L-Pyrrolysine

• Research compound only
• No established benefits in humans

• Potential research applications in protein engineering
• Theoretical applications in biotechnology

L-Pyrrolysine (Pyl) is often referred to as the ’22nd amino acid’ in the genetic code, following selenocysteine as the 21st. It is a specialized amino acid with a unique structure consisting of a pyrroline ring linked to the ε-amino group of L-lysine through an amide bond. Pyrrolysine is not used as a dietary supplement or therapeutic agent in humans; rather, it is a research compound of significant interest in biochemistry and molecular biology. The primary mechanisms and functions of pyrrolysine are observed in certain methanogenic archaea and bacteria, where it plays crucial roles in specific metabolic pathways.

In these organisms, pyrrolysine is genetically encoded by the UAG (amber) stop codon when a specific RNA structure called the Pyrrolysine Insertion Sequence (PYLIS) is present in the mRNA. This represents a natural expansion of the genetic code. The key mechanisms and functions of pyrrolysine include: 1) Methylamine metabolism: In methanogenic archaea, pyrrolysine is essential for enzymes involved in methylamine metabolism, particularly methyltransferases that catalyze the transfer of methyl groups from mono-, di-, and trimethylamine to produce methane. The pyrrolysine residue is located at the active site of these enzymes and is critical for their catalytic function.

2) Nucleophilic catalysis: The pyrroline ring of pyrrolysine provides unique chemical properties that enable nucleophilic catalysis in methyltransferase reactions. The electrophilic nature of the pyrroline carbonyl group facilitates the formation of covalent adducts with methylamines, which is a key step in the methyl transfer process. 3) Genetic code expansion: The natural incorporation of pyrrolysine through amber codon suppression has inspired synthetic biology approaches for genetic code expansion. Researchers have adapted the pyrrolysine incorporation machinery (consisting of a specialized tRNA and aminoacyl-tRNA synthetase) to incorporate various unnatural amino acids into proteins at specific sites, enabling new approaches for protein engineering and functional studies.

4) Protein structure stabilization: In proteins where it naturally occurs, pyrrolysine can contribute to protein stability through its unique structural properties. The pyrroline ring can participate in hydrophobic interactions and potentially form hydrogen bonds that help maintain protein tertiary structure. It’s important to note that pyrrolysine is not found in human proteins and has no known role in human physiology. Humans lack the genetic machinery for pyrrolysine incorporation, and there is no evidence that dietary supplementation with pyrrolysine would have any beneficial effects.

The interest in pyrrolysine is primarily scientific, focused on understanding natural genetic code variations and developing biotechnological applications based on this unique amino acid incorporation system. In research settings, synthetic pyrrolysine analogs and the pyrrolysine incorporation machinery are being explored for applications in protein labeling, creating proteins with novel functions, and developing new biocatalysts. These applications rely on the ability to site-specifically incorporate pyrrolysine or its analogs into proteins using genetic code expansion techniques.

L-Pyrrolysine is not used as a dietary supplement or therapeutic agent in humans. It is a specialized amino acid primarily found in certain methanogenic archaea and some bacteria, where it is incorporated into specific proteins involved in methylamine metabolism. There are no established dosages for L-pyrrolysine in humans as it is not considered a nutrient or therapeutic compound. In research settings, synthetic L-pyrrolysine and its analogs are used in controlled laboratory experiments for protein engineering and biotechnology applications, but these uses are strictly experimental and not related to human consumption or supplementation.

Any consideration of L-pyrrolysine as a supplement would be premature and unsupported by scientific evidence.

The bioavailability and absorption of L-pyrrolysine in humans has not been studied as it is not used as a dietary supplement or therapeutic agent. L-Pyrrolysine is a specialized amino acid primarily found in certain methanogenic archaea and some bacteria, where it is incorporated into specific proteins involved in methylamine metabolism. It is not naturally present in the human diet or produced endogenously in human tissues. Based on general principles of amino acid biochemistry, if synthetic L-pyrrolysine were to be administered orally (which is not done in practice), it would likely face several barriers to absorption: 1) The unique structure of pyrrolysine, with its pyrroline ring attached to lysine, would likely not be recognized by standard amino acid transporters in the human intestinal epithelium; 2) The compound might be subject to degradation by digestive enzymes in the gastrointestinal tract; 3) Even if absorbed intact, it would likely be metabolized in the liver rather than utilized for protein synthesis, as humans lack the specialized tRNA and aminoacyl-tRNA synthetase required for pyrrolysine incorporation into proteins.

