1. Introduction

The human gut microbiome is the microbial ecosystem containing billions of microbes and acts as an untapped resource for improving health (Fan & Pedersen, 2021). Microbiome functions include providing digestion and absorption of nutrients, modulatory role on immune response, and prevention or treatment of metabolic disease (Fan & Pedersen, 2021). Dysbiosis or loss of traditional microorganisms' composition is part of the etiology surrounding the aforementioned metabolic maladies (Boulangé et al., 2016). These diseases, affecting millions worldwide, carry a significant burden of morbidity and healthcare expenditure (Wang et al., 2021a). Traditional probiotics, such as Lactobacillus as well as Bifidobacterium species, exhibit health-promoting efficacy due to enhancement of gut barrier function as well as immunomodulation (Swanson et al., 2020). Nonetheless, transient benefits, variable efficacy, as well as non-specific action restrict traditional probiotics as therapies in complex metabolic disorders (Suez et al., 2019).

Synthetic biology, as a recently emerging interface of genetic engineering, systems biology, as well as bioinformatics, allows designing next-generation probiotics (NGPs) with custom-made functionalities (Bober et al., 2018). NGPs refer to custom-designed microbes designed for environmental trigger sensing, secretion of therapeutics molecules, or modulation of metabolic pathways with unprecedented selectivity (Yadav et al., 2023). Developments in tools such as CRISPR/Cas9, BioBricks, as well as in metabolic engineering have allowed designing NGPs with selective treatment of metabolically caused disorders (Chauhan, 2023). In this review, we juxtapose current advances in synthetic biology-led probiotic engineering with emphasis on applications in treating disorders of metabolism, delivery systems, as well as roadblocks in translating them clinically. We focus on determining the transformative potential of NGPs as well as delineating future directions of research in order to bypass current roadblocks.

2. Gut Microbiota and Metabolic Disorders

The host metabolism is controlled by the gut microbiota through the production of short-chain fatty acids (SCFAs), regulation of bile acids, and control of the immune system (Valdes et al., 2018). SCFAs, in particular butyrate, feed colonic epithelium cells, promote the barrier function of the gut, and regulate glucose as well as lipid metabolism (Canfora et al., 2019). Dysbiosis destroys these functionalities, contributing to metabolic disease. Reduced Akkermansia muciniphila is known in association with obesity as well as T2D, and dysregulated bile acid metabolism exacerbates NAFLD (Liu et al., 2021). PKU, a genetic disease caused by phenylalanine hydroxylase deficiency, produces accumulation of toxic phenylalanine, whose concentration can be controlled through gut microbes (Hillert et al., 2020).

Metabolic disorders pose a significant global healthcare problem. Obesity is present in over 650 million adult global citizens, predisposing them to T2D as well as cardiovascular disease (Boulangé et al., 2016). T2D, due to insulin resistance, is present in over 460 million, with NAFLD being present in 25% of those worldwide (Wang et al., 2021b). PKU, being rare, requires strict dietary control in an effort to prevent neurological regression (Charbonneau et al., 2020). Standard-issue probiotics, as useful as they demonstrate in general gastrointestinal health, have no selectivity in trying to target specific metabolic pathways; therefore, advanced engineering strategies are necessary (Azad et al., 2018).

3. Synthetic Biology Tools for Probiotic Engineering

Synthetic biology has innovatively fashioned next-generation probiotics (NGPs) with the promise of a set of advanced genetic tools with the potential of facilitating exact reprogramming of microbes towards therapeutically positive ends. With them, one can design microbes with custom-made functionalities such as those of secreting drugs, detecting disease-specific signatures, or managing host metabolism. By avoiding traditional drawbacks of probiotics, such as non-specific action as well as transitory behavior, synthetic biology enables scientists to design NGPs with improved             stability, manipulability, as well as potential towards therapeutics (McCarty & Ledesma-Amaro, 2019). In this paragraph, four of the most significant synthetic biology tools—CRISPR/Cas9 gene editing, BioBricks, as well as genetic circuits, remodeling of pathway remodeling of metabolism, as well as biosensors—should be studied in depth with reference to mechanisms of action, applications, as well as contributions towards designing towards treating metabolic disorders.

