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Metabolic engineering of Lactobacilli spp. for disease treatment

Microbial Cell Factories volume 24, Article number: 53 (2025) Cite this article

Abstract

Background

A variety of probiotics have been utilized as chassis strains and engineered to develop the synthetic probiotics for disease treatment. Among these probiotics, Lactobacilli, which are generally viewed as safe and capable of colonizing the gastrointestinal tract effectively, are widely used.

Main body of abstract

We review recent advancements in the engineering of Lactobacilli for disease treatment. Specifically, the Lactobacilli that are used for the construction of synthetic probiotics, the application of these engineered strains for diseases treatment, and the therapeutic outcomes of these engineered microbes are summarized in this review. Moreover, the applications of these engineered strains for disease treatment are categorized based on their engineering strategies. Of note, we compare the advantages and disadvantages of various engineering strategies and offer insights for the future development of genetically modified Lactobacillus strains with stable and safe properties.

Short conclusion

Our study comprehensively reviews researches on engineering diverse Lactobacillus strains for disease treatment, categorized by their engineering strategies, and emphasizes the importance of developing synthetic probiotics with stable and safe characteristics to enhance their therapeutic applications.

Background

Probiotics are live non-pathogenic microorganisms that can provide beneficial effects for the host when administered in proper amounts [1]. These microbes can deliver beneficial effects through multiple mechanisms, such as reducing intestinal pH, inhibiting the colonization and invasion of pathogenic organisms, and modulating host immune responses [2]. Based on these properties, numerous probiotics have been identified and utilized for the prevention and treatment of diseases [3]. However, the therapeutic application of many traditional probiotics is limited by several drawbacks, such as poor intestinal colonization, strain variability, and inadequate interaction with the host [4].

With advancements in synthetic biology tools and technologies, a wide range of probiotics have been employed as chassis strains and specifically engineered to enhance their therapeutic efficacy, e.g., Escherichia coli Nissle 1917, Clostridium butyricum, Saccharomyces boulardii, and the microbes belonging to genera Lactococcus, Lactobacillus, Bifidobacterium, and Bacteroides [5,6,7,8]. Among these probiotics, Lactobacillus strains, which are generally viewed as safe according to the U.S. Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA), have been extensively utilized in the food and medical industries [9]. Given the fact that Lactobacilli can colonize the intestine effectively to facilitate the mucosal targeting [10], a wide range of Lactobacillus strains have been employed as functional chassis for the development of synthetic probiotics, e.g., Lactobacillus plantarum (L. plantarum), Lactobacillus gasseri (L. gasseri), Lactobacillus johnsonii (L. johnsonii), Lactobacillus reuteri (L. reuteri), Lactobacillus paracasei (L. paracasei), Lactobacillus rhamnosus (L. rhamnosus), Lactobacillus jensenii (L. jensenii), Lactobacillus salivarius (L. salivarius), and Lactobacillus casei (L. casei), etc. Here, we summarize researches focused on engineering Lactobacilli and categorize the applications of these engineered strains for disease treatment based on their respective engineering strategies.

Surface displaying functional elements in Lactobacilli

Plasmid-based surface display

To develop effective vaccines for treating various diseases, numerous antigens or functional genes have been expressed using plasmid-based strategies and displayed on the surface of Lactobacilli. (Table 1; Fig. 1A).

Table 1 Plasmid-based surface display of functional elements in Lactobacilli
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Fig. 1
figure 1

Summary of the strategies used to display the functional elements on the cell surface of Lactobacilli. (A) Plasmid-based surface display of vaccines, functional elements for intestinal exclusion of viruses and pathogens, pharmaceutical compounds, and enzymes. (B) Genome integration-based surface display of vaccines, functional element for intestinal exclusion of virus, and pharmaceutical compounds

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Plasmid-based surface displaying antigens for treating virus infection-associated diseases

To treat human virus infection-related diseases, the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [11], the major structural protein Gag of human immunodeficiency virus (HIV) [12], and the E7 protein of human papillomavirus (HPV) type 16 (HPV16 E7) [13,14,15] had been selected as the functional antigens and displayed on the surface of different Lactobacill. These engineered microbes demonstrated high antigenicity and elicited robust antigen-specific immune responses, thereby enhancing the clearance of the aforementioned harmful viruses. Specifically, the engineered strain expressing Gag as a functional element induced antigen-specific IgA production and stimulated IFN-γ-producing cells via oral immunization [12]. For the HPV16 E7-displaying strain, it could increase the mean log titer of the serum IgG from 1.24 ± 0.24 to 3.15 ± 0.02, improve the E7-specific lymphocyte proliferative response (from 7.8 ± 0.9 to 11.0 ± 1.4), and enhance the E7-specific cytotoxic T lymphocyte (CTL) response (from 21 ± 5 to 510 ± 36 spot-forming cells (SFC)/106 cells) in C57BL/6 mice [13]. Moreover, immunization of TC-1 mouse tumor model with the HPV16 E7-expressing strain resulted in a substantial improvement in survival outcomes, elevating the survival rate from 0 to 50% [13]. Cervical intraepithelial neoplasia grade 3 (CIN3) is a mucosal precancerous lesion caused by high-risk human papillomavirus (HPV). Kei Kawana et al. evaluated the safety and clinical efficacy of an attenuated Lactobacillus casei strain 525 that expressed HPV16 E7 protein in patients with HPV16-associated CIN3 during a 9-week trial. It was noted that patients using 4–6 capsules/day showed increased E7-cell mediated immune response and exhibited pathological down-grade from CIN3 to CIN2 [15].

