Introduction

Major chronic diseases, including metabolic diseases, digestive diseases and neurodegenerative diseases, bring about serious negative effects on human health, further influence the quality of life. In the past decade, microecological therapies targeting unbalanced intestinal flora have attracted wide attention in the biomedical research community. Common microecological therapies mainly include prebiotics therapy, probiotics therapy and fecal microbiota transplantation (FMT) therapy. Prebiotics are a type of substrate that can be selectively utilized by host microorganisms and beneficial to host [1]. They can enrich specific microorganisms, such as Bifidobacterium, and produce metabolic end products to improve the body health [2]. Probiotics is a kind of microorganism that can bring health benefits to the host when given in sufficient [3]. Its main products, probiotic dietary supplements and over-the-counter drugs, have been used to reduce symptoms of gastrointestinal dysfunction, improve immune system function and regulate energy metabolism [4,5,6,7]. However, until now, the effectiveness of prebiotics and probiotics therapies has controversy in many application scenarios [8, 9]. Wilson et al. found that prebiotics, such as β-galactose oligosaccharide and guar gum, could not significantly improve the symptoms of irritable bowel syndrome and other functional bowel patients [10]. Costeloe et al. found that Bifidobacterium brig-001 had no significant effect in preventing necrotizing enterocolitis and late-onset septicemia in very premature infants [11]. In addition, recent experimental results also showed that rhamnose-Lactobacillus GG and other probiotics had no valid evidence in alleviating acute gastroenteritis caused by common pathogens such as rotavirus [12] and preventing ventilator-associated pneumonia in critically ill patients [13]. FMT refers to transplantation of functional flora, obtained from healthy people feces, into the gastrointestinal tract of patients, for the reconstruction of new intestinal flora to achieve the purpose of treating diseases [14]. In 2013, FMT was first written into the treatment guidelines for recurrent or refractory Clostridium difficile infection [15]. In the past ten years, FMT has achieved outstanding curative effect in the treatment of CDI. However, as an immature clinical treatment, the safety and reproducibility of FMT need to be further verified [16].

By virtue of synthetic biology technology, engineered probiotics expressing enzymes, cytokines, antimicrobial peptides, hormones, antibodies and other specific products under the stimulation of physiological or pathological signals in the intestine to improve the intestinal microenvironment may be a promising development direction for the treatment of chronic diseases [17]. Compared with traditional drug therapy, engineered probiotics have higher flexibility, specificity, predictability and controllability. At present, Lactobacillus, especially Lactococcus lactis, are often used as expression hosts to deliver therapeutic drugs due to their high safety, which has a wide range of clinical application value. In addition, Bacillus and Escherichia coli are also common chassis microorganisms in the process of recombinant living biological drugs research [18, 19]. In recent years, with the development of synthetic biology, the genetic toolbox of microbial in vivo therapy has been expanded.

In this paper, engineered probiotics targeting metabolic diseases, digestive diseases and neurodegenerative diseases are reviewed and explored. The application and regulatory role of engineered probiotics in these major chronic diseases, and the opportunities and challenges of using engineered probiotics to treat major chronic diseases are discussed.

Engineered probiotics and metabolic diseases

Metabolic diseases refer to a class of diseases caused by abnormal metabolism of glucose, protein, lipid and other nutrients in the body, including obesity, diabetes and non-alcoholic fatty liver disease, etc. [20]. In recent years, with changes in dietary habits and lifestyle, the incidence of metabolic diseases has been increasing year by year [21]. More and more studies have shown that metabolic diseases are related to intestinal microecological disorders. Many studies have demonstrated the efficacy of engineered probiotics in the treatment of metabolic diseases.

Engineered probiotics and diabetes

Diabetes is a type of metabolic diseases with hyperglycemia as the common phenotype, including type 1 diabetes (T1DM) and type 2 diabetes (T2DM) [22]. In addition to lifestyle and dietary habits, there is increasing evidence that intestinal flora plays an important role in the development of diabetes [23].

