Abstract
Engineered probiotics are a kind of new microorganisms produced by modifying original probiotics through gene editing. With the continuous development of tools and technology progresses, engineering renovation of probiotics are becoming more diverse and more feasible. In the past few years there have been some advances in the development of engineered probiotics that will benefit humankind. This review briefly introduces the theoretical basis of gene editing technology and focuses on some recent engineered probiotics researches, including inflammatory bowel disease, bacterial infection, tumor and metabolic diseases. It is hoped that it can provide help for the further development of genetically modified microorganisms, stimulate the potential of engineered probiotics to treat intractable diseases, and provide new ideas for the diagnosis of some diseases or some industrial production.
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Background
Probiotics have been studied for decades. The word "probiotics" first appeared in 1974, and, in 2001, the Food and Agriculture Organization of the United Nations and the World Health Organization defined probiotics as "live microorganisms which when administered in adequate amounts confer a health benefit on the host" [1, 2]. Probiotics play a great role in many aspects, such as preventing and treating various clinical diseases, improving the intestinal microenvironment, inducing immune regulation, preventing physiological stress, inhibiting the growth of pathogens, and improving the barrier function of the intestinal epithelium, etc. [3]. Probiotics are therefore of great interest to the scientific community.
In recent years, genome sequencing has become more affordable and some of the tools for editing and modifying microbial genomes have become more powerful, enable us to engineer probiotics according to our own ideas, so that we can develop customized probiotics [4]. By means of gene editing, probiotics can have a variety of beneficial properties, and can treat specific diseases, which is beneficial to human health. Genetic engineering of microbial strains such as probiotics is a promising research (Table 1). We are at a turning point in the research of probiotics. Engineered probiotics may become new ideas and new methods to solve some problems. This review briefly introduces the theoretical basis of gene editing technology and focuses on some recent engineered probiotics studies on diseases, including inflammatory bowel disease, bacterial infection, tumor and metabolic diseases. We hope to provide help for the further development of engineered probiotics.
Theoretical basis of engineered probiotics-gene editing
Through gene editing, the existing probiotics are modified to obtain the desired new probiotics. Such engineering modification allow us to directly verify whether the genetic material, proteins and functional roles of these novel microorganisms have been changed as desired. Gene editing technology continues to develop, from homologous recombination to the first-generation Zincfinger Nucleases (ZFNs) technology, to the second-generation Transcription Activator-like Effector Nucleases (TALEN) technology and to the more popular third-generation Clustered Regularly Spaced Short Palindromic Repeats CRISPR associated (CRISPR-Cas) technology in recent years [30]. Among them, ZFN and TALEN were selected as the method of the year by Nature Methods, CRISPR gene editing technology was selected as the best breakthrough of 2015 by Science news, and CRISPR-Cas9 technology won the 2020 Nobel Prize in Chemistry, which has a revolutionary impact on life sciences. This part will mainly give a brief introduction to these three generations of technologies.
These gene-editing tools introduce Double Strand Break (DSB) into target genes, DNA Repair is induced by an error-prone Non-homologous End Joining (NHEJ) pathway or Homology Directed Repair (HDR) [31]. ZFNs and TALEN are two artificial restriction endonucleases which use zincfinger DNA domains or TAL-effect DNA domains to edit or cut specific target DNA [32]. However, there have been some problems in the use of these two technologies, such as the lack of specificity of ZFN and easy introduction of non-targeted mutations [33]. The construction of standard ZFNs and TALENs is time-consuming and laborious [34]. Therefore, although ZFNs and TALENs have been used in gene editing of human, animal, and plant cells since 2002 and 2011, these limiting factors have hindered their wide application to a certain extent [35]. Since the end of 2012, researchers have turned their attention to the CRISPR technology, which has more powerful editing efficiency and is simpler and more flexible to use, so that the application of this technology brings a whole new direction to gene editing [36].
CRISPR and related Cas genes are considered to be part of the bacterial immune system, which is a self-protective sequence of bacteria and can provide phage resistance [37]. At the molecular level, bacteria integrate the virus gene from the first invasion into their CRISPR spacer, and when the virus invasions again, CRISPR transcribed to produce the precursor CRISPR RNA (pre-crRNA), which is further processed to contain crRNA that matches the viral gene sequence. CrRNA recognizes homologous sequences of viral genomes and mediates binding to viral genes and cleavage of Cas proteins [38, 39]. The CRISPR-Cas system is generally classified by the Cas protein gene adjacent to CRISPR. The currently reported CRISPR-Cas system is mainly divided into 2 classes, 6 types and 33 subtypes [40]. The nucleases Cas9 and Cas12a (Cpf1) in the 2 classes of Cas protein family are more widely used which are easy to edit to identify specific DNA sequences [41]. Among them, the type II CRISPR-Cas9 system is currently the more commonly used protein for genome editing [42].The CRISPR-Cas9 system is composed of Cas9 protein, crRNA and trans-activating RNA (tracrRNA). The Cas9 protein is the only protein with DNA catalytic activity among the many CaS proteins in Streptococcus thermophilus [43], It snips double-stranded DNA at sequence targets through crRNA matching [30]. By artificial design, crRNA and tracrRNA can be converted into small Guide RNA (sgRNA), which can guide Cas9 to perform site-specific DNA cutting, gene knockout and insertion [44].