Similar engineering strategies have been successfully implemented in the development of recombinant Lactobacilli for the treatment of porcine viral infection-associated diseases. Among these porcine viruses, the viral structural glycoprotein 5 (GP5) of porcine reproductive and respiratory syndrome virus (PRRSV) [16], the spike antigen of porcine transmissible gastroenteritis virus (TGEV) [17], the core neutralizing epitope (COE) antigen of porcine epidemic diarrhea virus (PEDV) [18,19,20], the E2 protein of classical swine fever virus (CSFV) [21], and the HINI HA1 protein of the swine infuenza A virus (swIAV) [22] were proved to be the functional antigens and displayed on the surface of Lactobacilli for disease treatment. The L. plantarum NC8-derived recombinant strain expressing the spike antigen of TGEV on its surface significantly enhanced B7 molecule expression on dendritic cells (DCs) and elicited robust immune responses, as demonstrated by significantly increased levels of IgG, secretory IgA, and the cytokines IFN-γ and IL-4 [17]. When the COE antigen of PEDV was displayed as functional element, the engineered strain elicited a potent serum IgG antibody response, resulting significantly enhanced PEDV-neutralizing activity (1:24) compared to control groups (< 1:2) following oral administration. Furthermore, the secretory IgA induced by this L. casei ATCC 393-derived recombinant strain elicited stronger neutralizing activity against PEDV (1:20 titer) compared to the control group (< 1:2) [18]. By displaying the core COE antigen of PEDV that conjugated with the M cell-targeting peptide (Col) and dendritic cell-targeting peptide (DCpep) on the surface of L. casei ATCC 393, the engineered strain provided stronger PEDV-neutralizing ability (1:36) than the control group (< 1:2) after oral administration [19]. To fight against TGEV and PEDV infection simultaneously, a L. casei ATCC 393-derived recombinant strain was constructed by displaying the D antigenic site of the TGEV spike (S) protein and the COE of the PEDV S protein on surface. Oral administration of the engineered strain significantly enhanced systemic immunity, as evidenced by elevated serum levels of anti-PEDV and anti-TGEV IgG antibodies, increased mucosal secretion of SIgA, and enhanced lymphocyte proliferation capacity [20]. Furthermore, oral immunization of pig with the L. plantarum HA33-1-derived recombinant strain that displaying the CSFV E2 protein in conjunction with thymosin α-1 could help it to fight against CSFV infection by eliciting the IgA-based mucosal, IgG-based humoral, and CTL-based cellular immune responses [21]. To fight against H1N1 virus infection, L. plantarum ZN3 was genetically engineered to express a surface-displayed fusion protein containing the H1N1 HA1 protein, DC-targeting peptide (DCpep), and M cell-targeting peptide, which was subsequently administered via oral gavage to mice challenged with H1N1 virus. This engineered strain could induce effective mucosal, cellular, and systemic immune responses in the intestine and upper respiratory airways, thus increasing the survival rate of mice from 0 to 60% [22]. Notably, intranasal immunization with the recombinant strain conferred complete protection, with all immunized mice surviving (100% survival rate), whereas the control mice succumbed to infection within 10 days post-challenge (0% survival) [22].

Furthermore, a series of Lactobacilli spp. have been engineered to provide vaccines for the avian viruses-related diseases. For example, the virial proteins 3M2e and HA2 of avian influenza virus (AIV) were fused together and displaced on the surface of L. plantarum NC8 [23]; the hemagglutinin 1 (HA1) subunit of the A/Aquatic bird/Korea/W81/2005 (H5N2) that fused with the Bacillus subtilis poly γ-glutamic acid synthetase A (pgsA) was surface displayed on L. casei [24]; the VP2 protein of infectious bursal disease virus (IBDV) and the Gp85 protein of J subgroup avian leukosis virus (ALV) were displayed on the surface of L. plantarum [25, 26]. The 3M2e-HA2 display strategy demonstrated remarkable efficacy, with immunization using the recombinant strain increasing survival rates in H9N2-challenged mice from 0 to 80% [23]. Utilizing the HA1-pgsA display strategy, the engineered strain administered through either oral or intranasal routes provided full protection against H5N2 infection (100% survival). In contrast, all control animals succumbed to infection between 8 and 9 days post-challenge [24]. For the VP2 displaying strategy, the survival rates of the vvIBDV-challenged chickens were increased from 0 to 100% [25]. The Gp85 display strategy significantly enhanced survival rates in ALV-J-challenged chickens, demonstrating marked protective efficacy against viral infection [26].