In research settings where pyrrolysine or its analogs are used for protein engineering through genetic code expansion, these compounds are typically introduced directly into cell culture systems or in vitro translation systems that have been genetically modified to express the pyrrolysine incorporation machinery from archaea or bacteria.

Not applicable for human supplementation, In research settings, various chemical modifications to improve stability, Protected forms for chemical synthesis, Specialized delivery systems for cell culture applications, Genetic engineering of the pyrrolysine incorporation machinery for improved efficiency in experimental systems

Timing recommendations are not applicable for L-pyrrolysine as

it is not used as a dietary supplement or therapeutic agent in humans. In research settings, the timing of L-pyrrolysine or analog administration is determined by specific experimental protocols and has no relevance to human supplementation practices. L-Pyrrolysine remains a compound of scientific interest primarily for understanding natural genetic code variations and for applications in protein engineering and biotechnology. Any consideration of L-pyrrolysine as a supplement would be premature and unsupported by scientific evidence.

• Unknown – no human studies have been conducted
• Not used as a supplement or therapeutic agent
• Potential toxicity has not been systematically evaluated
• As a non-standard amino acid, may potentially interfere with normal protein synthesis if incorporated inappropriately
• May potentially trigger immune responses as a foreign compound
• Possible allergic reactions to a non-human protein component
• Note: These are theoretical concerns as L-pyrrolysine is not used in humans

• Not applicable – not used as a supplement or therapeutic agent
• Should not be consumed by any individuals
• Pregnancy and breastfeeding – not studied, should be avoided
• Children – not studied, should be avoided
• Any medical condition – not studied, should be avoided
• Note: L-pyrrolysine is a research compound only and has no established medical uses

• Unknown – no studies have been conducted
• Not applicable as L-pyrrolysine is not used as a supplement or therapeutic agent
• Theoretical potential to interfere with protein synthesis pathways
• Possible interactions with aminoacyl-tRNA synthetase inhibitors (theoretical)
• Note: These are speculative as L-pyrrolysine is not used in humans

No upper limit has been established for L-pyrrolysine as it is not used as a dietary supplement or therapeutic agent in humans. L-Pyrrolysine is a specialized amino acid primarily found in certain methanogenic archaea and some bacteria, where it is incorporated into specific proteins involved in methylamine metabolism. It is not naturally present in the human diet or produced endogenously in human tissues. There are no human or animal studies evaluating the safety, toxicity, or tolerable upper intake levels of L-pyrrolysine.

In research settings, synthetic L-pyrrolysine and its analogs are used in controlled laboratory experiments for protein engineering and biotechnology applications, but these uses are strictly experimental and not related to human consumption. The compound should be handled according to standard laboratory safety practices for research chemicals. Given the lack of safety data and the absence of any established nutritional or therapeutic role in humans, consumption of L-pyrrolysine should be avoided. Any consideration of L-pyrrolysine as a supplement would be premature and potentially hazardous.

L-Pyrrolysine has no official regulatory status with the U.S. Food and Drug Administration (FDA) as a supplement ingredient or drug. It is not listed in the FDA’s Generally Recognized as Safe (GRAS) substances, nor is it approved as a food additive, dietary supplement ingredient, or pharmaceutical. The compound is not mentioned in the FDA’s Dietary Supplement Health and Education Act (DSHEA) regulations or subsequent guidance documents.

L-Pyrrolysine would likely be classified as a research chemical for laboratory use only. If it were to be marketed as a supplement ingredient (which is not currently the case), it would potentially be subject to New Dietary Ingredient (NDI) notification requirements, as it does not have a history of use in the food supply. However, given the lack of safety data and absence of established nutritional or therapeutic benefits in humans, it is highly unlikely that such a notification would be accepted. In research settings, L-pyrrolysine is handled according to standard laboratory safety practices for research chemicals, but it does not have specific FDA regulations governing its use in this context.

