Engineered n-lactoyl-phenylalanine producing bacteria to protect against obesity

Abstract

The present disclosure is directed to engineered probiotic bacteria capable of producing anti-obesity therapeutic molecules. More specifically, bacteria are engineered to express N-lactoyl-phenyalanine (Lac-Phe), an exercise inducible molecule capable of reducing food intake and suppressing obesity, driven by a high-fat diet. Also disclosed are methods of using the engineered bacteria to treat and protect individuals from obesity.

Description

[0001] DESCRIPTION

[0002] ENGINEERED N-LACTOYL-PHENYLALANINE PRODUCING BACTERIA TO PROTECT AGAINST OBESITY

[0003] REFERENCE TO RELATED APPLICATIONS

[0004] The present application claims the priority benefit of United States provisional application number 63 / 710,695, filed October 23, 2024, the entire contents of which are incorporated herein by reference.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0006] "This invention was made with government support under N00014-23-1-2874 awarded by the Department of Defense, Office of Naval Research, 2204402 awarded by the National Science Foundation, and the Department of Defense through the National Defense Science & Engineering Graduate Fellowship Program. The government has certain rights in the invention.

[0007] BACKGROUND

[0008] 1. Field of the Disclosure

[0009] The present disclosure relates generally to the fields medicine, disease and microbiology. More particularly, the disclosure relates to the generation and use of engineered bacteria to prevent and treat obesity.

[0010] 2. Background

[0011] Obesity is defined as the unhealthy accumulation of fat. It is a major risk factor for multiple debilitating and deadly diseases including cardiovascular disease, type 2 diabetes, cancer and neurodegeneration. Over the past two decades the global increase in the prevalence of obesity has led to an obesity pandemic. This increase in the rate of obesity has resulted in a rise in the incidences of obesity- associated co-morbidities. Current therapies to combat obesity either rely on highly variable dietary interventions that require strict adherence with inconsistent success rates, expensive pharmacologic interventions that require lifelong, costly, and frequent injections, or highly invasive surgical interventions that can have severe side effects. There is therefore an urgent need for the development of novel, safe and effective anti-obesity interventions.

[0012] SUMMARY

[0013] Thus, in accordance with the present disclosure, there is provided a microorganism that produces the metabolite A actoyl-phenylalanine (Lac-Phe) for use in the treatment of obesity or related metabolic disorders. Also provided is a microorganism engineered to overproduce, as compared to an otherwise equivalent non-engineered microorganism, A-lactoyl-phenylalanine (Lac-Phe). The microorganism may be supplemented with purified L(+)-lactic acid to boost Lac-Phe production, or supplemented with purified L(+)-phenylalanine to boost Lac-Phe production. The microorganism may be supplemented with purified L(+)-lactic acid and L(+)-phenylalanine to boost Lac-Phe production. The microorganism may express or overexpress, as compared to an otherwise equivalent non-engineered organism, L(+)-lactate dehydrogenase (IdhL) and / or carnosine dipeptidase 2 (CNDP2), any sequence homolog of these two genes, or any structural homolog of the protein products encoded by either of these two genes. The microorganism may express or overexpress, as compared to an otherwise equivalent non-engineered organism, bifunctional enzyme chorismate mutase / prephenate dehydratase (pheA). The microorganism may be supplemented with purified L(+)-lactic acid, L(+)-phenylalanine or both L(+)-lactic acid and L(+)-phenylalanine.

[0014] The microorganism may be Escherichia coli Nissle 1917, such as Escherichia coli Nissle 1917 further comprising both an introduced IdhL gene and an introduced CNDP2 gene, for example wherein in the IdhL and / or CNDP2 genes are carried in one or more episomal plasmids. The Escherichia coli Nissle 1917 may overexpress bifunctional enzyme chorismate mutase / prephenate dehydratase (pheA). The engineered Escherichia coli Nissle 1917 may be supplemented with L(+)-lactic acid, L(+)-phenylalanine or both L(+)-lactic acid and L(+)-phenylalanine. The amount of Lac-Phe produced by the microorganism may be up to 6888.309 ng / mL when measured in the supernatant of bacteria grown for 3 days in M9 media supplemented with 2mM MgSCU, O.lmM CaCh, 0.5% glucose and 0.2% casamino acids at 30°C in a shaking incubator. The Escherichia coli Nissle 1917 may comprise a knock-in of sfGFP into the chromosomal recA locus. The microorganism may further comprise a knockout mutation of an endogenous recA gene. The microorganism may be engineered to express a reporter gene such as “superfolder” GFP (sfgfp).

[0015] Also provided is a method of preventing weight gain in a subject or reducing the weight of an overweight or obese subject comprising administering to said subject the microorganism as described herein. The subject may be a mammal, a mouse, or a human. The subject may suffer from metabolic syndrome, type 2 diabetes, cardiovascular disease. The administering may be oral administration and / or may take place daily, every other day, every third day, twice a week, weekly, every other week, twice a month, or monthly. The subject may be further administered L(+)-lactic acid or L(+)-phenylalanine or both L(+)-lactic acid and L(+)-phenylalanine. The subject may be further administered at least one other anti-obesity drug. The subject may be further subjected to calorie restriction (low calorie diet: 800-1600 kcal / day or very low calorie diet: <800 kcal / day).