Apart from the above-mentioned diseases, the glycoprotein (G) of spring viremia of carp virus (SVCV) and ORF81 protein of koi herpesvirus (KHV) have been proved to be functional antigens and co-expressed on the surface of L. plantarum HA33-1 to provide protective immunity for cyprinid fish [27]. Compared to the control group, oral administration of the engineered strain elicited robust IgM production, conferring effective protection against viral challenge with 71% and 53% survival rates in vaccinated common carp and koi at 65 days post-infection [27].

Plasmid-based surface displaying vaccines for treating the parasites infection-associated diseases

For the parasites infection-associated diseases, L. plantarum NC8 was engineered to display the Eimeria tenella (E. tenella) -derived proteins (SO7, EtMic2, AMA1, and U6L5H2) as antigens on surface [28,29,30,31]. Immunizing chickens with these L. plantarum NC8-derived engineered strains could protect them from E. tenella challenge efficiently. For the EtMic2-displaying strain, the E. tenella infection-induced lesion scores of cecum was decreased from 3.75 ± 0.520 to 2.30 ± 0.506, the oocysts per gram of droppings (×106) was decreased from 1.44 ± 0.02 to 0.71 ± 0.04, while the anticoccidial index (ACI) was increased from 74.93 to 145.15 [28]. For the EtMic2 and AMA1-displaying strain, the body weight gain (BWG) of E. tenella-challenged chicken was increased from 210.50 ± 16.16 g to 313.71 ± 6.60 g, the lesion score in cecum was decreased from 3.83 ± 0.41 to 2.00 ± 0.63, the oocyst output (×105) was decreased from 9.50 ± 3.03 to 3.56 ± 1.30 [29]. For the recombinant strain that displaying SO7 that fused to DCpep, the body weight gain and serum antibody responses were increased in the E. tenella-challenged chicken, while the fecal oocyst shedding and pathological damage in cecum were decreased [30]. For the L. plantarum NC8-derived recombinant strain that displaying the eukaryotic initiation factor U6L5H2, the body weight gain of E. tenella-challenged chicken was increased from 83.32 ± 3.28 g to 101.57 ± 2.02 g, the average lesion score was decreased from 2.90 ± 0.42 to 1.79 ± 0.31, the oocyst output (×105) was decreased from 5.37 ± 0.43 to 1.35 ± 0.18, the ACI was increased from 109.90 to 168.28 [31]. Apart from Eimeria tenella infection-associated disease, the gp43 and nudix hydrolase (TsNd) of Trichinella spiralis (T. spiralis) were displayed on the surface of L. plantarum NC8 to provide effective vaccines against trichinellosis [32]. Immunizing the larval-challenged mice with the recombinant strain brought a 75.67% reduction of adult worms (AW) at 7 days post-infection (dpi) and 57.14% reduction of muscle larva (ML) at 42 dpi [32].

Plasmid-based surface displaying vaccines for treating the pathogen infection-associated diseases

To treat the pathogen infection-associated diseases, a recombinant L. plantarum strain that displaying the fusion antigen AgE6 (comprising Ag85B and ESAT-6) of Mycobacterium tuberculosis on surface was constructed and used for the treatment of tuberculosis [33]. The AgE6-displaying L. plantarum strain could not only induce antigen-specific proliferative responses in lymphocytes that purified from tuberculosis-positive donors, but also induce immune responses in mice after nasal or oral immunization [33]. As for the Clostridium perfringens (C. perfringens) infection-associated disease, a genetically engineered L. casei 393 was constructed by displaying the toxoid of C. perfringens α-toxin on surface. Oral administration of this engineered strain could improve the survival rates of C. perfringens-challenged mice (from 0 to 90%) by eliciting mucosal, humoral, and cellular immunity to neutralize the natural α-toxin of C. perfringens [34]. Besides, oral immunization of broiler chickens with the L caseia ATCC 393-derived recombinant strain that displaying the NetB toxin or the C-terminal domain of α-toxin from C. perfringens on surface could protect the chickens from C. perfringens-induced necrotic enteritis [35, 36]. In this strategy, the mean body weight change of the recombinant strain-immunized chickens (35.61%) were higher than that of the non-vaccinated chickens (24.13%) [36].