3.1 CRISPR/Cas9 Gene Editing

CRISPR/Cas9 is one such cornerstone of synthetic biology with unprecedented selectivity for genome editing of probiotic strains. CRISPR/Cas9 employs guide RNA in directing Cas9 nuclease towards any interesting DNA sequences, such that it allows site-specific insertion, deletion, or modification of genes (Zhou et al., 2020). CRISPR/Cas9 has been at the core of strain engineering of greater therapeutical potential, such as Lactobacillus as well as Escherichia coli Nissle 1917 (EcN). Lactococcus lactis has been genetically manipulated using CRISPR/Cas9 towards expression of insulinotropic peptides such as glucagon-like peptide-1(GLP-1) in sensitizing diabetic mouse model sensitivities towards insulin (Yadav et al., 2020). CRISPR/Cas9 precision allows tuning of expression such that there is stable expression of therapeutically desirable molecules with no adverse effect on the core operations of the microbe. Moreover, CRISPR-based tools such as base editing as well as CRISPR interference (CRISPRi) allow subtle modifications such as tuning of promoters in order to fine-tune the function of probiotics (Chauhan, 2023). These fine tunings avert problems of random mutagenesis of traditional strategies of genetic engineering, such as recurrent unpredictable results. With a mighty platform of fine genetic modifications, CRISPR/Cas9 is forging ways towards NGPs with custom designs towards specific disorders of metabolism, such as type 2 diabetes, as well as towards inflammatory bowel disease (IBD).

3.2 BioBricks and Genetic Circuits

BioBricks and genetic circuits may be thought of as a modular system of synthetic biology, whereby they can construct complex, programmatic systems in probiotic microbes.  BioBricks are standardized, interchangeable DNA sequences encoding for particular functions, like promoters, ribosome-binding sites, or protein-coding regions (Yamazaki et al., 2017). These genetic components can be combined to construct genetic circuits allowing NGPs to sense environmental stimuli and perform therapeutic activities. Genetic circuits have been engineered such that they can permit probiotics to sense disease-state metabolites, like thiosulfate in IBD or glucose in diabetes, and secrete anti-inflammatory peptides or insulinotropic peptides (Cui et al., 2021). One such interesting application is the use of Escherichia coli Nissle 1917, as it can be engineered such that it can detect markers of gut inflammation and secrete IL-10, an anti-inflammatory cytokine (Ozdemir et al., 2018). Modularity of BioBricks allows rapid prototyping as well as iterative design, like in constructing advanced functionalities of NGPs. Furthermore, genetic circuits can incorporate built-in feedback loops as well as toggle switches whereby they can keep gene expression dynamically, such that they can have accurate control of therapeutic output (Bober et al., 2018). Modularity then facilitates reproducibility as well as scaling of NGP engineering, such that it is a powerful tool in treating complex metabolic disorders.

3.3 Metabolic Pathway Reprogramming

Redesign of microbial metabolic pathways entails reforming microbial metabolic pathways towards the production of therapeutically valuable molecules or clearance of toxic metabolites, thus correcting metabolic imbalances in the host. It fine-tunes endogenous pathways or introduces new pathways to enhance the Therapeutic capacity of the probiotic (Lubkowicz et al., 2022). Lactobacillus reuteri, among others, has been genetically engineered to produce phenylalanine ammonia lyase (PAL), an enzyme involved in phenylalanine degradation, a toxic metabolite in phenylketonuria (PKU), decreasing serum levels of phenylalanine in up to 40% in preclinical systems (Adolfsen et al., 2021). Propionibacterium freudenreichii has been genetically altered to produce short-chain fatty acids such as butyrate, compounds that enhance enterocyte tightness as well as regulate fatty tissue metabolism in obese individuals, as well as in individuals with non-alcoholic fatty liver disease (NAFLD) (Mejía-Caballero et al., 2021). Carbon flux redirection is also involved in improving the yield of valuable metabolites such as propionate in glucose homeostasis regulation (Canfora et al., 2019). The flux balance approach, as well as synthetic pathways of metabolism, facilitate fine control of microbial catabolic activity towards efficacious production of drugs of interest. These advances resolve several shortcomings of conventional probiotics, such as minimal levels of valuable metabolites produced as seen in traditional probiotics, those being high-yield, selective production of valuable metabolites targeting particular disorders of metabolism.