The European Food Safety Authority (EFSA) has not evaluated L-pyrrolysine for use in food or supplements. The compound is not listed in the European Union’s approved food additives, novel foods, or supplement ingredients. Under European regulations, particularly Regulation (EC) No 258/97 concerning novel foods and novel food ingredients, L-pyrrolysine would likely be classified as a novel food ingredient if proposed for human consumption, requiring comprehensive safety evaluation before approval. Given the lack of history of consumption in the European Union before May 15, 1997, and the absence of safety data, it is highly unlikely that L-pyrrolysine would receive approval as a food or supplement ingredient under current EU regulations.

In research settings, L-pyrrolysine is subject to standard laboratory chemical regulations but does not have specific EFSA guidelines governing its use in this context.

Health Canada has not issued any specific regulations or guidance regarding L-pyrrolysine. The compound is not listed in the Natural Health Products Ingredients Database (NHPID) and has no monograph in the Natural Health Products Directorate (NHPD). Under Canadian regulations, particularly the Natural Health Products Regulations, L-pyrrolysine would likely be classified as a non-compliant ingredient for use in natural health products due to the lack of safety data and absence of traditional or established use. If proposed for use in supplements or natural health products, L-pyrrolysine would require a comprehensive safety assessment and pre-market approval, which would be unlikely to be granted given the current state of knowledge.

In research settings, L-pyrrolysine is subject to standard laboratory chemical regulations in Canada but does not have specific Health Canada guidelines governing its use in this context.

The Therapeutic Goods Administration (TGA) of Australia has not evaluated L-pyrrolysine for use in therapeutic goods, including supplements. The compound is not listed in the Australian Inventory of Chemical Substances (AICS) for general use, nor is it included in the TGA’s list of permitted ingredients for listed medicines (including supplements). Under Australian regulations, particularly the Therapeutic Goods Act 1989 and associated regulations, L-pyrrolysine would likely be classified as a non-approved ingredient for therapeutic goods due to the lack of safety data and absence of traditional or established use. If proposed for use in supplements or other therapeutic goods, L-pyrrolysine would require comprehensive safety assessment and pre-market approval, which would be unlikely to be granted given the current state of knowledge.

In research settings, L-pyrrolysine is subject to standard laboratory chemical regulations in Australia but does not have specific TGA guidelines governing its use in this context.

Globally, there are no known regulatory frameworks that specifically address L-pyrrolysine as a supplement ingredient or therapeutic agent. The compound remains primarily a research chemical of interest in biochemistry and molecular biology. In Japan, the Ministry of Health, Labour and Welfare has not evaluated L-pyrrolysine for use in Foods for Specified Health Uses (FOSHU) or other food categories. In China, the National Medical Products Administration (NMPA) has not approved L-pyrrolysine for use in health foods or other regulated products.

In Brazil, ANVISA (the Brazilian health regulatory agency) has not included L-pyrrolysine in its list of approved food additives or supplement ingredients. In India, the Food Safety and Standards Authority of India (FSSAI) has not evaluated L-pyrrolysine for use in foods or supplements. In Russia, the Federal Service for Surveillance in Healthcare (Roszdravnadzor) has not approved L-pyrrolysine for medicinal use or as a supplement ingredient. Across all major regulatory jurisdictions, the status of L-pyrrolysine is essentially the same: it is not approved for use in foods, supplements, or medications, and would require comprehensive safety evaluation before any such approval could be considered.

The compound remains exclusively a research chemical used in controlled laboratory settings.

L-Pyrrolysine is not available as a prescription medication in any jurisdiction worldwide. It is not approved for therapeutic use by any major regulatory agency, including the FDA, EMA, Health Canada, or TGA. The compound is not listed in any pharmacopeia (such as the United States Pharmacopeia, European Pharmacopoeia, or Japanese Pharmacopoeia) and has no established medical uses. L-Pyrrolysine is exclusively a research chemical used in biochemistry and molecular biology laboratories.

It is typically synthesized in small quantities for specific research purposes or purchased from specialized chemical suppliers that provide research-grade chemicals. These suppliers typically require institutional affiliation and research purpose declarations for purchase, though this is standard practice for research chemicals rather than a specific regulatory requirement for pyrrolysine. If L-pyrrolysine were to be developed for therapeutic applications in the future (which is not currently being pursued), it would require the full drug development and approval process, including preclinical studies, clinical trials, and regulatory review before becoming available by prescription. However, there is currently no scientific basis or commercial interest in developing L-pyrrolysine as a therapeutic agent.