[0016] In another embodiment, there is provided a method of producing Lac-Phe comprising culturing the microorganism of any one of claims 2-16 and purifying Lac-Phe secreted by said microorganism. Culturing may comprise fermentation. A further embodiment comprises a method of preventing weight gain in a subject or reducing the weight of an overweight or obese subject comprising administering the purified Lac-Phe produced according to the aforementioned method, such as by oral capsular or intravenous administration. The subject may be further administered at least one other anti-obesity drug; and / or wherein the subject is further subjected to calorie restriction (low calorie diet: 800-1600 kcal / day or very low calorie diet: <800 kcal / day).

[0017] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number.

[0018] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0020] FIG. 1. Lac-Phe levels secreted by wild-type and engineered bacteria after 3 days of growth in M9 media. Control = GFP-positive E. coli Nissle; Lac-Pheop= GFP-positive E. coli Nissle transformed with pAG54 (overexpressing both CNDP2 and IdhL)

[0021] FIG.2. E. coli Nissle with sfGFP knocked into the rec A locus of the genome produces green fluorescence as measured by flow cytometry.

[0022] FIG. 3. Lac-Phe levels secreted by engineered bacteria overexpressing IdhL and CNDP2 or engineered bacteria overexpressing IdhL, CNDP2 and pheA after 3 days of growth in M9 media. Lac-Pheop= GFP-positive E. coli Nissle transformed with pAG54 (overexpressing both CNDP2 and IdhL),- Lac-Pheop+ pheAOE= GFP-positive E. coli Nissle transformed with pAG54 (overexpressing both CNDP2 and IdhE) and pAG66 (overexpressing pheA).

[0023] FIG. 4. Lac-Phe levels secreted by engineered bacteria overexpressing IdhL and CNDP2 supplemented with various amounts of L-phenylalanine. Lac-Pheop= GFP-positive E. coli Nissle transformed with pAG54 (overexpressing both CNDP2 and IdhE).

[0024] FIG. 5. Lac-Phe levels secreted by wild-type and engineered bacteria after 3 days of growth in M9 media. Control = Wild-type BW25113 E. coli', Lac-Pheop= BW25113 E. coli transformed with pAG54 (overexpressing both CNDP2 and IdhL)

[0025] FIG. 6. Lac-Phe levels secreted by engineered bacteria overexpressing IdhL and CNDP2 supplemented with various amounts of L-phenylalanine. Lac-Pheop= BW25113 E. coli transformed with pAG54 (overexpressing both CNDP2 and IdhL).

[0026] FIG. 7A and FIG. 7B. Oil Red O fat staining demonstrates that engineered Lac-Phe producing bacteria reduce obesity-associated fat content in a C. elegans model of diet-induced obesity. Control = BW29665 transformed with pJTL019 and pMJS021 (control plasmids expressing GFP and dsRed, respectively); Lac-Pheop= E. coli BW29665 transformed with pAG16 and pAG25 (plasmids expressing IdhL and CNDP2 respectively).

[0027] FIG. 8. Lac-Phe levels secreted by wild-type and engineered bacteria after 3 days of growth in M9 media. IdhLOE = IdhL overexpression, CNDP2OE = CNDP2 overexpression. E. caballus = Equus caballus, H. sapiens = Homo sapiens, M. musculus = Mus musculus, E. coli = Escherichia coli.

[0028] FIG. 9. Lac-Phe levels secreted by wild-type and engineered bacteria after 3 days of growth in M9 media. IdhLOE = IdhL overexpression, CNDP2OE = CNDP2 overexpression, pheAOE = pheA overexpression. H. sapiens = Homo sapiens

[0029] FIG. 10. Mice fed a high-fat (obesogenic) diet gavaged with engineered Lac-Phe producing bacteria have reduced food intake. EcN vector = mice gavaged with GFP-positive E. coli Nissle transformed with pAG43 (empty plasmid); EcN Lac-Pheop= mice gavaged with GFP-positive E. coli Nissle transformed with pAG54 (overexpressing both CNDP2 and IdhL).

[0030] FIG. 11. Mice fed a high-fat (obesogenic) diet gavaged with engineered Lac-Phe producing bacteria have reduced body weight. EcN vector = mice gavaged with GFP-positive E. coli Nissle transformed with pAG43 (empty plasmid); EcN Lac-Pheop= mice gavaged with GFP-positive E. coli Nissle transformed with pAG54 (overexpressing both CNDP2 and IdhL).

[0031] FIG. 12. AgRP neurons of mice fed a high-fat (obesogenic) diet gavaged with engineered Lac-Phe producing bacteria have reduced firing frequency. EcN vector = mice gavaged with GFP-positive E. coli Nissle transformed with pAG43 (empty plasmid); EcN Lac-PheOP = mice gavaged with GFP-positive E. coli Nissle transformed with pAG54 (overexpressing both CNDP2 and IdhL).

[0032] FIG. 13. AgRP neurons of mice fed a high-fat (obesogenic) diet gavaged with engineered Lac-Phe producing bacteria are more hyperpolarized (harder to fire). EcN vector = mice gavaged with GFP-positive E. coli Nissle transformed with pAG43 (empty plasmid); EcN Lac-PheOP = mice gavaged with GFP-positive E. coli Nissle transformed with pAG54 (overexpressing both CNDP2 and IdhL).

