Engineers Create Bacteria That Can Synthesize an Unnatural Amino Acid

By University of DelawareJuly 19, 2023

Scientists have engineered bacteria to produce pN-Phe, a non-standard amino acid with potential medical applications. Future work will optimize this process and explore its potential in vaccines and immunotherapies.

Amino acids serve as the foundational elements of proteins, vital to the optimal functioning of biological structures. Proteins in all life forms are composed of 20 core amino acids<div class="cell text-container large-6 small-order-0 large-order-1"><div class="text-wrapper"><br />Amino acids are a set of organic compounds used to build proteins. There are about 500 naturally occurring known amino acids, though only 20 appear in the genetic code. Proteins consist of one or more chains of amino acids called polypeptides. The sequence of the amino acid chain causes the polypeptide to fold into a shape that is biologically active. The amino acid sequences of proteins are encoded in the genes. Nine proteinogenic amino acids are called "essential" for humans because they cannot be produced from other compounds by the human body and so must be taken in as food.<br /></div></div>" data-gt-translate-attributes="[{"attribute":"data-cmtooltip", "format":"html"}]">amino acids. However, nature offers an impressive variety of over 500 distinct amino acids. Additionally, a plethora of synthetic amino acids have been created by human ingenuity. These alternate amino acids hold promise in the development of innovative pharmaceuticals and therapeutic treatments.

Now, University of Delaware researchers in the lab of Aditya Kunjapur, assistant professor in the College of Engineering’s Department of Chemical and Biomolecular Engineering, have engineered bacteria to synthesize an amino acidAny substance that when dissolved in water, gives a pH less than 7.0, or donates a hydrogen ion." data-gt-translate-attributes="[{"attribute":"data-cmtooltip", "format":"html"}]">acid that contains a rare functional group that others have shown to have implications in the regulation of our immune system. The researchers also taught a single bacterial strain to create the amino acid and place it at specific sites within target proteins. These findings, published in Nature Chemical Biology, provide a foundation for developing unique vaccines and immunotherapies in the future.

The Kunjapur Lab uses tools from synthetic biology and genetic engineering to create micro-organisms that can synthesize different types of compounds and molecules, especially ones with functional groups or properties that are not well represented in nature.

In this study, the researchers focused on para-nitro-L-phenylalanine (pN-Phe), a non-standard amino acid that is neither one of the twenty standard amino acids nor has been observed in nature. Other research groups have utilized pN-Phe as a tool to stimulate the immune system to react to proteins that it typically ignores.

“The nitro chemical functional group has valuable properties and has been underexplored by folks who are trying to rewire metabolism,” Kunjapur said. “pN-Phe also has a nice history in the literature — it can be added onto a protein from a mouse, delivered back to mice, and the immune system will no longer tolerate the original version of that protein. That ability has promise for the treatment or prevention of diseases that are caused by rogue proteins that the immune system struggles to lock onto.”

Genetic code expansion methods allowed the researchers to increase the “alphabet” of available amino acids encoded by DNADNA, or deoxyribonucleic acid, is a molecule composed of two long strands of nucleotides that coil around each other to form a double helix. It is the hereditary material in humans and almost all other organisms that carries genetic instructions for development, functioning, growth, and reproduction. Nearly every cell in a person’s body has the same DNA. Most DNA is located in the cell nucleus (where it is called nuclear DNA), but a small amount of DNA can also be found in the mitochondria (where it is called mitochondrial DNA or mtDNA)." data-gt-translate-attributes="[{"attribute":"data-cmtooltip", "format":"html"}]">DNA. By coupling metabolic engineering techniques with genetic code expansion, the researchers were able to create a system that produces nitrated proteins autonomously.

“Because of the nitro functional group chemistry, the amino acid that we picked as our target for this project was unconventional, and many scientists within our field may not have expected that it could be made using biosynthesis,” Kunjapur said.

The next step for this research is to optimize their methods to synthesize higher amounts of nitrated proteins and expand this work into other microorganisms. The long-term goal is to further refine this platform for applications related to vaccines or immunotherapies, efforts that are supported by Kunjapur’s 2021 AIChE Langer Prize and the 2022 National Institutes of HealthThe National Institutes of Health (NIH) is the primary agency of the United States government responsible for biomedical and public health research. Founded in 1887, it is a part of the U.S. Department of Health and Human Services. The NIH conducts its own scientific research through its Intramural Research Program (IRP) and provides major biomedical research funding to non-NIH research facilities through its Extramural Research Program. With 27 different institutes and centers under its umbrella, the NIH covers a broad spectrum of health-related research, including specific diseases, population health, clinical research, and fundamental biological processes. Its mission is to seek fundamental knowledge about the nature and behavior of living systems and the application of that knowledge to enhance health, lengthen life, and reduce illness and disability." data-gt-translate-attributes="[{"attribute":"data-cmtooltip", "format":"html"}]">National Institutes of Health Director’s New Innovator Award. To further support this long-term goal, Kunjapur and Neil Butler, doctoral candidate and first author on this paper, co-founded Nitro Biosciences.

“I think the implications are interesting, in that you can take a bacterium’s central metabolism, its ability to produce different compounds, and with a few modifications you are able to expand its chemical repertoire,” said Butler. “The nitro functionality is rare in biology and absent from the standard 20 amino acids, but we showed bacterial metabolism is malleable enough that it can be rewired to create and integrate this functionality.”

Kunjapur added, “Bacteria are potentially useful drug delivery vehicles. We think we have created a tool that could leverage the ability of bacteria to produce target antigens within the body and exploit the ability of nitration to shine a light on those antigens at the same time.”

Reference: “A platform for distributed production of synthetic nitrated proteins in live bacteria” by Neil D. Butler, Sabyasachi Sen, Lucas B. Brown, Minwei Lin and Aditya M. Kunjapur, 15 May 2023, Nature Chemical Biology.DOI: 10.1038/s41589-023-01338-x

The research was funded by a grant from the National Science Foundation.