L-Pyrrolysine is not available as a dietary supplement or therapeutic agent for human consumption. It is exclusively a research chemical used in biochemistry and molecular biology laboratories. As a specialized research compound, synthetic L-pyrrolysine is extremely expensive, typically priced at several hundred to several thousand dollars per milligram from chemical suppliers that provide research-grade compounds. The high cost reflects the complex multi-step synthesis required, the specialized expertise needed for its production, the small scale of manufacturing, and the limited market consisting only of research laboratories.

Some pyrrolysine analogs used in protein engineering applications may be even more expensive, depending on their structural complexity and the difficulty of synthesis. For research institutions, the cost of pyrrolysine or its analogs represents a significant expense that is typically justified only for specific research projects focused on genetic code expansion, protein engineering, or studies of methanogenic archaea. It’s important to note that these price considerations are relevant only in the context of scientific research. There is no consumer market for L-pyrrolysine as a supplement, and any consideration of its cost-efficiency for health purposes would be inappropriate given the lack of safety data and absence of established benefits in humans.

Value comparison for L-pyrrolysine as a supplement is not applicable, as it is not used for this purpose. In research contexts, the value of pyrrolysine or its analogs must be evaluated based on their contribution to scientific knowledge or technological advancement rather than direct health benefits. Compared to other specialized research chemicals: L-Pyrrolysine is similarly priced to other highly specialized, non-standard amino acids used in research, reflecting the complexity of synthesis and limited market. It is generally more expensive than most standard biochemical reagents but comparable to other tools for genetic code expansion and protein engineering.

Compared to other approaches for protein modification: The pyrrolysine incorporation system represents one of several approaches for site-specific protein modification in research settings. Alternative methods include chemical conjugation strategies, enzymatic modifications, and other genetic code expansion systems. The relative value depends on the specific research application, with the pyrrolysine system offering advantages for certain types of modifications but being more costly than some alternatives. Compared to standard amino acids: L-Pyrrolysine is orders of magnitude more expensive than standard proteinogenic amino acids used in research, which typically cost a few dollars per gram rather than hundreds to thousands of dollars per milligram.

This price difference reflects both the complexity of synthesis and the specialized nature of its applications. It’s important to emphasize that these comparisons are relevant only in research contexts. There is no basis for comparing L-pyrrolysine to supplements or therapeutic agents, as it has no established role in these areas.

Bulk purchasing considerations for L-pyrrolysine are relevant only in research contexts, not for supplement use. In research settings, L-pyrrolysine is typically purchased in small quantities (milligrams) due to its high cost and specialized applications. Some chemical suppliers may offer modest discounts (typically 5-15%) for larger purchases, but these are still relatively small quantities by commercial standards. Research institutions conducting extensive work with pyrrolysine or its analogs sometimes establish collaborations with synthetic chemistry laboratories to produce the compounds in-house at lower cost than commercial purchase, though this requires specialized expertise.

For long-term research programs, some laboratories invest in developing more efficient synthetic routes or enzymatic production methods to reduce costs. However, even with these approaches, L-pyrrolysine remains an expensive research reagent. It’s worth noting that the stability limitations of L-pyrrolysine (see stability_information.json) make bulk purchasing potentially wasteful unless the compound will be used relatively quickly or stored under optimal conditions. These considerations have no relevance to supplement use, as L-pyrrolysine is not used as a supplement.

Insurance coverage for L-pyrrolysine is not applicable, as it is not used as a medication, supplement, or therapeutic agent in humans. No health insurance plans, including private insurance, Medicare, Medicaid, or national healthcare systems, provide coverage for L-pyrrolysine, as it has no approved medical uses. In research settings, the cost of L-pyrrolysine or its analogs would typically be covered by research grants, institutional funds, or departmental budgets rather than any form of insurance. These research expenses are subject to the normal funding mechanisms and accounting practices of scientific research institutions.

It’s important to emphasize that L-pyrrolysine remains exclusively a research compound with no established role in human health or medicine. Any consideration of insurance coverage for L-pyrrolysine as a health product would be inappropriate and potentially misleading.

L-Pyrrolysine is not used as a dietary supplement or therapeutic agent, so conventional shelf-life considerations for supplements do not apply. In research settings, synthetic L-pyrrolysine has limited stability due to its reactive functional groups, particularly the electrophilic pyrroline carbonyl group. As a research compound, lyophilized (freeze-dried) L-pyrrolysine is typically stored at -20°C or -80°C under inert gas (nitrogen or argon) to minimize degradation. Under these conditions, the compound may remain stable for 6-12 months, though gradual degradation can still occur.

