Engineered Bacteria Produce Record Nattokinase Levels and Curb Obesity in Mice

A fermented-food enzyme called nattokinase has intrigued researchers for years, but turning it into a practical supplement has been held back by expensive purification, limited scalability, and inconsistent yields from traditional fermentation. A new study published in the Journal of Animal Science and Biotechnology by Liu Y and colleagues takes a synthetic biology shortcut: engineer the bacterium that makes nattokinase to produce far more of it, then test whether the resulting formulation can counter obesity in mice. The findings are preliminary, but they push the field forward on two fronts simultaneously.

Engineered Bacillus subtilis Achieves Record Nattokinase Output

Nattokinase (NK) is a serine protease encoded by the aprN gene and naturally secreted by Bacillus subtilis during the fermentation of natto, the traditional Japanese soybean food. The mature enzyme is 275 amino acids long, weighs 27.7 kDa, and remains active across a wide pH range of 5.5 to 9.0. Traditional solid-state fermentation produces it inconsistently, and attempts to express NK in other hosts such as Lactobacillus lactis or Pichia pastoris have yielded disappointing activity levels.

The team chose B. subtilis as their chassis because, as research on Bacillus subtilis as an industrial microbial platform confirms, the bacterium has a well-characterized genome, secretes proteins efficiently, and produces no endotoxins or exotoxins. They drove aprN expression with Pspovg, a strong constitutive promoter, and fused a GFP tag to the gene so that high-performing clones could be sorted rapidly by flow cytometry rather than tested one at a time.

Screening identified transformant T3 as the top performer, which hit 1.18 × 10^5 U/mL in shake-flask cultures. When T3 was moved to a 5-liter bioreactor running high-density fed-batch fermentation, NK activity climbed to 4.19 × 10^5 U/mL, a 61% improvement over wild-type B. subtilis. The specific enzyme production rate during exponential growth was 119.5 U/OD/h. To make the enzyme usable in animal trials, the team mixed secreted NK with edible starch and freeze-dried it into a solid formulation containing 2 × 10^5 U per gram.

Nattokinase Reduced Fat Mass and Triglycerides in High-Fat-Diet Mice

To test the obesity angle, the researchers fed male C57BL/6J mice a high-fat diet for 52 days while gavaging them daily with NK at 10,000 U/kg body weight. Compared with unsupplemented high-fat-diet controls, NK-treated mice showed significantly lower body weight gain, body mass index, and Lee's index, a rodent measure of obesity. Perirenal and epididymal white adipose tissue weights both fell. Serum triglycerides dropped by 18.94%. Histological analysis using H&E staining confirmed that NK inhibited fatty vacuole formation in the liver and reduced fat accumulation in epididymal adipocytes.

Those metabolic effects align with earlier clinical evidence that nattokinase improves fasting insulin and glucose in people with obesity, suggesting the enzyme may have a broader metabolic role beyond its well-known fibrinolytic activity. That said, in this mouse study NK did not significantly change glucose tolerance on the oral glucose tolerance test, and total serum cholesterol was unaffected. The enzyme appears to act selectively on triglyceride metabolism and fat deposition rather than on the full lipid panel.

How the Gut Microbiome Responded to NK Treatment

Beyond metabolite changes, the team sequenced 16S rRNA from fecal samples to ask whether NK was reshaping the gut community. The high-fat diet had already reduced microbial richness and species diversity, as measured by Simpson and Shannon indexes, and NK did not restore those diversity metrics. At the phylum level, NK also failed to reverse the high-fat-diet-induced drop in Bacteroidota or the rise in the Firmicutes/Bacteroidota ratio.

The more informative signal came at the genus level. LEfSe analysis identified Blautia and Mucispirillum as enriched in the NK-treated group relative to unsupplemented high-fat-diet mice. Spearman correlation showed that genera elevated in the high-fat-diet group, particularly Colidextribacter, Lactobacillus, and Erysipelatoclostridium, correlated positively with obesity markers, while Bifidobacterium and Candidatus_Saccharimonas correlated negatively. NK appeared to shift the community in the direction of those negatively correlated taxa, at least partially.