[0033] FIG. 14. GFP-positive colonies extracted from mice feces gavaged with experimental and control bacteria seeded on AMP LB plates. W4 Fri = control strain collected on a Friday the day after the last gavage (Thursday); E2 Fri = experimental strain collected on a Friday after the last gavage (Thursday); W4 Mon = control strain collected on a Monday 4 days after the last gavage (Thursday); E2 Mon = experimental strain collected on a Monday 4 days after the last gavage (Thursday)

[0034] FIG. 15. Colony PCR of bacteria seeded as described in Fig. 9. El -8 = experimental strain; Wl-6 = control strain. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0035] As discussed above, obesity remains a major health challenge and the current methods of addressing this condition have limitations. The engineering of gut bacteria capable of producing anti -obesity therapeutic molecules offers a non-invasive and cost-effective way of combating obesity. Additionally, bacteria are inexpensive to make and maintain in large quantities. Recently, a metabolite called 7V-lactoyl-phenylalanine (Lac-Phe) was identified as an exercise inducible molecule capable of reducing food intake and suppressing obesity driven by a high-fat diet. The inventors hypothesized that probiotic bacteria that could secrete excess levels of Lac-Phe could protect individuals from obesity. Their approach is to (1) engineer Escherichia coli Nissle (EcN), a gutcolonizing human probiotic organism, to produce and secrete a physiologically relevant level of Lac-Phe, and (2) employ these organisms to protect animals fed a high fat diet from developing obesity, insulin resistance and liver steatosis. This approach does not require individuals to adhere to a strict diet and is relatively hands off since the bacteria can colonize the gut and establish a stable presence.

[0036] Probiotic E. coli Nissle (EcN) bacteria were transformed with plasmids that cause the overexpression of two genes, IdhL and CNDP2. The enzyme coded by the former promotes the conversion of pyruvate to L-lactic add while the latter promotes the conversion of L-lactic acid to N-lactoyl-phenylalanine (Lac-Phe) a compound found to have anti-obesity properties in mice studies. They also knocked into the recA locus a gene that encodes “supeifolder" GFP (sfgfp). This results in the knock-out of recA, which encodes a protein critical for homologous recombination and also gives the engineered bacteria the ability to produce sfGFP protein. The knockout of recA should further stabilize the transformed plasmids and the production of sfGFP allows for easy tracking the bacteria. To further boost Lac-Phe production by the engineered bacteria, the researchers introduced a plasmid for the overexpression of pheA. This gene encodes for an enzyme previously unknown to be involved in L-phenylalanine production. They also demonstrate that supplementation with purified L-phenyalanine can also increase Lac-Phe production by the engineered bacteria. Finally, they show that by using a similar setup in the laboratory E. coli strain BW25113 they can engineer bacteria to produce even higher amounts of Lac-Phe.

[0037] These and other aspects of the disclosure are described in detail below. I. Obesity

[0038] Obesity is a medical condition, sometimes considered a disease, in which excess body fat has accumulated to such an extent that it can potentially have negative effects on health. People are classified as obese when their body mass index (BMI) — a person's weight divided by the square of the person's height — is over 30 kg / m2; the range 25-30 kg / m2is defined as overweight. Some East Asian countries use lower values to calculate obesity. Obesity is a major cause of disability and is correlated with various diseases and conditions, particularly cardiovascular diseases, type 2 diabetes, obstructive sleep apnea, certain types of cancer, and osteoarthritis.

[0039] Obesity has individual, socioeconomic, and environmental causes. Some known causes are diet, physical activity, automation, urbanization, genetic susceptibility, medications, mental disorders, economic policies, endocrine disorders, and exposure to endocrine-disrupting chemicals.

[0040] While a majority of obese individuals at any given time attempt to lose weight and are often successful, maintaining weight loss long-term is rare. There is no effective, well-defined, evidence-based intervention for preventing obesity. Obesity prevention requires a complex approach, including interventions at societal, community, family, and individual levels. Changes to diet as well as exercising are the main treatments recommended by health professionals. Diet quality can be improved by reducing the consumption of energy-dense foods, such as those high in fat or sugars, and by increasing the intake of dietary fiber, if these dietary choices are available, affordable, and accessible. Medications can be used, along with a suitable diet, to reduce appetite or decrease fat absorption. If diet, exercise, and medication are not effective, a gastric balloon or surgery may be performed to reduce stomach volume or length of the intestines, leading to feeling full earlier, or a reduced ability to absorb nutrients from food.