CRISPR technology can be used not only for the study of loss of function such as CRISPR knock out (CRISPRKO) and CRISPR interference or inhibition (CRISPRi), but also for the screening study of gain of function such as CRISPR activation (CRISPRa) [30]. The development of gene editing technologies especially CRISPR provides essential supports for the emergence of the next generation of probiotics.
Research and application of engineered probiotics
Advances in gene editing technology provide new possibilities for gene editing of probiotics. Engineered probiotics have developed rapidly in recent years. Researchers have conducted a variety of biomedical studies on engineered probiotics for disease diagnosis and treatment. In particular, several engineered probiotics have also entered the clinical trial stage (Table 2). Scientists have also developed engineered microorganisms that are relevant to industrial applications. These studies are briefly discussed below.
Engineered probiotics and inflammatory bowel disease
Microbiota imbalance is closely related to the development of inflammatory bowel disease (IBD). Probiotics have been shown to improve the microbiome of the microbiota by changing the intestinal environment and inhibiting the growth of harmful bacteria, also, they can prevent inflammatory diseases from further affecting the host immune system, and have positive significance for the regulation of inflammation [45, 46]. Advances in gene editing technology have increased the link between probiotics and inflammatory bowel disease.
Specific probiotics have been designed to diagnose and treat IBD. Around 2017, researchers have used engineered bacteria to diagnose IBD [15,16,17]. Based on biomarkers such as thiosulfate, tetrasulfate, and nitric oxide (NO), they constructed stably genetically engineered bacteria as recognition elements of biosensors to diagnose IBD. In recent years, research on the use of engineered probiotics for the treatment of IBD has also emerged. Pichet Praveschotinunt and others of Harvard University genetically engineered E. coli Nissle 1917 (ECN) to produce a curly fiber matrix that promotes intestinal epithelial integrity, and the trefoil factor (TFF) component in the matrix promotes intestinal barrier function and epithelial repair [18]. Their study confirmed that the engineered ECN could in situ produce proteins that were protective against dextran sodium sulfate (DSS)-induced colitis model mice. This provides an engineered probiotic treatment for IBD. Engineered ECN is also used by many other researchers to treat intestinal-related inflammation. Researchers engineered ECN to express interleukin-10 (IL-10), ketone body (R)-3-hydroxybutyrate (3HB) and Schistosoma immunomodulatory protein Sj16 and other substances. These substances can protect the intestinal mucosa by promoting the growth of probiotics, inhibiting the growth of harmful bacteria, improving the intestinal microenvironment, and downregulating inflammatory response-related cells or proteins, so as to achieve the purpose of treating and relieving the symptoms of intestinal inflammation [12, 19, 28]. However, these studies have only made progress in mouse models. We believe that after further clinical trials, these engineered ECNs must have the opportunity to be used for the relief and treatment of human IBD. Benjamin M. Scott of the University of Maryland and Cristina Gutierrez-Vazquez of Harvard Medical School conducted a valuable study based on yeast. They developed a self-regulating engineered yeast probiotic that can express human P2Y2 purinergic receptors and they linked the activation of the probiotic’s P2Y2 receptor with the secretion of ATP-degrading enzymes, enabling it to sense pro-inflammatory molecules and neutralize pro-inflammatory molecules through self-regulation and secretion of corresponding proteins [20]. This self-regulating engineered yeast probiotic inhibits intestinal inflammation and reduces intestinal fibrosis and dysbiosis in IBD mouse models and we think their self-regulating strategy may provide a new way for engineered probiotics to treat IBD or other diseases. Liu Jinyao's team at Shanghai Jiaotong University School of Medicine in China synthesized a polydopamine nano immunosuppressant and used it to coat E. coli Nissle 1917 to inhibit the excessive immune response in the local tissues of mouse colitis and regulate the intestinal microbiota to promote the reversal of inflammation [21]. Liu et al.'s study showed that applying immunosuppressants on the surface of beneficial bacteria can promote improved colitis responses in mice. Through their research, can we engineer probiotics to express immunosuppressants to wrap themselves to treat inflammatory disease?
Engineered probiotics and bacterial infections
Bacterial infections have a great impact on people’s health and nearly one million people die from bacterial infections worldwide each year, and the lack of bacterial resistance and new antibiotics has become an obstacle to solving the problem of bacterial infections [47]. Researchers have found some new ways to alleviate this problem.
