increasingly sought after as a food resource for human consumption. Genetic tools for gene overexpression, suppression, subcellular localization and genome editing have been developed for the species over the past decade, and transgene expression has proven stable. Yet most published engineering efforts have introduced no more than three transgenes at a time, and the plasmid construction behind them remained laborious. That bottleneck is precisely what a MoClo system is designed to eliminate.

MoClo is built on the Golden Gate cloning technique, which exploits Type IIS restriction enzymes that recognize asymmetric DNA sequences and cut outside their recognition sites, generating defined overhangs. Because these overhangs can be assigned to specific positions in a genetic construct, multiple DNA fragments can be ligated together seamlessly in a single reaction tube, with no scar sequences left behind. The approach, first formalized in 2011, has since spawned tailored toolkits for bacteria such as Escherichia coli, the yeast Saccharomyces cerevisiae, the land plant Nicotiana benthamiana, and microalgae including Synechocystis, Synechococcus elongatus and Chlamydomonas reinhardtii. The new work extends this lineage to the stramenopile algae, adopting the syntax proposed by the plant synthetic biology community so that parts can, in principle, be exchanged across platforms.

The architecture of the Nannochloropsis toolkit follows the familiar three-level hierarchy of MoClo systems. Level 0 vectors carry individual genetic parts flanked by eight distinct fusion sites; these parts can be shuttled into level 1 vectors to build single transcriptional modules, and those modules can then be stacked into level 2 vectors carrying multiple expression cassettes. The 91 parts represent 39 unique genetic elements distributed across the standard framework and fall into clear functional categories: 12 promoters placed at three different positions, five terminators plus one polygenic terminator, five selectable markers, six signal peptides with two position options each, four reporter genes with five position options, five affinity tags, and one 2A peptide for expressing fused proteins from a single open reading frame.

Functional validation began with the selectable markers, which determine how efficiently transformed cells can be recovered. The team assembled four vectors pairing either the zeocin-resistance gene Ble from Streptoalloteichus hindustanus or the hygromycin B-resistance gene Hyg from Streptomyces hygroscopicus with two different promoter-terminator combinations: the RIBI promoter paired with the HS terminator, both of Nannochloropsis origin, and the VCP2 promoter paired with the FCPA terminator from the diatom Phaeodactylum tricornutum. Counting antibiotic-resistant colonies after electroporation revealed that the RIBI-HS combination outperformed VCP2-FCPA regardless of which marker was used, and reverse transcription quantitative PCR confirmed that the winning pair also drove higher transcription of the marker genes. The lesson, the authors note, is that promoter activity in this alga depends strongly on the terminator it is paired with, a phenomenon also documented in Chlamydomonas.

To quantify expression more precisely, the researchers turned to NanoLuciferase, a small and highly sensitive reporter. They built six level 2 vectors in which the Nluc gene was driven by three promoters, RIBI, VCP2 and the beta-tubulin promoter TUB, each paired with either the HS or FCPA terminator. Luminescence from transformed cultures correlated linearly with cell density, allowing quantitative comparison, and the spread between constructs was striking: otherwise identical constructs differing only in terminator produced dramatically different light output. The best-performing combination, pairing the TUB promoter with the FCPA terminator, yielded the highest luminescence, reinforcing the conclusion that promoter-terminator compatibility must be evaluated as a unit rather than assuming promoter strength is an intrinsic property.

Visualization tools received equally systematic treatment. The toolkit’s fluorescent protein set includes enhanced green fluorescent protein, enhanced yellow fluorescent protein and StayGold, a highly photostable and bright GFP variant that had never before been evaluated in Nannochloropsis. All three produced strong cytosolic fluorescence when expressed alone. The team then fused transit peptides to direct the reporters to specific compartments: chloroplast transit peptides from the glycine cleavage system L protein and from the violaxanthin/chlorophyll a-binding protein steered eGFP into the chloroplast, while the simian virus 40 nuclear localization sequence confined eYFP to a single nuclear spot. Crucially, dual-cassette constructs allowed two reporters to be targeted simultaneously, one to the chloroplast and one to the nucleus, demonstrating that the system supports co-localization studies within a single transformant. Five epitope tags, HA, 6xHis, Flag, Myc and StrepII, were also validated by immunoblotting, with the HA tag producing the strongest signal and four of the five tags being demonstrated in this organism for the first time.