Glucagon-like peptide-1 (GLP-1), a short incretin primarily produced by intestinal L-cells, reduces glucagon secretion, induces glucose-dependent insulin secretion, then decreases blood glucose [24]. In GLP-1 application, the short half-life is a major barrier [25]. In Zucker Diabetic Fatty (ZDF) rats, strain Lactococcus lactis transformed with the plasmid encoding GLP-1 resulting in a significant increase in insulin and decrease in blood glucose compared to the control [26]. The wide type GLP-1 was optimized with 8th serine substitution, which both were transformed into L. lactis. In db/db mice, the optimized L. lactis strain could significantly improve random glycemic control and reduce systemic inflammation [27]. Exendin-4 (Exd4) is a GLP-1 receptor agonist. Exd4 secreted by recombinant L. lactis enhanced glucose-dependent insulin synthesis and activated PI3-K/AKT signaling pathway [28].

Cytokines play a key role in connecting the risk factors that contribute to T1DM development and trigger the destructive action of T cells in pancreatic islets [29]. Recombinant L. lactis strain, controlling secretion of anti-inflammatory cytokine interleukin-10 (IL-10) and T1DM autoantigen GAD65370-57, could improve nonobese diabetes (NOD) by releasing proinsulin in the intestine of mice [30]. Combined expressed cytokines IL-4 and IL-10 in strain L. lactis MG1363 FnBPA+ was shown that the recombinant bacterial could reduce pancreatic islets-destruction and prevent hyperglycemia [31].

In addition, recombinant L. lactis expressing staphylococcal nuclease (SNase) was constructed to delay the onset of T1DM by exerting protective effects on pancreatic islets and relieving inflammation of the small intestine in NOD mice [32]; recombinant L. lactis expressing HSP65-6P277, mucosal administration of fusion protein of HSP65 with tandem repeats of P277, resulted in decreased blood glucose levels and reduced islet inflammation in NOD mice [33]; L. lactis secreting Lactobacillus sakei originated L-arabinose isomerase (L-AI) in fusion with the signal peptide usp45 had the ability to produce D-tagatose in vivo, showing good hypoglycemic effects on diabetic mice [34].

Engineered probiotics and obesity

Obesity is a chronic metabolic disease caused by multiple factors, which resulting in excessive accumulation of fat in the body for the harm of health [35]. Many studies have shown that the occurrence of obesity is closely related to the imbalance of intestinal flora. Further studies have shown that unbalanced intestinal flora can accelerate the formation of obesity by affecting the body’s energy intake and metabolism and stimulating pro-inflammatory response [36]. It may be a new approach to prevent and manage obesity to develop therapeutic methods for various factors affecting metabolism starting from intestinal microecology.

GLP-1 also can delay gastric emptying and control appetite [37]. Escherichia coli Nissle 1917 (EcN) secreting modified GLP-1 had potential beneficial effects on obesity, which may be related to the regulating of neuropeptide expression in the hypothalamus [38]. Engineered MG1363-pMG36e-GLP-1, constitutively secreted GLP-1, could improve obesity by increasing diversity of intestinal microbial and promoting oxidation of fatty acid probably [39].

In addition to GLP-1, the researchers also focused on oxyntomodulin (OXM), uncarboxylated or low-carboxylated OC (ucOC), fibroblast growth factor 21 (FGF21) and other hormone drugs for the treatment of obesity. A novel oral drug delivery system using Bifidobacterium as a carrier for the delivery of OXM and its analogue had been developed [40]. In overweight mice, strain B. longum transformed with OXM could reduce body weight, food intake, and level of plasma lipid. Strain Lactococcus lactis was engineered to produce and secrete recombinant mouse ucOC in the presence of the inducer nisin, which could treat obesity by triggering GLP-1 secretion in the small intestine [41]. Cao et al. synthesized L. lactis NZ3900/PNZ8149 system and realized the expression of human FGF21, which significantly reduced the body weight of mice and improved the overall homeostasis [42]. N-acyl phosphatidylethanolamines (NAPEs), precursors of N-acylethanolamides, are synthesized in the small intestine in response to reduce food intake and obesity [43]. Davies and his team have demonstrated that using Arabidopsis originated NAPEs synthase to modify EcN can significantly reduce body weight, hepatic triglycerides, fatty acid synthesis genes, and increase fatty acid oxidation genes in mice [44, 45]. In summary, the use of recombinant probiotics for in-situ delivery of anti-obesity drugs into the gut microbiota can potentially serve as an adjuvant therapy to retard development of obesity.