Apart from the strategies mentioned above, the α-β2-ε-β1 toxoid protein of C. perfringens had also been used as functional antigen and displayed on the surface of L. crispatus N-11 [37]. After booster immunization, the recombinant strain-immunized group showed higher levels of specific secretory IgA (SIgA) and IgY antibodies in the serum and intestinal mucus. Besides, the serum concentration of IFN-γ, lL-2, IL-4, IL-10, IL-12, and IL-17 were increased significantly in the same group [37]. To fight against S. pyogenes infection-associated diseases, the conserved region of streptococcal M6 protein (CRR6) was displayed on the surface of L. gasseri NM713. Oral administration of this engineered strain could induce systemic and mucosal immune responses to protect the host from S. pyogenes infection [38]. Specifically, after the nasal challenge of S. pyogenes, the mice that orally administered with the recombinant strain showed lower streptococcal infection (10%) and mortality (3.3%) rate as compared to the control group [38]. To prevent Aeromonas veronii (A. veronii) infection, L. casei was used as antigen deliver carrier and engineered to display the Aha1 of A. veronii that fused with the cholera toxin B subunit (CTB) or E. coli intolerant enterotoxin B subunit (LTB) as adjuvant on surface. Oral immunization of these engineered strains to carp protected them from A. veronii infection by stimulating the humoral and cellular immunity [39, 40]. For the Aha1-CTB displaying strain, it improved the survival rate of A. veronii-challenged carp from 0 to 64.29% [39]. Similarly, the Aha1-LTB displaying strain could increase the survival rate of A. veronii-challenged carp from 0 to 60.71% [40]. Furthermore, an engineered L. casei CC16 was constructed to surface display the MSH type VI pili B (MshB) of A. veronii as an antigen and cholera toxin B subunit (CTB) as a molecular adjuvant. Oral immunization of crucian carp with this L. casei CC16-derived engineered strain could protect it from A. veronii infection by improving the immune response [41]. Compared with the control group (about 10%), the survival rate of the recombinant strain-immunized crucian carp was increased to 60% [41]. To provide efficient vaccine for Vibrio mimicus (V. mimicus) infection, L. casei ATCC 393 was engineered to display the outer membrane protein K (OmpK) of V. mimicus as an antigen and cholera toxin B subunit (CTB) as the molecular adjuvant on surface. Oral administration of this engineered strain could protect Carassius auratus from V. mimicus infection by inducing humoral and cellular immunity [42]. At 10 days post V. mimicus challenge, the survival rate of the recombinant strain-immunized Carassius auratus was higher than that of the control group. As for the treatment of Fusobacterium nucleatum (F. nucleatum) infection associated inflammatory bowel disease (IBD), the FomA of F. nucleatum was surface-displayed on L. plantarum NC8. Oral immunization of mice with this engineered strain could decrease their mortality rate and body weight loss by inducing various immune responses to relieve F. nucleatum- or DSS-induced IBD [43]. To provide protective vaccine for Staphylococcus aureus (S. aureus) infection, the iron-regulated surface determinant protein B (IsdB) of S. aureus was displayed on the surface of L. reuteri WXD171. This engineered strain could induce mucosal responses in gut-associated lymphoid tissues and improve the survival rate of S. aureus-challenged mice from 10 to 70% [44]. To treat brucellosis, L. casei ATCC 393 was engineered to display the outer membrane protein OMP19 of Brucella species on surface. Oral administration of this engineered strain to mice could provide them with sufficient mucosal immune responses to resist the challenge of Brucella abortus 544 [45]. By assaying the CFU numbers of B. abortus 544 in spleen, the mice that orally immunized with OMP19-displaying strain showed higher degrees of protection (15-fold reduction of B. abortus 544 in spleen) as compared to the control group [45].

Plasmid-based surface displaying functional genes to facilitate the intestinal exclusion of viruses and pathogens

To treat rotavirus, an important pediatric pathogen for severe diarrhea, L. rhamnosus GG had been engineered to display the IgG-binding domain of protein G on surface. This engineered strain could fight against the rotavirus infection-induced diarrhea in mice by capturing rotavirus (simian strain RRV) via hyperimmune bovine colostrum antibodies (HBC-IgG) [46]. Compared with the usage of HBC alone, the combination usage of HBC antibodies and this engineered strain was more effective (10 to 100-fold increase) to reduce the prevalence, severity, and duration of diarrhea, thus decreasing the treatment costs considerably [46]. Similarly, displaying the two VHH fragments ARP1 and ARP3 on the cell surface of L. paracasei BL23 could facilitate the capture of rotavirus, thus reducing the diarrhea rate of rotavirus infection-induced mouse model [47]. To prevent L. monocytogenes infection-associated disease, L. casei ATCC 334 had been engineered to display the Listeria adhesion protein (LAP) on surface. This engineered strain could prevent the intestinal colonization of L. monocytogenes by occupying the surface presented LAP receptor Hsp 60 [48]. Compared with the control group, the number L. monocytogenes cells that adhered to the intestine were 100-fold lower in the mice that treated with the recombinant strain [48]. At 10 days post L. monocytogenes challenge, the surviving rate of the recombinant strain-treated mice (~ 92%) was higher than that of the control group (60%) [48]. Besides, displaying the invasion proteins internalins A and B (inlAB) of L. monocytogenes on the cell surface of L. casei ATCC 334 could protect Caco-2 cell from adhesion, invasion, and transcellular passage of L. monocytogenes [49]. In the adhesion assay, the recombinant strain reduced the adhesion of L. monocytogenes by 50% and 53.6% at 16 and 24 h, respectively, far more than that of the control group (8%) [49].