[0041] Obesity is a leading preventable cause of death worldwide, with increasing rates in adults and children. In 2022, over 1 billion people were obese worldwide (879 million adults and 159 million children), representing more than a doubling of adult cases (and four times higher than cases among children) registered in 1990. Obesity is more common in women than in men. Today, obesity is stigmatized in most of the world. Conversely, some cultures, past and present, have a favorable view of obesity, seeing it as a symbol of wealth and fertility. The World Health Organization, the U.S., Canada, Japan, Portugal, Germany, the European Parliament and medical societies, e.g. the American Medical Association, classify obesity as a disease. Others, such as the UK, do not. Obesity increases the risk of many physical and mental conditions. These comorbidities are most commonly shown in metabolic syndrome, a combination of medical disorders which include diabetes mellitus type 2, high blood pressure, high blood cholesterol, and high triglyceride levels. A substantial body of research supports an association between obesity and a chronic, “smoldering” inflammatory state. Obesity is associated with overproduction of inflammatory cytokines and chronic activation of inflammatory signaling pathways, including the NF-kB pathway. Chronic inflammation in adipose tissue is linked with the development of insulin resistance in skeletal muscle. Chronic activation of the NF-KB pathway has been shown to induce insulin resistance and NF-KB inhibition has been proposed as a therapeutic strategy for the treatment of Type 2 diabetes.

[0042] In a fashion analogous to the development of insulin resistance, obesity has been associated with the development of resistance to the action of leptin. Leptin, a peptide hormone, has complex biological effects but one important site of action is the mediobasal hypothalamus. This structure of the brain is known to exert control over feeding behavior and energy homeostasis. Recently, oxidative stress and activation of the NF-KB pathway in the hypothalamus were shown to be linked to hypothalamic insulin and leptin resistance. Activation of the antioxidant transcription factor Nrf2 is known to inhibit NF-KB activity, and Nrf2 activation by a semisynthetic triterpenoid has been reported to inhibit the development of obesity in mice fed on a high-fat diet.

[0043] One aspect of the present disclosure concerns new methods and reagents for the treatment and prevention of obesity. As discussed above, obesity is typically defined by body mass index (BMI) and may be further evaluated in terms of fat distribution via the waist-hip ratio and total cardiovascular risk factors. BMI is related to both percentage body fat and total body fat. BMI is calculated by dividing the subject’s mass by the square of his or her height (in metric units: kilograms / meters2). The definitions established by the World Health Organization (WHO) in 1997 and published in 2000 are listed below: BMI Classification s

[0044] k 18.5 underweight

[0045] : 18.5 24.9 normal weight |

[0046] 25.0 29.9 overweight

[0047] : 3().() 34.9 class I obesity ;

[0048] ?35.0-39.9 class II obesity

[0049] 40.0 class III obesity

[0050]

[0051] Thus, in one aspect, the present disclosure relates to the reduction of a person's classification from class III to a lower classification, the reduction of a person's classification from class II to a lower classification, the reduction of a person's classification from class I to a lower classification, and the reduction of a person's classification from class overweight to normal weight.

[0052] IL Probiotic Bacteria and Methods of Bacterial Engineering

[0053] A. Probiotic Bacteria

[0054] Probiotics are live microorganisms promoted with claims that they provide health benefits when consumed, generally by improving or restoring the gut microbiota. Probiotics are considered generally safe to consume, but may cause bacteria-host interactions and unwanted side effects in rare cases. There is some evidence that probiotics are beneficial for some conditions, such as helping to ease some symptoms of irritable bowel syndrome (IBS). However, many claimed health benefits, such as treating eczema, lack substantial scientific support.

[0055] The first discovered probiotic was a certain strain of bacillus in Bulgarian yoghurt, called Lactobacillus bulgaricus. The discovery was made in 1905 by Bulgarian physician and microbiologist Stamen Grigorov. The modern-day theory is generally attributed to Russian Nobel laureate Elie Metchnikoff, who postulated around 1907 that yoghurtconsuming Bulgarian peasants lived longer.

[0056] A growing probiotics market has led to the need for stricter requirements for scientific substantiation of putative benefits conferred by microorganisms claimed to be probiotic. Although some evidence claimed benefits are marketed towards using probiotic, such as reducing gastrointestinal discomfort, improving immune health, relieving constipation, or avoiding the common cold, such claims are strain-specific and cannot be extrapolated to other strains. As of 2019, numerous applications for approval of health claims by European manufacturers of probiotic dietary supplements have been rejected by the European Food Safety Authority for insufficient evidence of beneficial mechanism or efficacy.

[0057] An October 2001 report by the World Health Organization (WHO) defines probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host.” Following this definition, a working group convened by the Food and Agriculture Organization (FA0) / WH0 in May 2002 issued the Guidelines for the Evaluation of Probiotics in Food. A consensus definition of the term probiotics, based on available information and scientific evidence, was adopted after the aforementioned joint expert consultation between the FAO of the United Nations and the WHO. This effort was accompanied by local governmental and supra-governmental regulatory bodies’ requirements to better characterize health claims substantiations. That first global effort was further developed in 2010; two expert groups of academic scientists and industry representatives made recommendations for the evaluation and validation of probiotic health claims. The same principles emerged from those two groups as were expressed in the "Guidelines" of FAO / WHO in 2002. This definition, though widely adopted, is not acceptable to the European Food Safety Authority because it embeds a health claim that is not measurable. A group of scientific experts assembled in Canada in October 2013 to discuss the scope and appropriate use of the term "probiotic", adjusting the definition to be “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.”

[0058] Probiotics have received renewed attention in the 21st century from product manufacturers, research studies, and consumers. Their history can be traced to the first use of cheese and fermented products, which were well-known to the Greeks and Romans who recommended their consumption. The fermentation of dairy foods represents one of the oldest techniques for food preservation.