3.4 Biosensors

Biosensors are genetically constructed systems allowing NGPs in tracking disease-specific indicators of the gut ecosystem, as well as in eliciting disease-specific therapeutics. These systems have a sensor module, perceiving environmental stimuli, as well as an actuator module, eliciting gene expression (Ozdemir et al., 2018). Examples of biosensors have been integrated in Escherichia coli Nissle 1917 in order to detect inflammation indicators, for instance, nitric oxide or tumor necrosis factor-alpha (TNF-a), as well as elicit production of cytokines with anti-inflammatory activity, such as IL-10 production (Cui et al., 2021). In treating diabetes, biosensors have allowed for perceiving increased glucose concentration as well as for releasing GLP-1 with improved sensitization towards insulin (Zhou et al., 2020). Biosensors have been constructed further in order to detect indicators of dysbiosis of the gut, such as disrupted SCFA patterns, as well as elicit corrective metabolic pathways (Yadav et al., 2020). Construction of biosensors in designing quorum-sensing systems as well as synthetic promoters enhances selectivity as well as biosensor specificity, enabling in situ tracking as well as response in changing environments of the gut (Yamazaki et al., 2017). By constructing an adaptive as well as responsive platform, biosensors significantly enhance the accuracy as well as efficacy of NGPs, going beyond traditional probiotic passivity.

Therefore, integration of CRISPR/Cas9, BioBricks, reconstruction of catabolic pathways, as well as biosensors, has changed probiotic engineering such that one can design NGPs with unparalleled function as well as specificity. These tools enable one to design exact editing of genomes, construction of modular circuits, optimized production of metabolites, as well as temporal environmental monitoring, correcting classical probiotic shortcomings (McCarty & Ledesma-Amaro, 2019). By enabling treatments of chromosomally encoded, aimed metabolic diseases such as obesity, T2D, NAFLD, as well as PKU, such synthetic biology strategies have unprecedented potential in precision treatments. Incremental improvements in continuous tool development, as well as integrating them in solid microbial platforms, will further change the potential of NGPs as treatments, opening avenues towards personalized as well as efficient treatments. Figure 1 represents a summary of the synthetic biology toolkit for probiotic engineering.???????

4. Next Generation Probiotics in Metabolic Disorders

NFGLs have been designed to selectively inhibit certain pathways of metabolism with new therapies for NAFLD, T2D, obesity, and PKU.

4.1 Obesity

Obesity is caused by gut dysbiosis with lower SCFA-producing bacteria and higher inflammation (Canfora et al., 2019). NGPs have been developed as tools to restore microbial equilibrium and regulate metabolism. For instance, Escherichia coli Nissle 1917 (EcN) strain genetically engineered to produce butyrate increases the function of the gut barrier as well as cuts adipose tissue inflammation in obese mice (Wang et al., 2022). In a similar way, Akkermansia muciniphila strain genetically designed to overproduce mucin-degrading enzymes enhances glucose metabolism as well as decreases fat mass (Liu et al., 2021). In a 2024 clinical trial, Lactobacillus reuteri NCIMB 30242 strain genetically designed to produce conjugated linoleic acid (CLA) lowered LDL-cholesterol as well as body weight in obese individuals (Hasnain et al., 2024).