Plasmid-based surface displaying pharmaceutical compounds and enzymes

To endow the Lactobacilli with anti-inflammation ability, L. plantarum NC8 had been engineered to display murine IL-10 on the cell surface. This engineered strain could reduce the Poly(I: C)- or LPS-induced Th1 responses in RAW264.7 cells and decrease the expression of TNF-α, IFN-γ, IL-1β, and IL-6 [50]. Moreover, displaying the porcine IFN-λ3 on the cell surface of L. plantarum NC8 could inhibit the replication of TGEV and PEDV, thus reducing the prevalence of PEDV and TGEV viruses by 53% and 59%, respectively [51]. As to the functional enzyme, L. reuteri had been engineered to detoxify the fungal mycotoxins zearalenone (ZEN) by displaying the ZEN hydrolyzing enzyme lactonohydrolase on surface. This engineered strain was capable of hydrolyzing 2.5 mg/kg of ZEN-contaminated corn within 4 h [52].

Genome integration-based surface displaying vaccines, functional elements, and the pharmaceutical compound for the intestinal exclusion of virus

To guarantee the stable inheritance of functional genes, many researchers had tried to integrate the functional genes into the genome of Lactobacilli and then displayed these elements on cell surface (Table 2; Fig. 1B). For example, to provide effective vaccine for infectious bronchitis virus (IBV), the UTEpi C-A expression cassette containing the EpiC of IBV was integrated into the genome of L. salivarius TCMM17. This engineered strain could display EpiC on surface and served as a stable oral vaccine for the treatment of IBV [53]. To treat the rotavirus infection-induced illness, L. acidophilus NCFM was engineered to display the VP8* domain of the rotavirus EDIM VP4 capsid along with the adjuvants FimH and FliC on surface. Gavaging this engineered strain to BALB/cJ mice could reduce the fecal shedding of rotavirus antigen (4-fold) by inducing the immune responses [54]. To treat HIV infection, L. acidophilus ATCC 4356 was engineered to display the HIV-1 receptor CD4 on surface. This engineered strain could decrease the infection rate (57% reduction) of the HIV-1-challenged mice by adsorbing HIV-1 particles directly [55]. To construct a synthetic probiotic with antimicrobial activity, L. casei ATCC 393 was engineered to insert the lactoferricin (Lfcin) expression cassette at the thyA (thymidylate synthase) site. This engineered strain displayed Lactoferricin on surface, showed good antibacterial activity against Escherichia coli (40.05% inhibition) and Staphylococcus aureus (42.22% inhibition), and exhibited antiviral activity against PEDV (2-fold suppression of viral replication) [56].

Table 2 Genome Integration-based surface display of functional elements in Lactobacilli
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Lactobacilli-based secretion of functional elements

Plasmid-based secretion

Apart from displaying the functional elements on the surface of Lactobacilli, many functional elements were engineered to be secreted out of the cells for disease treatment (Table 3; Fig. 2A).

Table 3 Plasmid-based secretion of functional elements in Lactobacilli
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Fig. 2
figure 2

Summary of the strategies used to secret functional elements in Lactobacilli. (A) Plasmid-based secretion of vaccines, antibodies, pharmaceutical compounds, and enzymes. (B) Genome integration-based secretion of vaccines and pharmaceutical compounds

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Plasmid-based secretion of vaccines for treating virus infection

As reported, hemagglutinin (HA) had been proved to be an effective vaccine antigen against avian influenza virus (AIV). Thus, to provide effective vaccine for AIV, HA was co-expressed with the dendritic cell-targeting peptide (DCpep) in L. plantarum NC8. This engineered strain could improve the survival rate of AIV-infected mouse and chicken models by inducing robust immune responses [57]. To provide improved vaccines for classical swine fever virus (CSFV) and porcine parvovirus (PPV), L. casei ATCC 393 were engineered to co-express the CSFV-specific cytotoxic T lymphocyte (CTL) epitope 290 and the VP2 antigen of PPV. This engineered strain showed 86.7% effective protection for the CSFV-challenge pig, whereas pigs in the control group developed severe clinical signs of CSF [58]. To treat the disease caused by the infectious pancreatic necrosis virus (IPNV), L. casei ATCC 393 was engineered to secret the VP2 protein of IPNV as antigen. Oral administration of this engineered strain to juvenile rainbow trouts could prevent IPNV infection by inducing local and systemic immune responses. Compared to the control group, this recombinant strain showed more than 3-fold reduction in viral load [59].