Engineered probiotics and NAFLD

Nonalcoholic fatty liver disease (NAFLD) has type 2 diabetes mellitus, dyslipidemia, and metabolic syndrome risk factors, led to lipid deposition, in more serious cases, even inflammation and fibrosis in the liver. Ling Zhi 8 (LZ8) is an immunomodulatory protein, possessing a broad range of pharmacological effects [46]. The influence of recombinant L. lactis expressing LZ8 was evaluated in a cholesterol-fed rabbit model. The recombinant L. lactis could inhibit IL-1 expression, decrease fat droplet deposits and inflammatory cells infiltration, and improve liver function [47].

Engineered probiotics and phenylketonuria

Phenylketonuria (PKU) is a metabolic disease caused by biallelic mutations in the PAH gene that result in an inability to convert phenylalanine (Phe) to tyrosine, elevated blood Phe levels and severe neurological complications if untreated [48]. Expressing L-amino acid deaminase (LAAD) and phenylalanine ammonia lyase (PAL) based on ECN, designated as SYNB1618, catalyzed the deamination of Phe to the non-toxic product trans-cinnamate. Administration of SYNB1618 to the Pahenu2/enu2 PKU mouse model and healthy Cynomolgus monkeys, SYNB1618 reduced blood Phe concentration, independent of dietary protein intake [49]. Lately, SYNB1618 was studied in healthy volunteers and patients with PKU adult, which alleviated the symptoms of PKU with safety and tolerability [50]. Then, to optimize whole cell PAL activity, mutant PAL libraries was screened and used in the construction of SYNB1934, which demonstrated a better performance in PKU adult compared to SYNB1618 [51].

Intestinal microecology-based therapeutics targeting metabolic diseases are summarized in Table 1.

Table 1 Intestinal microecology-based therapeutics for metabolic diseases
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Engineered probiotics and digestive diseases

Digestive diseases are a group of chronic intestinal diseases that are frequent and common, covering inflammatory bowel disease (IBD) and colorectal cancer. Although the specific pathogenesis remains unclear, studies have shown that unbalanced intestinal flora is an important cause of digestive diseases [53, 54]. Engineered probiotic therapies targeting unbalanced gut flora show great potential in the prevention and treatment of digestive diseases.

Engineered probiotics and IBD

IBD, covering ulcerative colitis (UC) and Crohn’s disease (CD), is a chronic intermittent disorder characterized by epithelial barrier damage and intestinal inflammation [55]. The intestinal delivery of therapeutic agents targeting inflammatory factors is recognized as a latent effective therapy for the treatment of IBD. Secreting system of L. lactis expressing IL-10 [52] and tumor necrosis factor α (TNFα) [56] were constructed, respectively. Daily oral administration resulted in local release of IL-10 and anti-TNF-α nanoantibodies in the colon, avoided side effects caused by systemic administration, and effectively alleviated colon inflammation levels in mice. Through a combination of directed evolution and synthetic gene circuits, engineered yeast for the treatment of IBD was introduced. The engineered yeast, expressing a human P2Y2 purinergic receptor, could activate the expression and secretion of adenosine triphosphate diphosphatase, and then provide local inflammation treatment [57]. 3-hydroxybutyrate (3HB) was also synthesized in EcN, which realized the treatment of mice colitis by regulating the structure of intestinal flora and increasing the content of short-chain fatty acids [58]. In addition, recombinant probiotics can also treat IBD by delivering protein drugs, such as insulin-like growth factor, transforming growth factor, schistosome immunoregulatory protein Sj16, superoxide dismutase, cystatin, etc. [59,60,61].

The damaged intestinal mucosal barrier is also a potential target for IBD treatment. Trefoil factors (TFFs) and epidermal growth factor (EGF) are two important mucosal interventions that promote cell migration and repair mucosal wounds in a relatively short time. However, when administered orally, TFF and EGF are easily degraded by digestive enzymes and adhere to small intestinal mucus, resulting in decreased bioavailability. It had been proved that recombinant probiotics producing TFF [62, 63] and EGF [64] generated from EcN could promote wound healing of intestinal mucosa and play a protective role in colitis.