4.2 Type 2 Diabetes

T2D is related to decreased SCFA production in addition to disturbed bile acid signaling (Boulangé et al., 2016). NGPs designed towards the secretion of glucagon-like peptide-1 (GLP-1) enhance one’s sensitivity towards insulin. Lactococcus lactis genetically designed towards secretion of GLP-1, such as reduced levels of diabetic mouse models of fasting glucose (Zhou et al., 2020). Lactobacillus paracasei was one such strain genetically designed towards secretion of bile acid hydrolases, improving one’s metabolism of bile acid as well as insulin (Tian et al., 2024). These illustrate examples of NGPs’ capability of targeting selective pathways in T2D.

4.3 Non-Alcoholic Fatty Liver Disease (NAFLD)

NAFLD is characterized further as dysbiosis of the gut along with increased permeability of the gut, due to which noxious metabolites enter the liver (Wang et al., 2021b). Engineered Propionibacterium freudenreichii releasing vitamin B12 and SCFAs lowered hepatic steatosis in mouse models of NAFLD (Mejía-Caballero et al., 2021). Engineered Bacteroides fragilis releasing anti-inflammatory peptides lowered inflammation in the liver in preclinical studies as well (Silva et al., 2020). These NGPs offer specific interventions in restoring homeostasis of the gut-liver axis.

4.4 Phenylketonuria (PKU)

Phenylketonuria is due to defective phenylalanine metabolism, leading to neurological disease (Hillert et al., 2020). Engineered Lactobacoccus reuteri with PAL expression reduced mouse serum concentration of phenylanaline in mouse models of PKU by 40% (Adolfsen et al., 2021). An Escherichia coli Nissle 1917 strain genetically modified as a phenylanaline catabolizer had dose-dependent activity in human volunteer studies in good health (Kurtz et al., 2019). These demonstrate the potential of NGPs in enzyme replacement in low-prevalence disorders of metabolism. Table 1 provides an overview of Next-Generation probiotics for metabolic disorders.

5. Next Generation Probiotics Delivery Systems

Therapeutic action of next-generation probiotics (NGP) depends on their engraftment in one's gut, which they need to tolerate under unfavorable physiological environments in addition to competing with resident microbes. Stomach acid, bile salts, in addition to enzymes, create difficult obstacles towards viability of NGP, impairing their capabilities of colonizing one's gut in addition to releasing therapeutic cargos (Han et al., 2021). Moreover, colonization resistance of host-derived gut-derived microbiota, competing with nutrients in addition to occupying niche space, further reduces engraftment (Suez et al., 2019).

Synthetic biology thus has triggered the design of sophisticated delivery systems as tools for improving NGP stabilities, precision towards colonization, in addition to colonization efficiencies. These systems play central roles in verifying genetically designed microbes such as Lactobacillus in addition to Escherichia coli Nissle 1917 (EcN) arrive accurately at sites of action in one's gut in order achieve therapeutic action towards correcting disorders of metabolism such as obesity, disease of type 2 diabetes (T2D), in addition to disease of phenylketonuria (PKU).

Encapsulation is one such possible approach using biocompatible polymers in order to prevent gastric breakdown of NGPs. An example of chitosan-alginate matrix produces coatings of shielding around shielding protecting the probiotics along transit through the gastrointestinal tract, with release in the intestines of low acidic pH is one such situation (Song et al., 2022). Encapsulation increases survivability as well as colonization. An interesting example includes layer-by-layer encapsulation of Escherichia coli Nissle 1917, improving in preclinical models the gastrointestinal colonizing potential, maintaining function along with maintaining microbial viability (Kurtz et al., 2019). These encapsulation precautions of polymers of biocompatible polymers with similar detailed comparisons in polymer environmental chemistry in order to deploy sustainably materials such as chitosan in order to design green applications (Morici et al., 2022) can increase survivability of NGP in order to design sustainable delivery of drugs such as molecules of SCFAs or enzymes in order to target routes of metabolites.