Plasmid-based secretion of vaccines for treating parasite infection

To treat the Cryptosporidium parvum (C. parvum) infection-related disease, the P23 immunodominant surface protein of C. parvum was expressed stably in L. casei Zhang. Oral immunization of this engineered strain to mice could promote the clearance of C. parvum by inducing mucosal immune system and increasing the secretion of immunity factors, such as IgA, IL6, and IFN-γ [60].

Plasmid-based secretion of vaccines for treating pathogens infection

To provide effective oral vaccine against F4 + enterotoxigenic Escherichia coli (ETEC) infection, L. casei ATCC 393 was engineered to express FaeG, the main subunit of F4 (K88) fimbrial adhesin. Using the heat-labile enterotoxin A (LTAK63) and heat-labile enterotoxin B (LTB) as oral adjuvant, this engineered strain exhibited 100% protection against ETEC challenge and developed mild diarrhea for 2–3 days, whereas 80% of mice in the control group succumbed to the infection following viral challenge, exhibiting severe diarrhea that persisted for more than 12 days prior to mortality [61]. To provide protective immunity against Bacillus anthracis (B. anthracis), L. acidophilus NCFM was engineered to secret the protective antigen (PA) of B. anthracis that fused to a DC-binding peptide (DCpep). Oral administration of this genetically engineered strain conferred protection against B. anthracis infection (100% survival) in mice through the induction of both protective antigen (PA)-neutralizing antibodies and T cell-mediated immune responses [62]. To fight against the C. perfringens-derived necrotic enteritis, L. reuteri was engineered to secret nanobodies against NetB and α-toxin of C. perfringens. Oral administration of the recombinant strain to chickens demonstrated protective efficacy against necrotic enteritis, reducing both mortality rates (1.7- to 2.6-fold reduction) and pathological scores (2.5- to 3.6-fold reduction) [63].

Plasmid-based secretion of vaccines for treating neurodegeneration disease

Alzheimer’s disease (AD), one of the neurodegenerative disorder diseases, is a global health concern with huge implications for both the individuals and whole society. Gut microbiota contains the largest number of microbes in the intestine and has the potential to influence the host metabolism greatly. Thus, a healthy, balanced, and diverse gut microbiome is closely associated with the overall health of host. Recently, a plenty of results indicate that the dysbiosis of gut microbiome exerts a profound effect on the progression of neurological diseases, indicating the potential of treating these brain diseases through the gut-brain axis [64]. To alleviate AD, L. lactis subsp.

cremoris MG1363 was engineered to express the p62 protein of human beings. Oral administration of this engineered strain to the 3xTg-AD mice could benefit them by improving the memory function, modulating the neuronal proteolysis, and decreasing the AD typical signs [65]. For the recombinant strain gavaged group, the Aβ(1–42) peptide level was decreased by 42%; The activities of proteasome T-L and branched-chain amino acid preferring (BrAAP) were inhibited by 70% and 50%, respectively; The levels of protein oxidation products (3-NT and carbocyanine) were decreased by 1.37- to 1.78-fold; The levels of lipid peroxidation product (4-HNE) was decreased by 65%; The expression of anti-inflammatory cytokine (IL-10) was upregulated by 3-fold; The expression of pro-inflammatory cytokines (e.g., INF-γ, IL-1β, TNF-α, IL-2) were decreased by 2.0- to 3.25-fold [65].

Plasmid-based secretion of allergens for treating allergy

To prevent birch pollen allergy, L. plantarum NCIMB8826 Int-1 was engineered to produce the major birch pollen allergen Bet v 1. Systemic immunization of mice with this engineered strain could induce lower allergen-specific IgE and higher allergen-specific IgA. Besides, this engineered strain could activate the Th1-type immune responses effectively in mice and reduce the production of IL-5 significantly [66]. Furthermore, a recombinant L. plantarum NCL21 strain was constructed to express a major Japanese cedar pollen allergen Cry j 1 (Cry j 1-LAB). This engineered strain could provide prophylactic effect for the murine model of cedar pollinosis by suppressing the allergen-specific IgE response (2-fold reduction) [67].