Engineered probiotics and CRC

Colorectal cancer (CRC) originates in the colon or rectum [65]. Myrosinase was expressed in EcN, which successfully converted glucosinolate to raphanin with anticancer activity, inhibiting the proliferation of more than 95% cancer cells [66]. A new anticancer drug p8, isolated from Lactobacillus rhamnosus, was introduced into Pediococcus pentosaceus to construct a bacterial drug delivery system for CRC treatment. The results showed that the system had positive therapeutic effect in both CRC xenotransplantation and azoxymethane/DSS induced models [67]. Recombinant EcN, which converted glucose into anticancer drug 5-aminolevulinic acid (5-ALA), developed a new treatment for CRC in combination with photothermal therapy technology [68]. In conclusion, compared with traditional therapies, engineered probiotics have more innate advantages in CRC treatment, which can recognize cancer cells more intelligently and release ideal therapeutic components inside tumor tissues.

Intestinal microecology-based therapeutics targeting digestive diseases are summarized in Table 2.

Table 2 Intestinal microecology-based therapeutics for digestive diseases
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Engineered probiotics and neurodegenerative diseases

Neurodegenerative diseases are a group of complex psychological syndromes, mainly manifested as cognitive or behavioral disorders of different degrees [69]. At present, the pathogenesis of neurological diseases is not completely clear. Gut flora is considered as a potential target for treating neurological diseases. They are linked to the brain via a bidirectional gut-brain axis that influences brain development and regulates host behavior.

Engineered probiotics and AD

Alzheimer’s disease (AD) is a central nervous system disease characterized by progressive cognitive dysfunction and memory impairment [70]. Engineering probiotics that release brain beneficial substances in the intestine may be used as direct or auxiliary therapy for AD, becoming a new choice for clinical treatment of brain diseases. In addition to anti-sugar and fat reduction effects, GLP-1 can prevent the toxic effect of β-amyloid peptide by binding to receptors on the dendritic membrane or cell membrane of nerve cells, thereby preventing or reversing the neurodegenerative process. Engineered probiotic L. lactis cremoris with high GLP-1 expression had improved spatial learning and memory impairments, reduced inflammation in the brain and reduced beta-amyloid buildup in the hippocampus [71]. The improvement of these indexes was mainly related to the growth inhibition of enterococcus and other pathogens, and the destruction of phosphorylation of MAPKs and PI3K/Akt pathway [72]. Another study used L. lactis strain carrying pExu plasmid encoding the human p62 protein for the treatment of AD. By introducing pExu plasmid, memory was ameliorated, the ubiquitin-proteasome system was modulated, amyloid peptides level was reduced levels, and neuronal oxidative and inflammatory processes were diminished [73]. In conclusion, engineered probiotics have shown a broad prospect in the prevention and treatment of AD, but so far this kind of research has mostly stayed at the cellular level, so it is necessary to further carry out in vitro and in vivo verification and mechanism exploration for engineered probiotics.

Engineered probiotics and PD

Similarly with AD, Parkinson’s disease (PD) are also neurodegenerative diseases characterized by progressive degeneration of the central nervous system, and few medications are available to halt the progression of PD [74]. Via the probiotics effects of L. lactis MG1363, engineered strain MG1363-pMG36e-GLP-1 was constructed which continuously express GLP-1, and reduced motor dysfunction in PD mice [71, 75]. The possible mechanism targeted at the TLR4/NF-κB signaling pathway, which activating the Keap1/Nrf2/GPX4 signaling pathway to suppress ferroptosis by up-regulating FSP1 and down-regulating ACSL4. High-throughput sequencing results showed that recombinant strain reduced the abundance of Proteus and Enterococcus pathogens, and increased the abundance of Akkermansia, Akkermansia muciniphila, Sutterella and Oscillospira probiotics at the genus level.

Intestinal microecology-based therapeutics targeting neurodegenerative diseases are summarized in Table 3.

Table 3 Intestinal microecology-based therapeutics for neurodegenerative diseases
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Discussion

The human gut microbiota is a key target for the prevention and treatment of major chronic diseases. Many studies have emphasized the important role of prebiotics, probiotics and FMT therapies targeting intestinal microorganisms in the prevention and treatment of major chronic diseases. However, due to the low safety and poor controllability problems, the therapeutic effects of these traditional microecology-based treatments are not always reliable. As an alternative, engineered probiotics developed by synthetic biology technology show higher robustness and controllability, and has become an important component of the next generation of microecology-based treatments. To date, utilization of synthetic biology in intestinal microecology-based therapeutics has been applied in the treatment of metabolic diseases (e.g. diabetes, obesity, nonalcoholic fatty liver disease and phenylketonuria), digestive diseases (e.g. inflammatory bowel disease and colorectal cancer), and neurodegenerative diseases (e.g. Alzheimer’s disease and Parkinson’s disease). Brief schematic diagram is depicted in Fig. 1.

