Three birds with one stone, microorganisms that “eat” sugar substitutes are of great use

Although “erythritol” is still somewhat unfamiliar to the public, it has appeared more and more frequently in the ingredient list of beverages or foods. As a natural tetracarbon (C4) sugar alcohol, erythritol is widely found in fruits, vegetables and fermented foods. Because of its stable physical and chemical properties, high biological safety, certain sweetness and cannot be metabolized and absorbed by the human body, it is used as a natural sugar substitute (sweetener) in food production.

Recently, the team of Professor Li Jian of the School of Physical Science and Technology of ShanghaiTech University (hereinafter referred to as ShanghaiTech University) has constructed a number of E. coli bacteria that can use erythritol as the only carbon source. This “eating” sugar substitute microorganism was originally “born” for carbon reduction, but it has been unexpectedly discovered for a variety of uses, which is expected to have a positive impact on synthetic biology, metabolic engineering and in vivo therapy, food industry, and carbon recycling. The results have been published online in Advanced Science.

Professor Li Jian (former) and student Ba Fang. Photo courtesy of interviewee

Research aimed at “carbon reduction”

“The original intention of studying this issue was to reduce carbon.” Li Jian told China Science News, “Erythritol is a safe sugar substitute, which allows people to enjoy sweetness without worrying about rising blood sugar or excessive calorie intake.” Therefore, the original research goal was not to harness the energy of erythritol. ”

After the human body ingests glucose, fructose and other sugars, some of them will be absorbed, decomposed and metabolized by E. coli and other flora after entering the intestine to maintain the growth of intestinal flora. However, organisms and their internal flora generally lack metabolic pathways that can decompose erythritol, and this four-carbon sugar enters the human body or animals, and will be like a “passerby”, how to get in, and eventually as a “carbon waste”, in the form of “carbon source” excreted.

Compared with other synthetic sugar substitute products, erythritol has higher biological safety, and currently commercially available beverages and foods have gradually begun to use erythritol as sugar substitutes. Therefore, Li’s team wanted to figure out whether model bacteria (E. coli) could be used to metabolize erythritol.

“Although E. coli cannot metabolize erythritol, it can metabolize glucose or other sugars. At the same time, there are a small number of microorganisms that can metabolize erythritol in nature, and we want to isolate the gene clusters related to metabolizing erythritol in such microorganisms (multiple genes encoded together) and use it to modify E. coli. Let the enzyme proteins encoded by these gene clusters perform their respective functions, and gradually use erythritol catabolism. Ba Fang, the first author of the paper and a doctoral student at the School of Matter of the Shanghai University of Science and Technology, said.

To this end, the research team first screened microorganisms that could use erythritol from the natural environment, identified the erythritol metabolic pathway (five enzymes eryA, eryB, eryC, eryH, eryI), and then introduced this pathway into model E. coli cells to grow erythritol as the only carbon source in synthetic medium. Since then, through transcriptome analysis and metabolic engineering, the researchers have further improved the ability of E. coli to metabolize erythritol, and the density of the flora after continuous culture is close to that of glucose as the carbon source.

Metabolism of erythritol. Photo courtesy of interviewee

Discovering the “gene switch”

In the experiment, the researchers selected two strains of E. coli, including Nissle 1917, a harmless intestinal probiotic, as the starting strain. After modification, these E. coli bacteria can transport erythritol as an energy substance to the cell to break it down and enter other metabolic networks to maintain cell growth.

In a synthetic medium in the laboratory, these E. coli bacteria can be grown with erythritol as the only carbon source. In further research, the team found that there is a regulatory gene element in this metabolic pathway, which can realize the response and utilization of erythritol by E. coli as the only carbon source.

“That is, this particular E. coli can respond only in the presence of this carbon source (erythritol).” Li Jian added, “When erythritol enters the bacteria, it can initiate the expression of its downstream coding protein. In this way, we have a gene expression regulation method, which can be used to build various gene regulatory units to control the expression of another active protein like a switch. ”

Using this “on-off” function of the gene regulatory unit, people can express a therapeutic active protein or small molecule compound in probiotics. For example, in tumor treatment, people will use some live bacteria to “attack” tumor cells, if there is this regulatory ability, the live bacteria can be in a “dormant” state before they reach the tumor site to protect the normal cells of the human body from harm, and then “attack” when the live bacteria reach the tumor lesion.

“It’s equivalent to having a time bomb that you can start whenever you want.” Li Jian also emphasized, “Although it has been found that there are many possible application scenarios, such as biomedical treatment, we have not carried out research in this area, and we have only developed such a possible basic application system.” ”

Showcase multiple application scenarios

“This study is very innovative, paving the way for E. coli metabolism of erythritol, which is of great significance for expanding the range of carbon sources used by E. coli.” The reviewers of the paper believe that this idea of using four-carbon sugars by modifying E. coli has not been previously reported, which fills the gap between the carbon source spectrum between six carbons (such as glucose) and one carbon (such as carbon dioxide), and expands the scope of carbon source utilization. On the other hand, it can play a unique regulatory (response) role and can do more with E. coli.

Li Jian said that the engineered E. coli strain could be developed as a erythritol live detector to detect whether commercially available beverages contain sugar substitutes (erythritol). This in vivo assay is faster, more intuitive, and less expensive than traditional instrumental assays.

After the team constructed the erythritol metabolic pathway into the body of Nissle coli 1917, the bacteria can be grown in simulated intestinal fluids (in which erythritol is added as the only carbon source), which provides a research basis for the development of probiotics for live treatment with its host. More critically, the small molecule “switches” currently commonly used in the field of synthetic biology or microbial metabolism may cause harm to the human body. These small molecules are fine when experimented with in culture flasks, but they have many safety limitations when applied to humans. In contrast, erythritol and this strain of E. coli are safe and generally do not cause harm to the human body.

“This work uses the synthetic biology research idea of ‘design-build-verify-learn’ to realize the growth of E. coli using C4 compounds as the only carbon source for the first time, filling the research gap in related fields.” Our identification of erythritol metabolic pathways and their transcriptional regulators provides new standardized biological elements and gene circuits in the field of synthetic biology, which are expected to have a positive impact on synthetic biology, metabolic engineering, in vivo therapy, the food industry, and carbon recycling. Li Jian said. (Source: Zhang Shuanghu, China Science News)

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