Plasmid-based secretion of antibodies

To treat AIV, L. paracasei was engineered to secret the 3D8 single-chain variable fragment (scFv), which could bind and hydrolyze nucleic acids. Gavaging this engineered strain to chickens could lower virus shedding significantly and protect them from H9N2 avian influenza virus [68]. To fight against Clostridium difficile (C. difficile) infection, an engineered L. paracasei BL23 was constructed by expressing two neutralizing anti-TcdB VHH fragments (VHH-B2 and VHH-G3). Gavaging this engineered strain to the C. difficile spore-challenged hamsters could improve their survival rate from 0 to 50% [69].

Plasmid-based secretion of pharmaceutical compounds

To facilitate the exclusion of C. perfringens, L. johnsonii FI9785 was engineered to export the cell wall-hydrolysing endolysin (CP25L) of phage. Compared with the wild-type L. johnsonii, the numbers of C. perfringens were 2- to 2.6-log less when it was co-cultured with this engineered strain [70]. To protect the intestine from irradiation, an engineered L. reuteri 6475 was constructed to release the murine IL-22, which was functioned as an irradiation protector. Oral administration of this engineered strain to irradiated mice could modulate the cytokines level in the serum and intestine, thus improving the survival rate of irradiation-treated mice from 10 to 60% at day 30 [71]. Apart from protecting the host intestine from irradiation, this IL-22-secreting strain could also reduce the fatty liver disease of high-fat diet-fed mice by reducing the liver weight ratio (22.3% decrease) and liver triglyceride (4.6-fold decrease) [72]. Additionally, oral administration of this recombinant strain could reduce the liver damage of the alcoholic liver disease mouse model by increasing the expression level of Reg3g in small intestine, decreasing the expression level of Cxcl1 and Cxcl2, and reducing the number of the bacteria that translocated to liver [73]. To treat hypertension, the angiotensin converting enzyme inhibitory peptide (ACEIPs) was overexpressed in L. plantarum NC8. Oral administration of this recombinant strain could treat hypertension effectively in spontaneously hypertensive rats. Compared with the control group, the systolic blood pressure (SBP) of the engineered L. plantarum-treated group was decreased from 184.810 ± 4.305 mmHg to 167.111 ± 3.418 mmHg at day 15. Besides, the serum triglyceride was decreased from 1.213 ± 0.176 mM to 0.750 ± 0.181 mM [74]. To treat rheumatoid arthritis (RA), L. reuteri 647 was engineered to secret the Kv1.3 potassium blocker ShK-235 (LrS235). Gavaging this engineered strain to the rat model of rheumatoid arthritis could reduce the mean score of arthritis from 25 ± 2 to 4 ± 1(84% reduction) [75].

Plasmid-based secretion of functional enzymes

To suppress Pseudomonas aeruginosa (P. aeruginosa) infection, L. plantarum WCFS1 was engineered to secrete PelAh, the hydrolase domain of glycoside hydrolase PelA from P. aeruginosa, to degrade the biofilm of P. aeruginosa [76]. The cultures and supernatants of this engineered strain exhibited 80% and 85% reduction in biofilm biomass of P. aeruginosa, respectively [76]. To provide safe and efficient strategy for the treatment of ulcerative colitis (UC), L. paracasei F19 was engineered to produce palmitoylethanolamide (PEA) by expressing the N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) of human beings. Co-administration of this engineered strain with palmitate to the DSS-induced colitis mice could decrease the disease activity index (DAI) score (71% reduction), spleen weight (62% reduction), colitis histopathological score (47% reduction), and MPO activity (56% reduction), while increase the colon length (1.13-fold increment) and colonic expression of zonula occludens (ZO-1) (5.43-fold increment) and occluding (3.97-fold increment). Besides, the colonic level of iNOS (80% reduction), COX-2 (75% reduction), and IL-1β (63% reduction) and the plasma level of NO (79% reduction), PGE2 (74% reduction), IL-1β (81% reduction), and TNF-α (86% reduction) were decreased in colitis mice that were orally administrated with this engineered strain and palmitate [77]. To treat diabetic retinopathy, L. paracasei ATCC 27092 was engineered to secret the angiotensin converting enzyme 2 (ACE2), which was fused with the non-toxic subunit B of cholera toxin to facilitate transmucosal transportation. The secreted ACE2 could degrade angiotensin II and reduce inflammation and oxidative stress. Oral administration of this engineered strain could alleviate diabetic retinopathy by reducing the number of acellular capillaries, blocking the retinal ganglion cell loss, and decreasing the expression of retinal inflammatory cytokines [78]. To treat hyperoxaluria, L. plantarum WCFS1 was engineered to secrete the oxalate decarboxylase (OxdC) of Bacillus subtilis to degrade oxalate. Gavaging this engineered strain to male wistar albino rats with hyperoxaluria could reduce their serum uric acid (34% reduction), urinary oxalate excretion (40% reduction) and CaOx crystal deposition [79]. To treat phenylketonuria (PKU), the phenylalanine lyase gene of Anabaena variabilis (AvPAL) was codon-optimized and expressed in L. reuteri 100–23 C. Gavaging this engineered strain to PAHenu2 mouse model of PKU could reduce their blood Phe concentrations [80]. Besides, L. plantarum CM_PUJ411 was engineered to secret human phenylalanine hydroxylase (PAH), an enzyme that can metabolize Phe. Assisted with the signal peptide GI1 or GI2, the secreted PAH could transport through the cell monolayer of Caco-2 cells to reduce the Phe levels (28% reduction) [81].