The demonstration that ties the toolkit together is metabolic: the modular assembly of keto-carotenoid biosynthetic pathways. Canthaxanthin and astaxanthin are red-pigmented carotenoids with powerful antioxidant properties and established markets in food, feed and nutraceuticals. The researchers assembled a beta-carotene ketolase from Chlamydomonas, targeted to the chloroplast, to produce canthaxanthin, and a two-enzyme module combining that ketolase with a beta-carotene hydroxylase from Haematococcus lacustris to produce astaxanthin. Transformants turned visibly orange, accumulating 4.5 milligrams of canthaxanthin or 2.8 milligrams of astaxanthin per gram of dry weight, comparable to yields previously achieved with traditional cloning. Interestingly, the engineered strains accumulated more keto-carotenoid under normal light than under high light, the opposite of the stress-induced production seen in natural producers like Haematococcus, likely because high light depletes the beta-carotene precursor pool in Nannochloropsis. Canthaxanthin was even detected in isolated thylakoid membranes, at 4.4 moles per 100 moles of chlorophyll a, and transcriptomic analysis showed that the engineered accumulation perturbed endogenous carotenoid gene expression only mildly, with fold changes below 1.5.

The practical implications extend well beyond pigments. Because assembly is modular, the authors estimate that researchers can now design and construct complex constructs carrying up to six distinct genes in a single level 2 vector within days rather than weeks, and the availability of multiple selectable markers means more than one level 2 vector can be introduced at once, theoretically allowing over ten genes to be stacked. The toolkit also proved portable: introducing the canthaxanthin module into Nannochloropsis gaditana, a close industrial relative, likewise yielded substantial canthaxanthin. Combined with existing CRISPR-Cas genome editing systems and high-throughput microfluidic screening, the toolkit positions Nannochloropsis as a genuine sunlight-driven chassis for carbon-neutral biomanufacturing, in which captured CO2 becomes feedstock for lipids, carotenoids and other high-value biochemicals grown in seawater rather than on arable land.

There remain honest limits. Astaxanthin yields in the engineered Nannochloropsis still fall far short of the roughly four percent of cell dry weight that stressed Haematococcus cultures can reach, and the authors point to strengthening the plastidial MEP pathway and beta-carotene supply as the logical next engineering targets. But the structural obstacle, the absence of a fast, standardized, expandable assembly system, has now been removed. With 91 validated parts, a shared syntax compatible with the broader plant and algal synthetic biology community, and demonstrated performance across two Nannochloropsis species, the toolkit converts what was once a craft-based cloning exercise into an engineering discipline, opening the door to rapid design-build-test cycles for anyone hoping to turn these tiny marine cells into photosynthetic factories.

Subject of Research: Development of a modular cloning toolkit for metabolic engineering of the microalga Nannochloropsis

Article Title: A modular synthetic biology toolkit unlocks metabolic engineering of the industrially relevant alga Nannochloropsis

Article References: A modular synthetic biology toolkit unlocks metabolic engineering of the industrially relevant alga Nannochloropsis. (n.d.). https://doi.org/10.1007/s44307-026-00096-w

Image Credits: AI Generated

DOI: 10.1007/s44307-026-00096-w

Keywords: synthetic biology, Nannochloropsis, MoClo, Golden Gate cloning, metabolic engineering, microalgae, canthaxanthin, astaxanthin, carotenoid biosynthesis, CO2 valorization, genetic toolkit, algal biotechnology