Some researchers have successfully developed engineered probiotics. The American team of Arun K. Bhunia designed an engineered Lactobacillus casei strain that can produce Listeria adhesion protein [22]. This strain is colonized in the intestine of mice, competitively reducing the colonization of Listeria mucosa and systemic transmission, protecting mice from fatal infections. They can also enhance the intestinal immune regulation function by accumulating intestinal mucosal regulatory T cells, CD11c+ dendritic cells and natural killer cells. The method of engineering L. casei ATCC334 to reduce Listeria infection and protect the intestinal tract is worth learning. Some researchers have designed and constructed an engineered probiotic based on E. coli Nissle 1917, which can specifically target and kill the two most common vancomycin-resistant Enterococcus, and significantly reduce the number of Enterococcus faecalis and Enterococcus faecium in the feces of model mice [29]. Some researchers have engineered Saccharomyces boulardii so that the engineered bacteria secrete a fusion protein (ABAB) that can neutralize 4 different Clostridium difficile toxins [5]. The preventive administration of these bacteria can significantly relieve the inflammation and tissue damage related to the infection of Clostridium difficile in the mouse intestines mucosa, therefore reduce the mortality of these mice. In addition, researchers have developed some engineered probiotics to better prevent and prevent and diagnose bacterial infections. Some researchers have genetically modified Lactobacillus lactis to produce engineered probiotics that recognize the cholera autoinducer 1 (CAI-1) produced by Vibrio cholerae and cause color changes in feces [11]. Another researcher has constructed an engineered lactic acid bacteria that can be used to detect real-time changes in autoinducer peptide-I (AIP-I) produced by Staphylococcus aureus [27]. The researchers also engineered E. coli Nissle 1917 to specifically kill pathogens by detecting the autoinducer N-acyl homoserine lactone (AHL) of Pseudomonas aeruginosa and releasing antibiotics and anti-biofilm enzymes [26]. This series of studies may provide a new direction for the treatment and prevention of drug-resistant bacterial infections. It is hoped that the safety and effectiveness of these engineered probiotics can be further confirmed, and these products can serve the clinic as soon as possible.
Engineered probiotics and tumors
The discussion about the treatment of cancer by bacteria has existed for decades [48, 49]. Some probiotics can induce anti-cancer effects by enhancing the apoptosis of cancer cells and preventing oxidative stress [50,51,52]. With the development of modern gene editing technology, it has become possible to design probiotics that can play a positive role in fighting cancer. Researchers are constantly searching and developing new probiotics to fight tumors.
In the study of Candice R. Gurbatri and others at Columbia University, researchers used synthetic biology methods to modify bacteria and obtained an engineered E. coli strain subtype called "SLIC" [6]. SLIC colonizes tumor cells, when it proliferates to a certain extent, it can spontaneously lyse and then release PD-L1 and CTLA-4 nano antibodies. So that SLIC can promote T cell activation, increase memory T cells, and enhance anti-tumor immune response, thereby effectively inhibiting tumor growth, causing tumor regression, and inhibiting tumor metastasis. Their research has positive significance for enhancing the effect of tumor treatment and inhibiting tumor cell proliferation. In a similar study, researchers in China engineered E. coli Nissle 1917 to target the angiogenic inhibitor TUM-5 and tumor suppressor p53 to the anaerobic tumor region [7]. This treatment significantly inhibited the growth of transplanted tumor in mice, and their idea of using the tumor tropism of some bacteria to target tumors provides a direction for the use of anti-tumor drugs. An engineered E. coli Nissle 1917 that can be targeted to colonize tumors has also been published recently. It can convert the metabolic waste produced by tumors into L-arginine that enhances the anti-tumor immune response, and can effectively enhance the therapeutic effect of programmed cell death protein-1 and its ligand inhibitors on mouse tumors [8]. An engineered E. coli Nissle 1917 that can achieve precise tumor treatment in vivo through the control of light color changes has also been successfully constructed(9). These studies have provided some help for the treatment of tumors. Now there are two engineering probiotics related to the treatment of tumors in clinical trials (NCT04167137 and NCT04025307). The most notable of these is the SYNB1891 strain obtained by the engineering of E. coli Nissle 1917 by Synlogic (NCT04167137). This strain can produce cyclic di-AMP to stimulate the interferon gene pathway, and then trigger innate immunity by activating antigen-presenting cells to present tumor antigens [10]. Their phase1 clinical trials are underway, and we believe that the emergence of this engineered probiotic drug will be seen clinically in the near future.
Engineered probiotics and metabolic diseases
The content of metabolism-related substances is generally in a relatively stable state in the body. When the biochemical process in the body is hindered, the accumulation or lack of certain metabolites can cause diseases [53,54,55]. These diseases are difficult to treat, and often require long-term control of living and eating habits, which brings serious economic burdens and life pressures to patients [51]. Engineered probiotics can break down or transform these accumulated substances to help treat such diseases.
Phenylketonuria (PKU) is caused by mutations in the phenylalanine hydroxylase gene. Patients with phenylalanine hydroxylase deficiency cannot effectively decompose phenylalanine, causing phenylalanine to accumulate in the blood, which seriously affects the patients’ health [53]. The researchers expressed the phenylalanine lyase gene in Lactobacillus reuteri 100-23C, so that the engineered probiotic strain can produce phenylalanine lyase, which can reduce the blood phenylalanine of the PKU mouse model [24]. Their experiment provides an idea for the treatment of phenylketonuria with engineered probiotics. However, the strain may colonize the host body for a long time, which is also a problem waiting to be solved. Synlogic also conducted a similar study. They inserted the genes encoding phenylalanine ammonia lyase and l-amino acid deaminase into the genome of E. coli Nissle 1917, and constructed a strain of E. coli named SYNB1618 [25]. This engineered probiotic strain can consume phenylalanine in the gastrointestinal tract and will not colonize the intestinal tract. It has clinical safety (NCT03516487). And they also extended the model used to predict the therapeutic potential of SYNB1618 based on this study, which combined in vitro experiments and knowledge of human physiology to predict the degree of plasma phenylalanine reduction by SYNB1618 [56]. This facilitates the use of the biologic SYNB1618. Follow-up clinical trials for SYNB1618 are also underway (NCT04534842 and NCT04984525), and it is hoped that this bioactive drug can bring good news to patients with phenylketonuria.