[0059] The original modern hypothesis of the positive role played by certain bacteria was first introduced by Russian scientist and Nobel laureate Elie Metchnikoff, who in 1907 suggested that it would be possible to modify the gut microbiota and to replace harmful microbes with useful microbes. Metchnikoff, at that time a professor at the Pasteur Institute in Paris, proposed the hypothesis that the aging process results from the activity of putrefactive (proteolytic) microbes producing toxic substances in the large bowel. Proteolytic bacteria such as Clostridia, which are part of the normal gut microbiota, produce toxic substances including phenols, indols, and ammonia from the digestion of proteins. According to Metchnikoff, these compounds were responsible for what he called “intestinal autointoxication,’’ which would cause the physical changes associated with old age.

[0060] At that time, milk fermented with lactobacillales were known to inhibit the growth of proteolytic bacteria because of the low pH produced by the fermentation of lactose. Metchnikoff had also observed that certain rural populations in Europe, for example in Bulgaria and the Russian steppes, who lived largely on milk fermented by lactic-acid bacteria, were exceptionally long-lived. Based on these observations, Metchnikoff proposed that consumption of fermented milk would "seed" the intestine with harmless lactic-acid bacteria and decrease the intestinal pH, and that this would suppress the growth of proteolytic bacteria. Metchnikoff himself introduced in his diet sour milk fermented with the bacteria he called "Bulgarian Bacillus" and believed his health benefited. Friends in Paris soon followed his example and physicians began prescribing the sour-milk diet for their patients.

[0061] Bifidobacteria was first isolated from a breastfed infant by Henry Tissier, who also worked at the Pasteur Institute. The isolated bacterium named Bacillus bifidus communis was later renamed to the genus Bifidobacterium. Tissier found that bifidobacteria are dominant in the gut microbiota of breast-fed babies and he observed clinical benefits from treating infant diarrhea with bifidobacteria.

[0062] During an outbreak of shigellosis in 1917, German professor Alfred Nissle isolated a strain of Escherichia coli from the feces of a soldier who was not affected by the disease. Methods of treating infectious diseases were needed at that time when antibiotics were not yet available, and Nissle used the E. coli Nissle 1917 strain in acute gastrointestinal infectious salmonellosis and shigellosis. Since that time, it has been widely studied as a probiotic and several marketed probiotics include it and naturally colonizes the human intestines and has positive probiotic properties.

[0063] In 1920, Rettger and Cheplin reported that Metchnikoff s “Bulgarian Bacillus,” later called Lactobacillus delbrueckii subsp. bulgaricus, could not live in the human intestine. They conducted experiments involving rats and human volunteers, feeding them with Lactobacillus acidophilus. They observed the disappearance of the pathogenic protist Balantidium coli as well as of other gas-producing bacteria. Rettger further explored the possibilities of L. acidophilus, and reasoned that bacteria originating from the gut were more likely to produce the desired effect in this environment. In 1935, certain strains of L. acidophilus were found very active when implanted in the human digestive tract. Contrasting antibiotics, probiotics were defined as microbially derived factors that stimulate the growth of other microorganisms. In 1989, Roy Fuller suggested a definition of probiotics that have been widely used: “A live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance.” Fuller's definition emphasizes the requirement of viability for probiotics and introduces the aspect of a beneficial effect on the host.

[0064] The term “probiotic” originally referred to microorganisms that have effects on other microorganisms. The concept of probiotics involved the notion that substances secreted by one microorganism stimulated the growth of another microorganism. The term was used again to describe tissue extracts that stimulated microbial growth. The term probiotics was taken up by Parker, who defined the concept as, “Organisms and substances that have a beneficial effect on the host animal by contributing to its intestinal microbial balance.” Later, the definition was greatly improved by Fuller, whose explanation was very close to the definition used today. Fuller described probiotics as a “live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance.” He stressed two important claims for probiotics: the viable nature of probiotics and the capacity to help with intestinal balance.

[0065] In the following decades, intestinal lactic-acid bacterial species with alleged health-beneficial properties were introduced as probiotics, including Lactobacillus rhamnosus, Lactobacillus casei, and Lactobacillus johnsonii.