Another new delivery system consists of nanoparticles, able to deliver NGPs in controlled as well as in target-specific modes in defined regions of the gut. Lipid nanoparticles, e.g., can shelter probiotics against inactivation as well as deploy them accurately in the small intestine or colon, in regions of greatest activity requirement (Cui et al., 2021). In 2024, it was shown that ursolic acid nanoparticles can function as carriers of Lactobacillus strains in diabetic mice with sustained release of glucagon-like peptide-1 (GLP-1) in order to enhance susceptibility to insulin (Tian et al., 2024). Payload stabilization as well as tissue-specific targeting are strengths of nanoparticles, suitable in general as carriers of complex NGPs designed as expressers of metabology-assaying enzyme activity or of peptides of anti-inflammatory action. These systems conform as well with green chemistry design principles in using biodegradable materials with minimized environmental effect at manufacture as well as at deployment (Mejía-Caballero et al., 2021).

Time-release products invoke in assistance a third approach in maximizing the delivery of NGPs as well as therapeutic effect. In products in capsules or hydrogel, products release sustained activity of the probiotic in one's intestines, allowing long-term colonization with sustained longevity of therapeutic effect (Mugwanda et al., 2023). By controlling release kinetics, time-release products avoid problems of rapid clearance presented in the gut environment, such that there is time in which there is a chance whereby NGPs can engraft in contact with the host microbiome. Examples of such products vary from products in time-release capsules releasing Lactobacillus reuteri genetically modified in order to manufacture phenylalanine ammonia lyase (PAL), initiating catabolism of phenylalanine in animal models of PKU (Adolfsen et al., 2021). These products maximize precision as well as longevity of treatments with NGPs, correcting errors in conventional probiotics, most of which colonize as long-term colonists of one's intestines (Suez et al., 2019).

6. Challenges in Developing NGPs and Clinical Translation

Despite such revolutionary potential, NGPs have tremendous development as well as translation obstacles in the clinical domain. These span technical, biological, as well as regulatory platforms, requiring new solutions in order to have safe as well as effective therapeutic benefits. Overcoming such hurdles is crucial in transferring bench-to-bedside synthetic biology-led probiotics.

Biocontainment is extremely vital as uncontrolled release of genetically designed organisms in an environmental setting will have unintended ecological impacts. Synthetic biology devices such as kill switches, along with auxotrophic strains, have already been designed in such a manner that NGOIs cannot survive outside of a host (Ozdemir et al., 2018). Auxotrophic strains cannot survive outside of host-specific nutrients, whereas kill switches induce microbial suicidal behavior in response to a lack of some of the gastric cues, thereby evading environmental persistence (Bober et al., 2018). Examples include an auxotrophic Escherichia coli Nissle 1917 strain developed as a candidate in treating PKU with good biocontainment in a preclinical research setting (Kurtz et al., 2019). These measures reduce the likelihood of release of GMOs in alignment with ethics as well as regulations of deployment of synthetic biology (National Academies of Sciences, Engineering, and Medicine, 2018).

Scalability is another essential issue because it is hard to scale up industrial-scale production of NGPs without compromising genetic stability in addition to treatment effectiveness. Large-scale fermentation needs optimized bioreactor designs of bioreactors with synthetic medium in order to have steady function as well as microorganism growth (Wang et al., 2013). Optimization of bioprocess design, such as high-cell-density bioprocesses with synthetic nutrient combinations, is resolving these issues in order to maximize yield as well as cost reduction (Mejía-Caballero et al., 2021). An example of scaling Propionibacterium freudenreichii genetically engineered with function of SCFA products with NAFLD treatment had potential in similar ways as optimizing bioprocesses in order to relate this research with sustainable production protocols of polymer environmental chemistry, with feedstocks of wastes being utilized in order to maximize cost reduction as well as environmental impact minimization (Morici et al., 2022).