In solution, L-pyrrolysine is significantly less stable, with noticeable degradation occurring within hours to days depending on temperature, pH, and exposure to oxygen. For research applications requiring longer-term storage, L-pyrrolysine is often kept as protected derivatives that mask the reactive functional groups, which can then be deprotected immediately before use. Some pyrrolysine analogs designed for research applications have modified structures to improve stability while maintaining functional properties for protein incorporation. It’s important to note that these stability considerations are relevant only for research applications and have no bearing on supplement use, as L-pyrrolysine is not used as a supplement.

For research-grade L-pyrrolysine:, Store as lyophilized powder at -20°C or -80°C, Keep under inert gas (nitrogen or argon) when possible, Protect from moisture using desiccants, Shield from light, particularly UV radiation, Avoid repeated freeze-thaw cycles, For solutions (research use only):, Prepare fresh solutions immediately before use when possible, Use degassed solvents to minimize oxidation, Store solutions at -20°C for short periods if necessary, Buffer at slightly acidic pH (5-6) for improved stability, Note: These conditions are for research applications only, not for supplement storage

Oxidation: The pyrroline ring is susceptible to oxidation, particularly at the carbon-carbon double bond, Hydrolysis: The amide bond connecting the pyrroline moiety to lysine can undergo hydrolysis, especially under acidic or basic conditions, Nucleophilic attack: The electrophilic pyrroline carbonyl group can react with nucleophiles (including water, alcohols, thiols, and amines), Heat: Elevated temperatures accelerate all degradation reactions, Light: UV and visible light can catalyze oxidation and other degradation pathways, pH extremes: Both acidic and basic conditions can accelerate hydrolysis of the amide bond, Enzymatic degradation: Susceptible to proteases and other enzymes that may cleave the amide bond or modify the pyrroline ring, Metal ions: Certain metal ions may catalyze oxidation or other degradation reactions, Microbial contamination: Can lead to enzymatic degradation in solutions, Note: These factors are relevant for research applications only, not for supplement stability

L-Pyrrolysine exhibits limited stability in aqueous solutions due to its reactive functional groups. The stability is highly dependent on several factors: pH significantly affects stability, with moderate stability observed at slightly acidic pH (5-6) and increased degradation rates at both lower and higher pH values. The electrophilic pyrroline carbonyl group is particularly susceptible to nucleophilic attack by water and other nucleophiles present in solution. Temperature has a major impact, with degradation rates approximately doubling for every 10°C increase in temperature.

Solutions should be kept cold (0-4°C) during handling and frozen for any storage. Oxygen exposure accelerates oxidative degradation, particularly of the pyrroline ring. Degassing solutions and maintaining an inert atmosphere can improve stability. The presence of reducing agents can sometimes improve stability by preventing oxidation, though they may also react with the pyrroline carbonyl group.

Buffer composition can affect stability, with phosphate buffers generally providing reasonable stability at appropriate pH. Metal contaminants in buffers can catalyze degradation reactions and should be minimized. Concentration also plays a role, with more dilute solutions typically showing faster relative degradation rates. For research applications requiring L-pyrrolysine in solution, it is generally recommended to prepare fresh solutions immediately before use and to keep them cold and protected from light and air.

If storage is necessary, aliquoting and freezing at -20°C or below under inert gas provides the best stability, though some degradation will still occur over time. These considerations are relevant only for research applications, as L-pyrrolysine is not used as a supplement or therapeutic agent.

Natural Sources

Synthetic Production Methods

Quality Indicators

Sustainability Considerations

L-Pyrrolysine has no history of traditional use in any medical system or cultural practice. Unlike many other amino acids and nutritional compounds that have been used traditionally for various health purposes, pyrrolysine was only discovered in 2002 and has never been part of the human diet or traditional medicine. The amino acid is naturally present only in certain methanogenic archaea and some bacteria, organisms that are not traditionally consumed by humans or used in medicinal preparations. These microorganisms typically inhabit extreme environments such as deep-sea hydrothermal vents, anaerobic sediments, and the digestive tracts of ruminants, making them inaccessible to traditional healers or early nutritionists.