Mutations in the alanine glyoxylate aminotransferase gene can cause hyperoxalic acid. The accumulation of glyoxylic acid can lead to the increase of oxalate and even the production of urinary stones [54]. Synlogic has also constructed an orally engineered E. coli strain called SYNB8802, which can ingest oxalic acid in the gastrointestinal tract to reduce urinary oxalic acid levels and reduce kidney damage caused by hyperoxaluria. Results from a Phase 1a study of this trial demonstrated the strain's safety profile, and the later trials are still in progress (NCT04629170). Novome has also developed a drug named NOV-001 for the treatment of intestinal hyperoxalic acid. This drug is composed of engineered strain NB1000S and plant-derived polysaccharide NB2000P. It is now undergoing clinical trials to verify its efficacy (NCT04909723). In our opinion, engineered probiotics could be a good way to treat diseases in combination with other drugs.
Ammonia is a highly neurotoxic metabolite. Impaired ammonia clearance occurs when urea cycle enzyme deficiency or liver cell dysfunction occur. If hyperammonemia is not treated in time, coma or even death may occur [55]. Researchers genetically modified the oral probiotic E. coli Nissle1917 to obtain an engineered strain named SYNB1020. This strain can convert ammonia into L-arginine, reduce the ammonia content in the intestine of a mouse model of hyperammonemia, and improve the survival rate of mice [14]. However, in patients with cirrhosis, SYNB1020 was discontinued due to lack of efficacy (plasma ammonia AUC) compared to placebo (NCT03447730). Clinical trials in patients with urea cycle disorders are still underway (NCT03179878), hoping to produce a good result.
Researchers have also developed engineered probiotics for some other metabolic-related diseases. Some researchers have constructed an engineered commensal Lactobacillus gasseri that secretes glucagon-like peptide-1 (GLP-1) to improve diabetes [13]. GLP-1 secreted by this strain can reprogram intestinal cells into insulin-producing cells, thereby reducing hyperglycemia in mice. Similar studies have shown an engineered EcN-GM that induces GLP-1 expression, which may have a beneficial effect on obesity, hyperglycemia and liver steatosis [23]. Precigen ActoBio has genetically modified Lactococcus lactis to obtain a engineered bacteria named AG019. AG019 can deliver proinsulin and interleukin-10 to the gastrointestinal mucosa tissues, reduce or eliminate the damage to pancreatic β cells, and potentially stabilize or improve endogenous insulin production. Their experiment Also in progress (NCT03751007).
Engineered bacterias and industrial production
Engineered probiotics can not only be used for the diagnosis and treatment of some diseases, but also be closely related to industrial production. Jaewoo Son and others of the Korea Academy of Science and Technology used the CRISPRi system to develop an engineered Leuconostoc citrate [57]. The strain down-regulated the expression of two genes and introduced a co-expression operon to increase the production of riboflavin. Recently, researchers conducted similar studies on E. coli LS31T to increase the production of riboflavin [58]. Thus the industrial production of riboflavin, as a food additive, may find a new direction. Photosynthetic cyanobacteria convert carbon dioxide into monosaccharides, and some researchers have designed an engineered E. coli that can further convert the products of photosynthetic cyanobacteria into 2,3-butanediol [59]. The study of this engineered microbe is worth digging deeper, and perhaps in the future it can really provide propellant for rocket launches and be used as return material for interstellar travel, just as researchers designed. Studies have reported an engineered E. coli strain that can produce benzoic acid from plant-derived glucose [60]. Their research may provide a safer new idea for the production of benzoic acid as a preservative. Some researchers engineered Clostridium ljungdahlii to produce isopropanol, 3-hydroxybutyric acid, ethanol and other substances [61], which research provides conditions for the use of single-carbon source materials to produce a large number of chemicals. There are also some studies providing a platform for the production of some industrial substances such as L-citrulline, flavan-3-ols, β-carotene, etc. by designing E. coli, lactic acid bacteria and yeast into engineered bacteria [62,63,64,65,66].
Other research on engineered bacterias
Zbiotics has added an acetaldehyde dehydrogenase gene that decomposes acetaldehyde in Bacillus subtilis, which can further convert the acetaldehyde converted from ethanol into acetic acid to reduce the harm of alcohol. They used the spores of this strain to conduct a 90-day repeated administration toxicological evaluation on mice, and proved that the bacterium has no side effects [67]. Zbiotics is the first company in the world to produce and sell genetically engineered probiotic products. Although their products are already on sale, the safety of this bacteria in the human body may still need to be demonstrated. To use CRISPR for genetic modification of microorganisms. Some researchers have constructed engineered Lactobacillus plantarum WCFS1 to produce n-acetylglucosamine [68]. Some researchers have found that the modified Brucella ATCC MYA-796 can ferment galactose faster [69, 70]. There are also more and more researchers constructing gene editing platforms for engineered bacteria such as Corynebacterium glutamicum, Candida parapsilosis, Lactobacillus casei, Lactobacillus reuteri, Bifidobacterium, Clostridium butyricum, and Escherichia coli [71,72,73,74,75,76,77,78]. They successfully applied CRISPR-related technology to the genetic modification of microorganisms. Their research will certainly provide help for the application of this technology in engineered probiotics, and the engineering of probiotics based on this technology will surely make it easier to produce the desired engineered probiotics.