[0066] B. Bacterial Engineering

[0067] Bacteria can be genetically engineered by combining DNA synthesis with molecular cloning to construct extrachromosomal vectors such as plasmids or episomes, or linear or circular DNA elements that can be integrated into the chromosome thereby modifying the chromosome sequence. These customized DNA vectors can be transformed into bacteria via methods such as conjugation, electroporation, and chemical transformation. The presence of the vector can be selected for via addition of antibiotic against which an antibiotic resistance marker encoded on the vector provides resistance, the ability of the vector to complement an auxotrophy such as the lack of an amino acid biosynthesis gene, or other means. The engineered vector can carry genetic expression cassettes for genes of interest, such as enzymes that convert native or supplemented substrates to products of interest. Those vectors can be maintained in vitro and in vivo via antibiotic selection, though antibiotic selection may not be necessary for use in an in vivo environment. The probiotic bacterium E. coli Nissle 1917 can be purchased under the commercial trade name Mutaflor from pharmacies or other vendors of consumer foods and goods. Other strains of E. coli bacteria can be sourced from repositories such as the American Type Culture Collection. Other bacterial organisms can be procured from similar repositories. Gene and protein sequences can be obtained from publicly available databases including the NCBI. Every gene or protein sequence in these databases is generally associated with an accession number. The accession numbers for the protein sequences used in the experiments described below are: Bacillus coagulans IdhL ADN38376; Equus caballus CNDP2: XP_023503757; E. coli Nissle / ? / / zM:CAD6006359; Homo sapiens CNDP2:NP_001161971. These sequences may need to be codon optimized for expression in the organism of interest. Codon optimization can be performed using computational tools such as those offered by DNA synthesis companies. Codon optimized genes can be synthesized by said companies. Plasmids and plasmid parts can be obtained from repositories such as Addgene. For example, the backbone for the plasmid, pAG54, was obtained from Addgene (Plasmid #: 117852).

[0068] III. Formulation and Administration

[0069] The present disclosure provides pharmaceutical compositions. Such compositions comprise a prophylactically or therapeutically effective amount of the engineered probiotic organisms, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0070] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, intra-rectal, vaginal, topical or delivered by mechanical ventilation.

[0071] Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0072] Generally, ingredients of compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0073] Genetically engineered bacteria can be administered to a subject orally as a liquid, a concentrated suspension, a semi-solid or solid inside of a digestible pill, or inside of a capsule such as a hydrogel that is stable for longer periods of time in the body.

[0074] IV. Examples

[0075] The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. Example 1 - Materials and Methods

[0076] Plasmid and strain construction: The sequences of the genes encoding for CNDP2 and IdhL were taken from a publicly available database (NCBI) containing the Bacillus coagulans. Equus caballus, and human genomes. The inventors then codon optimized these sequences for E. coli expression and sent them to Twist Bioscience for synthesis. The inventors placed the CNDP2 and IdhL genes under the control of the constitutively active promoters J23110 and J23100, respectively. They used the high-copy number ColEl / pMBl / pBR322 / pUC origin of replication to increase the number of plasmids produced by the cell and hence, the level of expression of the genes. The plasmids, named pAG54 (E. caballus version of CNDP2) and pAGlll (H. sapiens version of CNDP2), also carry the ampicillin-resistance gene as a selection marker. All cloning was carried out using Gibson assembly. The inventors knocked superfolder GFP (sfgfp) into the recA locus using the CRISPR-Cas9 system as previously described (Jiang et al., 2015). All transformations into E. coli Nissle strains were carried out by electroporation. All transformations into other E. coli strains were carried out chemically.

[0077] The sequence for pheA was taken from the E. coli Nissle genome available on NCBI. The inventors placed it under the control of the constitutively active promoter apFAB295 on a plasmid with a medium-copy number pl 5 A origin of replication. The plasmid, named pAG66, also carries the chloramphenicol-resistance gene as a selection marker. They transformed this plasmid into the E. coli Nissle strain carrying pAG54 or pAGlll. To further boost Lac-Phe production, the inventors also introduced a plasmid for the overexpression of pheA which promotes L-phenylalanine productions.

[0078] The preferred strain comprises IdhL (from Bacillus coagulans), CNDP2 (from Homo sapiens) and pheA (from Escherichia coli). In addition to sfGFP knocked into the recA locus may also be included.

[0079] Measurement of Lac-Phe secreted by bacteria: Control GFP-positive E. coli Nissle bacteria (GFP+ EcN) and GFP+ EcN carrying the CNDP2 and IdhL overexpressing plasmid, pAG54, were grown overnight in LB media in a 37°C shaker. The next day, bacteria were diluted 1:100 in M9 minimal media supplemented with 2mM MgSCU, O.lmM CaCh, 0.5% glucose and 0.2% casamino acids and grown for 2 hours in a 37°C shaker until the culture’s OD reached approximately 0.2-0.3. The bacteria were then diluted 1:1000 in M9 minimal media supplemented as described above and grown for 3 days in a 30°C shaker in a tube that has been tightly capped. For L-phenylalanine supplementation experiments, the media was also supplemented with 0 mM, 0.5 mM, 1 mM or 2 mM L-phenylalanine. The bacteria were then spun down at 4000xg for 10 minutes at 4°C and the supernatant was transferred to a clean, prelabelled, microcentrifuge tube. The samples were then either immediately analyzed for Lac-Phe levels by Liquid Chromatography-Mass Spectrometry (LCMS) or stored in -20°C and analyzed by LCMS at another date.

[0080] Caenorhabditis elegans fat experiments: Gravid N2 C. elegans worms were bleached and the embryos rocked in S-buffer at room temperature for 18 hours to synchronize the hatchlings. After estimating the concentration of worms, ~200 worms were seeded on 6cc NGM plates (in the presence or absence of lOmg / mL of fructose) that had been seeded with the appropriate bacteria (BW29665 transformed with pMJS021 and pJTLO 19 control plasmids or BW29665 transformed with IdhL-overexpressing plasmid, pAG16, and CNDP2-overexpressing plasmid, pAG025 which overproduce Lac-Phe). These bacteria were prepared as described above with the exception that after centrifugation at 20°C, the pellet was resuspended in S-buffer and then seeded on NGM plates (200pL per plate). After seeding, worms were grown at 20°C for roughly 48-55 hours until they reached the young adult stage which was determined by observing the worms under a microscope. The worms were then washed off the plate, fixed in 60% isopropanol and then stained with 0.5% Oil Red O overnight at 25°C. The next day, stained worms were washed 3 times in S-buffer with 0.01% Triton-X before being mounted on a slide and imaged using a Nikon Al microscope.