Fig. 1
figure 1

Brief schematic diagram of engineered probiotics for the treatment of major chronic diseases

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In addition, other chronic diseases need to pursue for intervention strategy which suitable for the application of synthetic biology. For example, depression, as one common cause of suicide, has no direct and effective treatment currently [76]. Studies have shown a significant negative correlation between gamma-aminobutyric acid (GABA) and depression [77]. A recombinant E. coli, synthesizing GABA by colocating glutamate synthase, glutamate decarboxylase and GABA transporter, is constructed [78], which may be as a potential therapeutics for the treatment of depression.

Except for application of synthetic biology in production of corresponding medicaments directly, the utilization of synthetic biology can promote the colonization and growth of engineered probiotics in the gut, which may enhance the therapeutic effect of engineered probiotics. By using synthetic biology to display ligands on the surface of probiotics and express bio-membrane formation or adhesin [79] can improve the adhesiveness of engineered probiotics on intestinal mucosa and improve the intestinal colonization of engineered probiotics, thereby protecting engineered probiotics from stomach acid and improving their robustness. Combinatorial engineering probiotics simultaneously producing corresponding medicaments and protecting factors may grow up a new strategy for intestinal microecology-based therapeutics for chronic diseases.

At present, using synthetic biology, the customized transformation of single bacteria has been preliminarily realized. However, considering the problems such as low load, weak anti-interference ability and limited execution of complex function of single engineered probiotics, artificial intestinal flora is expected to become a new generation of microecological therapy. The application of synthetic biology to the construction of artificial intestinal flora has a unique advantage, which can modify microorganisms according to the intestinal microecological environment of different individuals to build synthetic intestinal flora, and detect a variety of disease markers to develop precise diagnostic products, so as to carry out personalized treatment and maintain intestinal health without causing damage to other cells or tissues. But in fact, until now, it is just our beautiful vision. In practice, the research of synthetic microflora targeting intestinal microecology, then for the treatment of chronic diseases still faces a series of opportunities and challenges.

Although synthetic biology plays a unique role in the application of intestinal microecology-based therapeutics for major chronic diseases, there are still many problems to be solved. How to improve production of corresponding medicaments, how to ensure the safety of engineered probiotics, and how to standardize the construction strategy have not been systematically proved. The development of CRISPR-Cas [80] and Mobile-CRISPRi [81] and other tools may provide effective methods for the metabolic engineering to engineer probiotics to increase production. Furthermore, this kind of research only remains at the application stage of how to use synthetic biology in intestinal microecology-based therapeutics in cellular level and model mice, which has not been really used in the clinical treatment of major chronic diseases. In the future, it is necessary to deepen the crossover of synthetic biology, microecology and other disciplines, make full use of the advantages of each discipline to further develop the construction of probiotics biosynthesizing corresponding medicaments, and finally design a universal method to help the development of human health.

Conclusion

Correcting intestinal microecological imbalance has become one of the core strategies to treat chronic diseases. Some traditional microecology-based therapies targeting intestine, such as prebiotic therapy, probiotic therapy and fecal microbiota transplantation therapy, have been used in the prevention and treatment of clinical chronic diseases, which still facing low safety and poor controllability problems. The development of synthetic biology technology has promoted the development of intestinal microecology-based therapeutics for chronic diseases, which exhibiting higher robustness and controllability, and become an important part of the next generation of microecological therapy. Evidence proposed that synthetic biology has been applied in the intestinal microecology-based therapeutics for chronic diseases, covering metabolic diseases (e.g. diabetes, obesity, nonalcoholic fatty liver disease and phenylketonuria), digestive diseases (e.g. inflammatory bowel disease and colorectal cancer), and neurodegenerative diseases (e.g. Alzheimer’s disease and Parkinson’s disease). This review summarizes the application of synthetic biology to construct engineered probiotics for the improvement of intestinal microecology to treat major chronic diseases and discusses the opportunities and challenges in the above process, providing clinical possibilities of engineered probiotics applied in microecological therapies.