Prospects
The emerging gene-editing methods used to develop synthetic engineered probiotics have great potential for the treatment and diagnosis of different types of human diseases, and engineered probiotics may become a new method for the treatment of cancer, inflammation, infection and some other diseases. The engineered probiotics designed by the researchers are more effective, have fewer side effects and are more affordable than traditional treatments or wild-type strains, benefiting more patients and families. The advantages of engineering probiotics such as stability, specificity, preference, low cost, and relative safety may make them a new choice for the treatment of many of the diseases mentioned above and more. However, the application of engineered probiotics still faces a series of challenges. For example, many microorganisms have few genetic tools that can be manipulated, and the microorganisms currently used for research design are only a small part of the vast family of microorganisms, which leads to the restriction of probiotic modification to a few strains. There are still a few engineered probiotics that go from the laboratory to the clinic, and even if they enter clinical trials, it cannot be guaranteed that it can achieve the intended effect, which hinders the efforts of some researchers to construct engineered probiotics. Some argue that genetically modified organisms (GMOs) are harmful, preventing the effective deployment of engineered probiotic therapies.
Conclusions
Despite these limitations in the research and application of engineered probiotics, we cannot ignore their potential role in improving human health. Success and failure are inevitable in the research and development of engineered probiotics. We need more research to find the possibility of success, not only in the research of various probiotic modification tools, but also in-depth understanding of disease pathology, so as to make it easier for us to design the desired probiotics. The efficacy of engineered probiotics is a problem that we must consider, but before these probiotics undergo clinical transformation, safety issues must be resolved. It is necessary to ensure that the guidelines established by various health regulatory agencies are followed to design and construct safe and effective probiotics. We believe that with the increasing understanding of the human microbiome and disease mechanisms, the rapid development of gene editing technology, safe, stable and effective engineered probiotics will surely shine in many fields.
Availability of data and materials
Not available.
Change history
24 May 2022
A Correction to this paper has been published: https://doi.org/10.1186/s12934-022-01820-6
References
Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, et al. Expert consensus document The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol. 2014;11 (8):506–14.
Binda S, Hill C, Johansen E, Obis D, Pot B, Sanders ME, et al. Criteria to Qualify Microorganisms as “Probiotic” in Foods and Dietary Supplements. Front Microbiol. 2020;11:1662.
Suez J, Zmora N, Segal E, Elinav E. The pros, cons, and many unknowns of probiotics. Nat Med. 2019;25 (5):716–29.
O’Toole PW, Marchesi JR, Hill C. Next-generation probiotics: the spectrum from probiotics to live biotherapeutics. Nat Microbiol. 2017;2:17057.
Chen K, Zhu Y, Zhang Y, Hamza T, Yu H, Saint Fleur A, et al. A probiotic yeast-based immunotherapy against Clostridioides difficile infection. Sci Transl Med. 2020;12:567.
Gurbatri CR, Lia I, Vincent R, Coker C, Castro S, Treuting PM, et al. Engineered probiotics for local tumor delivery of checkpoint blockade nanobodies. Sci Transl Med. 2020;12:530.
He L, Yang H, Tang J, Liu Z, Chen Y, Lu B, Intestinal probiotics E. coli Nissle, et al. as a targeted vehicle for delivery of p53 and Tum-5 to solid tumors for cancer therapy. J Biol Eng. 1917;2019 (13):58.
Canale FP, Basso C, Antonini G, Perotti M, Li N, Sokolovska A, et al. Metabolic modulation of tumours with engineered bacteria for immunotherapy. Nature. 2021;598 (7882):662–6.
Huizhuo P, Lianyue L, Gaoju P, Chunli H, Baona L, Yingying Z, et al. Engineered NIR light-responsive bacteria as anti-tumor agent for targeted and precise cancer therapy. Chem Eng J. 2021;426: 130842.
Leventhal DS, Sokolovska A, Li N, Plescia C, Kolodziej SA, Gallant CW, et al. Immunotherapy with engineered bacteria by targeting the STING pathway for anti-tumor immunity. Nat Commun. 2020;11 (1):2739.
Mao N, Cubillos-Ruiz A, Cameron DE, Collins JJ. Probiotic strains detect and suppress cholera in mice. Sci Transl Med. 2018;10:445.
Yan X, Liu XY, Zhang D, Zhang YD, Li ZH, Liu X, et al. Construction of a sustainable 3-hydroxybutyrate-producing probiotic Escherichia coli for treatment of colitis. Cell Mol Immunol. 2021;18 (10):2344–57.
Duan FF, Liu JH, March JC. Engineered commensal bacteria reprogram intestinal cells into glucose-responsive insulin-secreting cells for the treatment of diabetes. Diabetes. 2015;64 (5):1794–803.