[0081] Mouse model fat experiments: 8-week-old mice are treated with antibiotics for 1 week to clear their gut and promote the colonization of the engineered bacterium. Mice are then gavaged with either control or engineered Lac-Phe producing bacteria daily over a 1-week period. Body weight and food intake is measured before the first gavage, at the end of the last gavage and weekly over a period of 10 weeks. At the end of the experiment an electrophysiology assay was performed to assess AgRP neuronal activity.

[0082] Example 2 - Results

[0083] E. coli Nissle 1917, a known probiotic, was transformed with a plasmid containing constructs for the constitutive expression of two genes required for Lac-Phe production: IdhL (obtained from the Bacillus coagulans genome) and CNDP2 (obtained from the Equus caballus genome). The gene IdhL encodes for an enzyme that promotes the conversion of pyruvate to L-lactate and CNDP2 encodes for an enzyme that promotes the conversion of L-lactate and L-phenylalanine to Lac-Phe. The inventors also knocked in a gene that encodes sfgfp into the recA locus of the transformed bacteria. This results in the knock-out of recA, which encodes a protein critical for homologous recombination, while simultaneously giving the engineered bacteria the ability to produce sfGFP. The knockout of recA is expected to further stabilize the plasmid, and the production of sfGFP allows tracking of the bacteria by fluorescence measurement.

[0084] As shown in FIG. 1, engineered E. coli Nissle secrete Lac-Phe 103-fold more than wildtype E. coli Nissle. The amount of Lac-Phe produced by the engineered bacteria is physiologically relevant. It is close to the amount of Lac-Phe observed in the serum of exercising mice (~2.1 pM produced by bacteria vs ~2 pM in the serum of exercising mice (Li et al., 2022). As shown in Fig. 8, the human version of CNDP2 leads to the highest increase in Lac-Phe production.

[0085] sfGFP was knocked into this bacterium to allow easy tracking of the bacteria in the gut and feces of animals (FIG. 2). The researchers also demonstrate that the overexpression of pheA, in addition to IdhL and CNDP2 overexpression, increases Lac-Phe production to 1540 ± 61.16 (S.E.) ng / mL (6.5 pM), a significant increase compared to the amount produced by E. coli Nissle bacteria overexpressing IdhL and CNDP2 alone (FIG. 3). Furthermore, supplementation of bacteria overexpressing IdhL and CNDP2, with L-phenylalanine can significantly increase Lac-Phe production reaching 2,662 ± 35.48 (S.E.) ng / mL (11.22 pM) (Fig. 4). Adding pheA overexpression to the strain comprising human version of CNDP2 also leads to a larger increase in Lac-Phe production (see Fig. 9). Finally, performing the same manipulations in the laboratory E. coli strain BW25113 results in an even bigger increase in Lac-Phe production demonstrating the potential of engineering E. coli bacteria for Lac-Phe production using the setup described. BW25113 bacteria overexpressing IdhL and CNDP2 produce 1517 ± 62.78 (S.E.) ng / mL (6.4 pM) of Lac-Phe (Fig. 5) and this can be further increased to 6761 ± 69.64 (S.E.) ng / mL (28.5 pM) by supplementing the media with 2mM L-phenyl alanine (Fig. 6).

[0086] Oil Red O is a red dye that stains fat and increased Oil Red O staining means increased fat levels. FIGS. 7A-B shows engineered Lac-Phe producing bacteria reduce fat accumulation in C. elegans fed a high-fructose diet, a model for diet-induced obesity.

[0087] As shown in Fig. 10, mice fed a high-fat diet gavaged with engineered Lac-Phe producing bacteria have reduced food intake. As shown in Fig. 11, mice fed with high-fat diet gavaged with engineered Lac-Phe producing bacteria have reduced body weight.

[0088] The engineered bacteria reduce AgRP neuronal activity in mouse DIO model. Fig. 12 demonstrates that AgRP neurons of mice fed a high-fat (obesogenic) diet gavaged with engineered Lac-Phe producing bacteria have reduced firing frequency. Fig. 13 demonstrates that AgRP neurons of mice fed a high-fat (obesogenic) diet gavaged with engineered Lac-Phe producing bacteria are more hyperpolarized (harder to fire).

[0089] In mice that have been gavaged twice a week, GFP -positive bacteria can be detected in the feces of mice after the last gavage and before the first gavage of the week (4 days after the last gavage) demonstrating that the bacteria can survive in the mouse gut for at least a few days (see Fig. 14). Additionally, these mice were confirmed to have bacteria carrying the correct plasmids after going through the mouse gut (4 days after the last gavage) (see Fig. 15).