Furthermore, even if these organisms had been consumed, the amount of pyrrolysine would be negligible and would not be absorbed or utilized by humans, who lack the genetic machinery for pyrrolysine incorporation into proteins. There are no historical records, ethnobotanical reports, or traditional medical texts that mention compounds that could be retrospectively identified as pyrrolysine or its derivatives. Any claims of traditional use of pyrrolysine for health purposes would be historically inaccurate and scientifically unfounded.

L-Pyrrolysine was discovered in 2002 by researchers studying methanogenic archaea, particularly Methanosarcina barkeri. The discovery came about through investigations of an unusual genetic feature: certain genes involved in methylamine metabolism contained an in-frame amber (UAG) stop codon that did not terminate protein synthesis as expected. This led to the identification of a specialized tRNA that could recognize this UAG codon and incorporate a previously unknown amino acid, which was subsequently identified as pyrrolysine. The discovery was reported in two landmark papers published in Science in 2002: one by Srinivasan, James, and Krzycki demonstrating the existence of a specialized tRNA that could decode UAG as an amino acid, and another by Hao et al.

identifying the structure of this amino acid as pyrrolysine. This discovery was significant in the field of molecular biology as it represented only the second known expansion of the genetic code beyond the standard 20 amino acids, following the earlier discovery of selenocysteine. The complete chemical structure of pyrrolysine was determined to be 4-methyl-pyrroline-5-carboxylate (MPC) in amide linkage to the ε-amino group of L-lysine. In 2004, Hao and colleagues reported the first chemical synthesis of pyrrolysine and determined the crystal structure of pyrrolysine bound to its cognate tRNA synthetase.

The biosynthetic pathway for pyrrolysine was elucidated in 2011 by Gaston et al., who demonstrated that pyrrolysine is synthesized from two molecules of lysine through a series of enzymatic reactions catalyzed by the products of the pylB, pylC, and pylD genes. Since its discovery, pyrrolysine has remained primarily a subject of basic research in biochemistry and molecular biology, with no development as a therapeutic agent or supplement.

Since its discovery in 2002, L-pyrrolysine has remained exclusively a research compound with no evolution into therapeutic or nutritional applications. The primary focus of pyrrolysine research has been in understanding its natural role in certain microorganisms and in developing biotechnological applications based on its unique incorporation mechanism. In the early years following its discovery (2002-2005), research focused on characterizing the basic biochemistry of pyrrolysine, including its structure, the mechanism of its incorporation into proteins, and its role in methylamine metabolism in methanogenic archaea. From approximately 2005-2010, there was increasing interest in the evolutionary aspects of pyrrolysine usage, including the distribution of pyrrolysine-utilizing organisms in various environments and the evolutionary origin of the pyrrolysine genetic code expansion.

During this period, researchers also began exploring the potential of the pyrrolysine incorporation machinery for biotechnological applications. From 2010 onwards, a major focus has been on adapting the pyrrolysine incorporation system (particularly the pyrrolysyl-tRNA synthetase and tRNAPyl pair) for genetic code expansion in synthetic biology. This has led to the development of numerous pyrrolysine analogs that can be incorporated into proteins at specific sites, enabling applications such as protein labeling, introduction of post-translational modification mimics, and creation of proteins with novel functions. Recent research (2015-present) has focused on optimizing the efficiency and specificity of pyrrolysine analog incorporation, expanding the range of organisms in which this technology can be applied, and developing new applications in protein engineering and biotechnology.

Throughout this evolution, there has been no development of pyrrolysine as a supplement or therapeutic agent, and no clinical studies have been conducted to evaluate potential health effects in humans. The compound remains of interest primarily to researchers in biochemistry, molecular biology, and synthetic biology.

No clinical trials involving L-pyrrolysine supplementation or therapeutic applications are currently registered or ongoing, Research on L-pyrrolysine is primarily conducted in basic science laboratories focusing on protein engineering, synthetic biology, and understanding microbial metabolism, Some ongoing research explores the use of the pyrrolysine incorporation machinery for introducing unnatural amino acids into proteins for various biotechnological applications, Studies investigating the evolutionary significance of pyrrolysine and the distribution of pyrrolysine-utilizing organisms in various environments, Research on optimizing the pyrrolysine incorporation system for improved efficiency in protein engineering applications