Kurtz CB, Millet YA, Puurunen MK, Perreault M, Charbonneau MR, Isabella VM, et al. An engineered E coli Nissle improves hyperammonemia and survival in mice and shows dose-dependent exposure in healthy humans. Sci Transl Med. 2019;11:475.
Daeffler KN, Galley JD, Sheth RU, Ortiz-Velez LC, Bibb CO, Shroyer NF, et al. Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation. Mol Syst Biol. 2017;13 (4):923.
Riglar DT, Giessen TW, Baym M, Kerns SJ, Niederhuber MJ, Bronson RT, et al. Engineered bacteria can function in the mammalian gut long-term as live diagnostics of inflammation. Nat Biotechnol. 2017;35 (7):653–8.
McKay R, Ghodasra M, Schardt J, Quan D, Pottash AE, Shang W, et al. A platform of genetically engineered bacteria as vehicles for localized delivery of therapeutics: Toward applications for Crohn’s disease. Bioeng Transl Med. 2018;3 (3):209–21.
Praveschotinunt P, Duraj-Thatte AM, Gelfat I, Bahl F, Chou DB, Joshi NS. Engineered E. coli Nissle 1917 for the delivery of matrix-tethered therapeutic domains to the gut. Nat Commun. 2019;10 (1):5580.
Wang L, Liao Y, Yang R, Zhu Z, Zhang L, Wu Z, et al. An engineered probiotic secreting Sj16 ameliorates colitis via Ruminococcaceae/butyrate/retinoic acid axis. Bioeng Transl Med. 2021;6 (3): e10219.
Scott BM, Gutierrez-Vazquez C, Sanmarco LM, da Silva Pereira JA, Li Z, Plasencia A, et al. Self-tunable engineered yeast probiotics for the treatment of inflammatory bowel disease. Nat Med. 2021;27 (7):1212–22.
Li J, Hou W, Lin S, Wang L, Pan C, Wu F, et al. Polydopamine Nanoparticle-Mediated Dopaminergic Immunoregulation in Colitis. Adv Sci (Weinh). 2021;89:e2104006.
Drolia R, Amalaradjou MAR, Ryan V, Tenguria S, Liu D, Bai X, et al. Receptor-targeted engineered probiotics mitigate lethal Listeria infection. Nat Commun. 2020;11 (1):6344.
Ma J, Li C, Wang J, Gu J. Genetically Engineered Escherichia coli Nissle 1917 Secreting GLP-1 Analog Exhibits Potential Antiobesity Effect in High-Fat Diet-Induced聽Obesity Mice. Obesity (Silver Spring). 2020;28 (2):315–22.
Durrer KE, Allen MS, Hunt Herbing I. Genetically engineered probiotic for the treatment of phenylketonuria (PKU); assessment of a novel treatment in vitro and in the PAHenu2 mouse model of PKU. PLoS ONE. 2017;12 (5):e0176286.
Puurunen MK, Vockley J, Searle SL, Sacharow SJ, Phillips JA, Denney WS, et al. Safety and pharmacodynamics of an engineered E. coli Nissle for the treatment of phenylketonuria: a first-in-human phase 1/2a study. Nat Metab. 2021;3 (8):1125–32.
Hwang IY, Koh E, Wong A, March JC, Bentley WE, Lee YS, et al. Engineered probiotic Escherichia coli can eliminate and prevent Pseudomonas aeruginosa gut infection in animal models. Nat Commun. 2017;8:15028.
Lubkowicz D, Ho CL, Hwang IY, Yew WS, Lee YS, Chang MW. Reprogramming Probiotic Lactobacillus reuteri as a Biosensor for Staphylococcus aureus Derived AIP-I Detection. ACS Synth Biol. 2018;7 (5):1229–37.
Cui M, Pang G, Zhang T, Sun T, Zhang L, Kang R, et al. Optotheranostic Nanosystem with Phone Visual Diagnosis and Optogenetic Microbial Therapy for Ulcerative Colitis At-Home Care. ACS Nano. 2021;15 (4):7040–52.
Geldart KG, Kommineni S, Forbes M, Hayward M, Dunny GM, Salzman NH, et al. Engineered E. coli Nissle 1917 for the reduction of vancomycin-resistant Enterococcus in the intestinal tract. Bioeng Transl Med. 2018;3 (3):197–208.
Zhang M, Eshraghian EA, Jammal OA, Zhang Z, Zhu X. CRISPR technology: The engine that drives cancer therapy. Biomed Pharmacother. 2021;133:e45.
Yadav M, Shukla P. Efficient engineered probiotics using synthetic biology approaches: A review. Biotechnol Appl Biochem. 2020;67 (1):22–9.
Yadav R, Kumar V, Baweja M, Shukla P. Gene editing and genetic engineering approaches for advanced probiotics: A review. Crit Rev Food Sci Nutr. 2018;58 (10):1735–46.
Puchta H. Applying CRISPR/Cas for genome engineering in plants: the best is yet to come. Curr Opin Plant Biol. 2017;36:1–8.