[0090] The inventors will further optimize the Lac-Phe producing E. coll Nissle strain. The inventors will knock out several genes (adhE, pta, IdhA, lldP) that may lead to increased flux towards L-lactic acid production: higher L-lactic acid levels may lead to higher Lac-Phe levels.

[0091] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

[0092] V. References

[0093] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0094] Li et al., Nature, 606:785-790 (2022).

[0095] Chen et al., JCI, 124(8): 3391-3406 (S2014).

Claims

WHAT IS CLAIMED IS;1. A microorganism that produces the metabolite A-lactoyl-phenylalanine (Lac-Phe) for use in the treatment of obesity or related metabolic disorders.

2. A microorganism engineered to overproduce, as compared to an otherwise equivalent non-engineered microorganism, A-lactoyl-phenylalanine (Lac-Phe).

3. The microorganism of claim 1 or claim 2, supplemented with purified L(+)-lactic acid to boost Lac-Phe production, or supplemented with purified L(+)-phenylalanine to boost Lac-Phe production.

4. The microorganism of claims 1 and 2, supplemented with purified L(+)-lactic acid and L(+)-phenylalanine to boost Lac-Phe production.

5. The microorganism of claim 2, wherein the microorganism expresses or overexpresses, as compared to an otherwise equivalent non-engineered organism, L(+)-lactate dehydrogenase (IdhL) and / or camosine dipeptidase 2 (CNDP2), any sequence homolog of these two genes, or any structural homolog of the protein products encoded by either of these two genes.

6. The microorganism of claim 5, wherein the microorganism expresses or overexpresses, as compared to an otherwise equivalent non-engineered organism, bifunctional enzyme chorismate mutase / prephenate dehydratase (pheA).I. The microorganism of claim 5 or claim 6, supplemented with purified L(+)-lactic acid, L(+)-phenylalanine or both L(+)-lactic acid and L(+)-phenylalanine.

8. The microorganism of claim 1 or claim 2, wherein the microorganism is Escherichia coli Nissle 1917.

9. The microorganism of claim 8, wherein the Escherichia coli Nissle 1917 further comprises both an introduced IdhL gene and an introduced CNDP2 gene.

10. The microorganism of claim 9, wherein in the IdhL and / or CNDP2 genes are carried in one or more episomal plasmids.I I. The microorganism of any one of claims 8-10, wherein the Escherichia coli Nissle 1917 overexpresses bifunctional enzyme chorismate mutase / prephenate dehydratase (pheA).

12. The microorganism of any one of claims 8-11, wherein the engineered Escherichia coli Nissle 1917 is supplemented with L(+)-lactic acid, L(+)-phenylalanine or both L(+)-lactic acid and L(+)-phenylalanine.

13. The use of claim 2, or the microorganism of any one of claims 2-11, wherein the amount of Lac-Phe produced is up to 6888.309 ng / mL when measured in the supernatant of bacteria grown for 3 days in M9 media supplemented with 2mM MgSC , 0.1 mM CaCh, 0.5% glucose and 0.2% casamino acids at 30°C in a shaking incubator.

14. The microorganism of any one of claims 7-12, wherein the Escherichia coli Nissle 1917 comprises a knock-in of sfGFP into the chromosomal recA locus.

15. The microorganism of claim 2, further comprising a knock-out mutation of an endogenous recA gene.

16. The microorganism of claim 2, wherein the microorganism is engineered to express a reporter gene such as “superfolder” GFP (sfgfp).

17. A method of preventing weight gain in a subject or reducing the weight of an overweight or obese subject comprising administering to said subject the microorganism of any one of claims 2-16.

18. The method of claim 17, wherein the subject is a mammal, a mouse, or a human.

19. The method of claim 17 or claim 18, wherein the subject is overweight.

20. The method of claim 17 or claim 18, wherein the subject is obese.

21. The method of claim 17 or claim 18, wherein the subject suffers from metabolic syndrome, type 2 diabetes, cardiovascular disease.

22. The method of any one of claims 18-21, wherein the administering is oral administration.

23. The method of any one of claims 17-22, wherein the administering takes place daily, every other day, every third day, twice a week, weekly, every other week, twice a month, or monthly.

24. The method of any one of claims 17-23, wherein the subject is further administered L(+)-lactic acid or L(+)-phenylalanine or both L(+)-lactic acid and L(+)-phenylalanine.

25. The method of any one of claims 17-24, wherein the subject is further administered at least one other anti-obesity drug.

26. The method of any one of claims 17-25, wherein the subject is further subjected to calorie restriction (low calorie diet: 800-1600 kcal / day or very low calorie diet: <800 kcal / day).

27. A method of producing Lac-Phe comprising culturing the microorganism of any one of claims 2-16 and purifying Lac-Phe secreted by said microorganism.

28. The method of claim 27, wherein culturing comprises fermentation.

29. A method of preventing weight gain in a subject or reducing the weight of an overweight or obese subject comprising administering the purified Lac-Phe produced according to the method of claim 27 or 28 to the subject, such as by oral capsular or intravenous administration.

30. The method of claim 29, wherein the subject is further administered at least one other anti-obesity drug; and / or wherein the subject is further subjected to calorie restriction (low calorie diet: 800-1600 kcal / day or very low calorie diet: <800 kcal / day).

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