Vitamin K₂ in a New Way: How a “Cheese” Microbe Taught Scientists to Make Vitamins Cheaper and More Eco-Friendly

A team from Rice University has found out why Lactococcus lactis bacteria (the very same safe “workhorse” of cheeses and kefir) stubbornly refuse to produce too much of the precursor of vitamin K₂ — and how to carefully “remove the limiters”. It turned out that cells balance between benefit (quinones are needed for energy) and toxicity (their excess triggers oxidative stress). Scientists have assembled a super-sensitive biosensor, “threw wires” into the synthesis pathways and connected a mathematical model. Conclusion: two “curtains” interfere at once — the built-in regulation of the pathway and the lack of the initial substrate; plus, even the order of genes on DNA is important. If you adjust three knobs together (substrate → enzymes → gene order), the output ceiling can be raised. The work was published in mBio on August 11, 2025.

Background of the study

• Why does everyone need vitamin K₂? Menaquinones (vitamin K₂) are important for blood clotting, bone health, and probably blood vessels. Demand for supplements is growing, and classic chemical synthesis is expensive and not the greenest. The logical solution is to make K₂ by fermentation on safe food bacteria.
• Why Lactococcus lactis? It is the workhorse of the dairy industry, with GRAS status. It is easy to cultivate, safe, and already used in food – the perfect base for turning the microbe into a vitamin biofactory.
• Where is the real dead end? The K₂ biosynthesis pathway goes through reactive quinone intermediates. On the one hand, they are needed by the cell (energy, electron transfer), but on the other hand, in excess they become toxic (oxidative stress). Therefore, even if you “tweak” the enzymes, the cell itself sets limits on the flow rate.
• What was missing before.
  • Accurate measurements of unstable intermediate metabolites - they are difficult to "catch" with standard methods.
  • Understanding whether low output is due to pathway regulation, lack of initial substrate, or… the often overlooked architecture of the operon (the order of genes on the DNA).
• Why this work. The authors needed:
  1. create a sensitive biosensor to finally measure the “slippery” intermediates;
  2. assemble a model of the entire cascade and find out where the real “bottlenecks” are;
  3. to test how three knobs at once affect the release - substrate supply, levels of key enzymes and the order of genes - and whether it is possible to break through the natural ceiling by turning them in concert.
• Practical sense. If you understand where exactly the microbe "slows itself down," you can design strains that produce more vitamin with the same resources, and make production cheaper and more environmentally friendly. This is also useful for other pathways where "useful" quinones are on the verge of toxicity - from vitamins to drug precursors.

What exactly did they do?

• An invisible intermediate product was caught. The precursor from which all forms of vitamin K₂ (menaquinone) are assembled is very unstable. To "see" it, a custom biosensor was made in another bacterium - the sensitivity increased thousands of times, and simple laboratory equipment was enough for measurements.
• They twirled the genetics and compared it with the model. The researchers changed the levels of key enzymes of the pathway and compared the actual release of the precursor with the model's predictions. While the model considered that the substrate was "infinite", everything diverged. It was worth considering the depletion of the start, and the predictions "fell" into place: we are running into not only enzymes, but also raw materials for the pathway.
• The role of DNA "architecture" was found. Even the order of the genes of the enzyme cascade affects the level of the unstable intermediate product. The rearrangement gave noticeable shifts - this means that evolution also uses the geometry of the genome as a regulator.

Key findings in simple terms

• L. lactis maintains just enough precursor to survive and grow without going into toxicity. Simply “adding enzymes” doesn’t help if there isn’t enough substrate: it’s like putting in more cookie sheets without adding flour.
• The production "ceiling" is set by two things together: the internal regulation of the pathway and the availability of the source. Plus on top of all this is the order of genes in the operon. Tuning three levels at once allows you to go beyond the natural limit.

Why is this necessary?

• Vitamin K₂ is important for blood clotting, bones, and probably vascular health. Currently, it is obtained by chemical synthesis or extraction from raw materials - this is expensive and not very environmentally friendly. Engineering safe food bacteria gives a chance to make K₂ by fermentation - cheaper and "greener".
• Understanding where the “brakes” in the synthesis pathway are is a map for producers: it is possible to create strains that produce more vitamin on the same amount of feed and area, and in the future, even probiotics that synthesize K₂ directly in the product or in the intestines (strictly under regulation, of course).

Quotes

• "Vitamin-producing microbes have the potential to transform nutrition and medicine, but first we need to decipher their internal 'emergency stopcocks,'" says co-author Caroline Aho-Franklin (Rice University).
• “When we took into account substrate depletion, the model finally matched the experiment: the cells hit a natural ceiling when the source runs out,” adds Oleg Igoshin.

What this means for the industry - point by point

• Tools: Now there is a biosensor for fine control and a model that correctly calculates "bottlenecks". This speeds up the "design → check" cycle.
• Scaling strategy: Don't chase one "super enzyme". Tweak three knobs: substrate feed → enzyme levels → gene order. This way, you have a higher chance of breaking through the natural limit.
• Tolerability: The benefit/toxicity balance principles for quinones apply to other microbes and pathways, too, from vitamins to antibiotics: too many reactive intermediates and growth falls.

Where is caution?

This is fundamental work on safe food bacteria and in laboratory conditions. There are still questions before the workshop: strain stability, regulation for "functional" products, scaling economics. But the roadmap - where to turn and what to measure - already exists.

Summary

To make more vitamin from a microbe, it’s not enough to just “give gas” to an enzyme — it’s also important to supply fuel and assemble the right wiring. The mBio study shows how to tweak substrate, genes, and regulation together to turn Lactococcus lactis into a green K₂ factory — and make vitamins cheaper and cleaner.

Source: Li S. et al. The growth benefits and toxicity of quinone biosynthesis are balanced by a dual regulatory mechanism and substrate limitations, mBio, August 11, 2025. doi.org/10.1128/mbio.00887-25.