The authors propose that NK may improve obesity-related metabolic disorder partly through modulation of specific intestinal flora, but the mechanism linking enzyme ingestion to microbiome composition changes is not yet established.

Open Questions About Heart Weight and Mechanism

The study surfaces several caveats worth noting. NK treatment increased heart weight and heart index compared with both the control and high-fat-diet groups, an unexpected finding that the authors flag as requiring further investigation. Whether this reflects a beneficial cardiac remodeling effect, a stress response, or something else is unknown.

The precise mechanisms by which NK reduces fat accumulation remain unclear. The enzyme is best understood as a fibrinolytic agent that degrades fibrin and related proteins; how that activity connects to triglyceride metabolism or adipocyte size is not yet worked out. Comparing activity values across nattokinase studies is also complicated by the fact that different labs use different assay methods, making absolute yield numbers hard to place in a broader context.

It is also worth noting that a randomized controlled trial in healthy, low-risk adults found nattokinase had no significant effect on subclinical atherosclerosis progression, which is a reminder that promising animal and high-risk-population data do not always translate across contexts.

What Comes Next for Nattokinase Research

The production side of this work is arguably the most immediately actionable result. A 61% yield improvement in a 5-liter bioreactor, combined with a freeze-dried solid formulation, means that larger animal studies and eventually human trials become economically feasible. Ongoing randomized controlled trials are already probing whether nattokinase can slow subclinical atherosclerosis and cognitive decline in healthy adults over three years, which will provide a cleaner test of the enzyme's cardiovascular claims independent of obesity.

For the obesity application specifically, the next steps the authors identify involve clarifying the heart weight finding, tracing the fat-metabolism pathway NK appears to engage, and determining whether the gut microbiome shift is a cause or a byproduct of the metabolic improvements. Until those mechanistic questions are answered, the mouse data should be read as hypothesis-generating rather than practice-changing. What the study does convincingly demonstrate is that synthetic biology can solve the supply problem that has long limited nattokinase research, and that the enzyme's anti-obesity effects in rodents are real enough to justify the follow-up work.

Evidence rating

INVESTIGATIONAL — 71/100

Caveats:

• All metabolic and microbiome findings are from a mouse model only — results may not translate to humans.
• Small group sizes (n=6–8 per group) limit statistical power and generalizability.
• Gut microbiota changes are correlational; no causal mechanism linking nattokinase to specific microbial shifts has been established.
• No funding source was disclosed, so independence of the research cannot be confirmed.
• Glucose tolerance was not significantly improved by nattokinase in this study, and an unexplained increase in heart index was observed, warranting further safety investigation.

This study combines a genetic engineering and fed-batch fermentation report with a mouse dietary intervention (n=6–8 per group). The metabolic and gut microbiota findings are derived entirely from a high-fat diet mouse model, so they establish association rather than causation and cannot be directly extrapolated to humans. The microbiome results have not been independently replicated, and no funding source was disclosed.

References

1. Yilin Liu, Lei Zhu, Tingyu Yao, Yanfeng Wang, Chunyan Xie, Le Gao (2026). Engineered Bacillus subtilis for high-yield nattokinase production and therapeutic potential in obesity management. Journal of Animal Science and Biotechnology. https://doi.org/10.1186/s40104-026-01438-3
2. Shiying Wu, Xiuwen Sun, Tingwen Fan, et al. (2025). Metabolic Engineering Strategy for Bacillus subtilis Producing MK-7. Foods. https://doi.org/10.3390/foods14234150
3. Leopoldo M Cobos, Yanira Sanchez-De La Torre, Karen L Herbst, Karen Beltran (2020). SAT-618 Nattokinase to Improve Insulin Sensitivity and Weight Loss in Women with Obesity +/- Diabetes. Journal of the Endocrine Society. https://doi.org/10.1210/jendso/bvaa046.145
4. Nattokinase atherothrombotic prevention study: A randomized controlled trial – PubMed. NIH. https://pubmed.ncbi.nlm.nih.gov/34947967/
5. Nattokinase Atherothrombotic Prevention Study | ClinicalTrials.gov. clinicaltrials.gov. https://clinicaltrials.gov/study/NCT02080520