Patent Analysis: Memphis Meats and Glutamine Synthetase
Memphis Meats’ patent application, Compositions and Methods for Increasing the Efficiency of Cell Cultures Used for Food Production, discusses a set of genetic modifications beneficial for large scale cell production. Some of the modifications were discussed in previous patent applications—for example, activating the Yes-Associated Protein 1 to disable contact inhibition, or knocking out a cyclin-dependent kinase inhibitor to immortalize the cell line. The most interesting new genetic modification they discuss is overexpressing glutamine synthetase (GS), an enzyme that can turn the metabolite ammonia to the nutrient glutamine.
A major challenge in large scale cell cultures is controlling the levels of ammonia (NH3)—a molecule naturally created during cellular respiration that can be toxic to cells at high levels [1]. In the body, ammonia is filtered through the bloodstream and disposed of in urine, or it’s recycled as a nitrogen source. However, in cell culture these mechanisms are unavailable. GS helps solve this problem by taking some of the excess ammonia and converting it into glutamine, an important amino acid and alternative energy source. By overexpressing this enzyme, Memphis Meats can control ammonia levels, while simultaneously increasing levels of an amino acid that can be reused in other proteins.
Another interesting result from their experiment is that when glutamine is included in the media, GS overexpression has no effect. This is likely because glutamine levels can be too high. Glutamine is highly unstable, and often degrades into its constituent glutamate [2] and ammonia, meaning that unused glutamine can increase ammonia levels over time. Therefore, the lowest ammonia levels occur when there is GS overexpression and no glutamine in the media.
The benefits of this system are:
Lowered ammonia levels.
Increased glutamine levels. Glutamine levels will also be more stable since degraded glutamine can be replaced over time.
Glutamine is no longer needed in the media. This will lower media costs somewhat, although glutamine is not a major cost driver.
It will also lower risk of contamination from external glutamine sources.
The potential drawbacks are:
Higher levels of glutamine synthetase could cause an undesirable decrease in glutamate levels, another important amino acid. Glutamate is also a precursor to other amino acids like proline, so lower glutamate levels could affect overall amino acid balance.
Glutamine synthetase overexpression requires genetic modification, which could be detrimental from a regulatory or consumer acceptance standpoint.
As some genes express more than one protein, overexpression of glutamine synthetase may alter expression of other proteins or affect downstream pathways.
GS expression systems have some historical precedent for various applications. For example, since most cells cannot make glutamine, GS expression system can be used as a selectable marker for cell-line development. Additionally, overexpression of GS has been used in several studies to enhance nitrogen metabolism in plants [3] and animals [4].
Footnotes
[1] The underlying mechanism of ammonia toxicity is not entirely understood. Some research suggests that accumulation of ammonia can affect electrochemical balance, cause cytoplasm acidification, and result in intracellular enzyme inactivation and apoptosis.
[2] Glutamate is itself an important neurotransmitter, and can serve as a precursor for other amino acid synthesis.
[3] In some studies, GS overexpression had favorable effects on ammonia levels and nitrogen metabolism, and also made plants more resistant to environmental changes such as salt, drought, and cold stress. However, there are also drawbacks such as lower grain yield, and decreased amino acid production in seeds.