Tang X, Lowder LG, Zhang T, Malzahn AA, Zheng X, Voytas DF, et al. A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants. Nat Plants. 2017;3:17018.
Manghwar H, Lindsey K, Zhang X, Jin S. CRISPR/Cas System: recent advances and future prospects for genome editing. Trends Plant Sci. 2019;24 (12):1102–25.
Bak RO, Gomez-Ospina N, Porteus MH. Gene Editing on Center Stage. Trends Genet. 2018;34 (8):600–11.
Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007;315 (5819):1709–12.
McGinn J, Marraffini LA. Molecular mechanisms of CRISPR-Cas spacer acquisition. Nat Rev Microbiol. 2019;17 (1):7–12.
Mekler V, Kuznedelov K, Severinov K. Quantification of the affinities of CRISPR-Cas9 nucleases for cognate protospacer adjacent motif (PAM) sequences. J Biol Chem. 2020;295 (19):6509–17.
Makarova KS, Wolf YI, Iranzo J, Shmakov SA, Alkhnbashi OS, Brouns SJJ, et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol. 2020;18 (2):67–83.
Liu R, Liang L, Freed EF, Gill RT. Directed Evolution of CRISPR/Cas Systems for Precise Gene Editing. Trends Biotechnol. 2021;39 (3):262–73.
Burgess DJ. CRISPR screens beyond Cas9. Nat Rev Genet. 2020;21 (5):273.
Adli M. The CRISPR tool kit for genome editing and beyond. Nat Commun. 2018;9 (1):1911.
Knott GJ, Doudna JA. CRISPR-Cas guides the future of genetic engineering. Science. 2018;361 (6405):866–9.
Tsai YL, Lin TL, Chang CJ, Wu TR, Lai WF, Lu CC, et al. Probiotics, prebiotics and amelioration of diseases. J Biomed Sci. 2019;26 (1):3.
Shamoon M, Martin NM, O’Brien CL. Recent advances in gut Microbiota mediated therapeutic targets in inflammatory bowel diseases: Emerging modalities for future pharmacological implications. Pharmacol Res. 2019;148: 104344.
Rappuoli R, Bloom DE, Black S. Deploy vaccines to fight superbugs. Nature. 2017;552 (7684):165–7.
Yaghoubi A, Khazaei M, Jalili S, Hasanian SM, Avan A, Soleimanpour S, et al. Bacteria as a double-action sword in cancer. Biochim Biophys Acta Rev Cancer. 2020;1874 (1): 188388.
Moghimipour E, Abedishirehjin S, Baghbadorani MA, Handali S. Bacteria and Archaea: A new era of cancer therapy. J Control Release. 2021;338:1–7.
Badgeley A, Anwar H, Modi K, Murphy P, Lakshmikuttyamma A. Effect of probiotics and gut microbiota on anti-cancer drugs: Mechanistic perspectives. Biochim Biophys Acta Rev Cancer. 2021;1875 (1): 188494.
Aggarwal N, Breedon AME, Davis CM, Hwang IY, Chang MW. Engineering probiotics for therapeutic applications: recent examples and translational outlook. Curr Opin Biotechnol. 2020;65:171–9.
Sehrawat N, Yadav M, Singh M, Kumar V, Sharma VR, Sharma AK. Probiotics in microbiome ecological balance providing a therapeutic window against cancer. Semin Cancer Biol. 2021;70:24–36.
Hillert A, Anikster Y, Belanger-Quintana A, Burlina A, Burton BK, Carducci C, et al. The Genetic Landscape and Epidemiology of Phenylketonuria. Am J Hum Genet. 2020;107 (2):234–50.
Zheng R, Li Y, Wang L, Fang X, Zhang J, He L, et al. CRISPR/Cas9-mediated metabolic pathway reprogramming in a novel humanized rat model ameliorates primary hyperoxaluria type 1. Kidney Int. 2020;98 (4):947–57.
Soria LR, Brunetti-Pierri N. Targeting autophagy for therapy of hyperammonemia. Autophagy. 2018;14 (7):1273–5.
Charbonneau MR, Denney WS, Horvath NG, Cantarella P, Castillo MJ, Puurunen MK, et al. Development of a mechanistic model to predict synthetic biotic activity in healthy volunteers and patients with phenylketonuria. Commun Biol. 2021;4 (1):898.
Son J, Jang SH, Cha JW, Jeong KJ. Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of Leuconostoc citreum and Its Application for Engineering Riboflavin Biosynthesis. Int J Mol Sci. 2020;21:16.
Liu S, Hu W, Wang Z, Chen T. Rational Engineering of Escherichia coli for High-Level Production of Riboflavin. J Agric Food Chem. 2021;69 (41):12241–9.
Kruyer NS, Realff MJ, Sun W, Genzale CL, Peralta-Yahya P. Designing the bioproduction of Martian rocket propellant via a biotechnology-enabled in situ resource utilization strategy. Nat Commun. 2021;12 (1):6166.
Luo ZW, Lee SY. Metabolic engineering of Escherichia coli for the production of benzoic acid from glucose. Metab Eng. 2020;62:298–311.