Safety regulation is a complex obstacle since artificial cells pose long-term moral concerns with human health as well as environmental impact. Regulatory processes of NGPs are still increasing, with rigorous testing processes of safety prior to inhibiting unintended immunological reactions or disrupting the gut microbiome (National Academies of Sciences, Engineering, and Medicine, 2018). Clinical trials, such as those of SYNB1618, an Escherichia coli Nissle-derived NGP of PKU, revealed safety in stage I studies but await licensure before they can be prescribed routinely (Perreault et al., 2024). Regulatory complexity, especially of live biotherapeutics, defines the need for harmonious regulations in order to clear paths towards market entry with patient security in place (Swanson et al., 2020).

Colonization resistance of resident host microbiota further inhibits NGP engraftment because resident microbes compete against niche space for nutrients, reducing strain survival (Han et al., 2021). Accordingly, research studies prebiotics as well as artificial consortia of microbes in an effort to enhance NGP colonization. Prebiotics such as inulin, selectively nourish engineered probiotics, while artificial consortia of microbes design systems of synergistic microbes in an effort to enhance NGP engraftment (Zhou et al., 2024). As just one example, an artificial consortium of Lactobacillus, as well as strains of Bifidobacterium, improved colonization of genetically manipulated Lactococcus lactis in mouse models of diabetes in an effort to produce elevated levels of GLP-1 in animal models of disease (Tian et al., 2024). These substitutes improve NGP ecological fitness in an effort to yield long-term therapeutic gains.

Individual variation in gut microbiome composition is one final challenge because of variations in microbial diversity among individuals, as they can impact NGP efficacy (Suez et al., 2019). Personalized strategies using multi-omics tools to create an individual microbiome profile are being conceptualized in order to design NGPs specific to patient needs (Hettich et al., 2013). Metagenomic sequencing can point towards microbial deficits in T2D individuals, thus directing the design of NGPs in order to restore deficient pathways of metabolism, such as SCFA production (Liu et al., 2021). Personalized NGPs have potential in enhancing therapeutic efficacy but need sophisticated diagnostic tools as well as large-scale clinical verification.

7. Future Directions

For enabling the complete potential of NGPs, future research must focus on the highest points. First, patient-specific NGPs tailored based on one's microbiome signature can maximize efficacy, utilizing multi-omic devices such as metagenomics and metabolomics in designing patient-specific therapies (Hettich et al., 2013). Second, combination therapies of NGPs with prebiotics, postbiotics, or heritage drugs can act in a synergistic combination in outcome enhancement. An example is a combination of engineered Lactobacillus with GLP-1 agonists in maximizing diabetes treatment (Zhou et al., 2024). Third, next-generation genetic devices such as universal genetic platforms can streamline NGP prototyping with quick construction of novel strains (Yamazaki et al., 2017). Fourth, larger-scale human trials must be carried out in order to determine NGP safety as well as efficacy in diverse population cohorts, capitalizing on initial advances such as SYNB1618 in treating PKU (Hasnain et al., 2024). Fifth, sustainable production using waste-based feedstocks as well as green power can reduce cost as well as environmental impact, learning polymer environmental chemistry lessons in green chemistry principles (Morici et al., 2022). These avenues will lead NGPs towards clinical as well as industrial success, remodeling the face of treating metabolic disorders.

8. Conclusion

Synthetic biology-based NGPs pave a new wave of precision, target-specific treatments of metabolic disorders. Engineered Lactobacoccus reuteri as well as Escherichia coli Nissle 1917 strains, as well as Akkermansia muciniphila, have demonstrated potential in preclinical as well as initial-stage human studies in human obesity, T2D, NAFLD, as well as PKU. CRISPR/Cas9, BioBricks, as well as delivery systems advancements, bolster functionality as well as the potential of NGPs. Biocontainment, scale-up, as well as regulation concerns ought to be handled in order for them to realize maximum potential. Multidisciplinary research as well as personalized therapies will redefine NGPs towards clinical as well as commercial realization, rewriting the precision of metabolic disorders.

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