Jia D, He M, Tian Y, Shen S, Zhu X, Wang Y, et al. Metabolic Engineering of Gas-Fermenting Clostridium ljungdahlii for Efficient Co-production of Isopropanol, 3-Hydroxybutyrate, and Ethanol. ACS Synth Biol. 2021;10 (10):2628–38.
Sun C, Li G, Li H, Lyu Y, Yu S, Zhou J. Enhancing Flavan-3-ol Biosynthesis in Saccharomyces cerevisiae. J Agric Food Chem. 2021;69 (43):12763–72.
Jiang S, Wang D, Wang R, Zhao C, Ma Q, Wu H, et al. Reconstructing a recycling and nonauxotroph biosynthetic pathway in Escherichia coli toward highly efficient production of L-citrulline. Metab Eng. 2021;68:220–31.
Liu L, Yang D, Zhang Z, Liu T, Hu G, He M, et al. High-Efficiency Genome Editing Based on Endogenous CRISPR-Cas System Enhances Cell Growth and Lactic Acid Production in Pediococcus acidilactici. Appl Environ Microbiol. 2021;87 (20): e0094821.
Durmusoglu D, Al’Abri IS, Collins SP, Cheng J, Eroglu A, Beisel CL, et al. In Situ Biomanufacturing of Small Molecules in the Mammalian Gut by Probiotic Saccharomyces boulardii. ACS Synth Biol. 2021;10 (5):1039–52.
Jin Y, Yu S, Liu JJ, Yun EJ, Lee JW, Jin YS, et al. Production of neoagarooligosaccharides by probiotic yeast Saccharomyces cerevisiae var boulardii engineered as a microbial cell factory. Microb Cell Fact. 2021;20 (1):160.
Appala Naidu B, Kannan K, Santhosh Kumar DP, Oliver JWK, Abbott ZD. Lyophilized B subtilis ZB183 Spores: 90-Day Repeat Dose Oral (Gavage) Toxicity Study in Wistar Rats. J Toxicol. 2019;2019:3042108.
Zhou D, Jiang Z, Pang Q, Zhu Y, Wang Q, Qi Q. CRISPR/Cas9-Assisted Seamless Genome Editing in Lactobacillus plantarum and Its Application in N-Acetylglucosamine Production. Appl Environ Microbiol. 2019;85:21.
Liu JJ, Zhang GC, Kong II, Yun EJ, Zheng JQ, Kweon DH, et al. A Mutation in PGM2 Causing Inefficient Galactose Metabolism in the Probiotic Yeast Saccharomyces boulardii. Appl Environ Microbiol. 2018;84:10.
Liu JJ, Kong II, Zhang GC, Jayakody LN, Kim H, Xia PF, et al. Metabolic Engineering of Probiotic Saccharomyces boulardii. Appl Environ Microbiol. 2016;82 (8):2280–7.
Liu J, Wang Y, Lu Y, Zheng P, Sun J, Ma Y. Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum. Microb Cell Fact. 2017;16 (1):205.
Lombardi L, Turner SA, Zhao F, Butler G. Gene editing in clinical isolates of Candida parapsilosis using CRISPR/Cas9. Sci Rep. 2017;7 (1):8051.
Song X, Huang H, Xiong Z, Ai L, Yang S. CRISPR-Cas 9 (D10A) Nickase-Assisted Genome Editing in Lactobacillus casei. Appl Environ Microbiol. 2017;83:22.
Barrangou R, van Pijkeren JP. Exploiting CRISPR-Cas immune systems for genome editing in bacteria. Curr Opin Biotechnol. 2016;37:61–8.
Zuo F, Zeng Z, Hammarstrom L, Marcotte H. Inducible Plasmid Self-Destruction (IPSD) Assisted Genome Engineering in Lactobacilli and Bifidobacteria. ACS Synth Biol. 2019;8 (8):1723–9.
Goh YJ, Barrangou R. Portable CRISPR-Cas9 (N) System for Flexible Genome Engineering in Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus paracasei. Appl Environ Microbiol. 2021;87:6.
Zhou X, Wang X, Luo H, Wang Y, Wang Y, Tu T, et al. Exploiting heterologous and endogenous CRISPR-Cas systems for genome editing in the probiotic Clostridium butyricum. Biotechnol Bioeng. 2021;118 (7):2448–59.
Kan A, Gelfat I, Emani S, Praveschotinunt P, Joshi NS. Plasmid Vectors for in Vivo Selection-Free Use with the Probiotic E coli Nissle 1917. ACS Synth Biol. 2021;10 (1):94–106.
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We thank all the authors for their efforts in writing this article, and we thank all those who have been read or quoted by us.
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This work was supported by The National Nature Science Foundation of China (Grant No. 32170119, No. 31870135, No. 81860653 and No. 31600116) and Key Laboratory of Non-coding RNA and Drugs of Higher Education in Sichuan Province (No. FB19-05).
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JM and YL wrote the main manuscript text; CY, SD and XJ made substantial contributions to conception and design; XL, FC and XW took part in drafting the article or revising it critically for important intellectual. All authors read and approved the final manuscript.
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Ma, J., Lyu, Y., Liu, X. et al. Engineered probiotics. Microb Cell Fact 21, 72 (2022). https://doi.org/10.1186/s12934-022-01799-0
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DOI: https://doi.org/10.1186/s12934-022-01799-0