Genome integration-based secretion of vaccines and pharmaceutical compounds

Until now, a series of functional elements had been integrated into the genome of Lactobacilli and secreted out of cell to treat diseases (Table 4; Fig. 2B). For example, to fight against PEDV, the PEDV S1 gene was integrated into the genome of alanine racemase-deficient L. paracasei△Alr HLJ-27 strain. Oral administration of this engineered strain could activate the mucosal, humoral, and cellular immune responses in mice and piglets effectively. The piglet challenging experiment results indicated that L. paracasei△Alr HLJ-27 administration could endow the piglets with resistance against PEDV LJB2019 infection by decreasing the PEDV copy number, maintaining intact villi structure, and relieving the inflammatory status [82]. To provide potent vaccine for C. botulinum and B. anthracis infection simultaneously, L. acidophilus NCFM was engineered to secret the host receptor-binding domain of the heavy chain of C. botulinum serotype A and the anthrax protective antigen of B. anthracis. This engineered strain was a promising candidate for the development of mucosal vaccines against botulism and anthrax [83]. To treat diabetes, L. gasseri ATCC 33323 was engineered to secret the inactive full-length form of GLP-1(1–37), which could stimulate the conversion of intestinal epithelial cells to insulin-secreting cells [84]. The diabetic rats that fed with this engineered strain developed insulin-producing cells in the upper intestine and showed high insulin levels and glucose tolerance [85].

Table 4 Genome Integration-based secretion of functional elements in Lactobacilli
Full size table

Engineering vaginal Lactobacilli for disease treatment

As reported, L. crispatus, L. jensenii, L. gasseri, and L. iners are the four main vaginal Lactobacilli and play important roles in sustaining the health of females. Although most of the vaginal Lactobacilli exhibit beneficial properties, the application of these microbes as probiotics is limited by the deficiency of appropriate delivery systems [86]. Here, the current strategies that used for engineering vaginal Lactobacilli for disease treatment are reviewed (Table 5).

Table 5 Engineering vaginal Lactobacilli for disease treatment
Full size table

Plasmid-based surface displaying functional element

To prevent the heterosexual transmission of HIV, L. jensenii 1153 was engineered to display the two-domain of high-affinity HIV-binding protein CD4 (2D CD4) on surface. The 2D CD4 molecules were distributed uniformly on the bacterial surfaces and could be recognized by the conformation-dependent anti-CD4 antibody [87]. Thus, this engineered strain might prevent the HIV transmission by adsorbing the HIV particles directly.

Plasmid-based secretion of functional elements

To treat intrauterine adhesions (IUA), L. crispatus MH175 was engineered to secret the murine CXCL12, which could recruit immune cells to promote the tissue regeneration and repair. Vaginal application of this engineered strain could decrease the levels of pro-inflammatory factors IL-1β and TNF-α in the serum and uterine tissues of IUA mice, inhibit the inflammatory and fibrotic signalling pathways in uterine tissue [88]. Besides, the engineered L. crispatus-treated mice could restore the vaginal microbiota composition of IUA mice by increasing the abundance of Lactobacillus and decreasing the abundance of Klebsiella microbes [88]. To treat human immunodeficiency virus (HIV) infection, L. plantarum ATCC 14917 and L. gasseri ATCC 9857 were engineered to secret the HIV-1 fusion inhibitors FI-3 to facilitate the neutralization of primary HIV-1 isolates and SHIV-162P3. For the HIV-1 isolates, the FI-3 expressing L. plantarum could reduce the viral infection of five HIV-1 isolates by 71–98%. Furthermore, the Lactobacillus-derived FI-3 could achieve 72% inhibition of SHIV-162P3 infection [89]. L. jensenii 1153 was engineered to secret the anti-HIV-1 chemokine RANTES and a CCR5 antagonist analogue as live HIV-1 blockers. The L. jensenii-derived wild-type RANTES could inhibit the acute HIV-1 infection, with IC50s reached 0.54 nM against HIV-1BaL and 1.14 nM against HIV-1SF162. Differently, although the Lactobacillus-derived C1C5 RANTES was devoid of proinflammatory activity, it showed lower anti-HIV-1 activity (IC50s of 5.00 nM for HIV-1BaL and 4.8 nM for HIV-1SF162) as compared to the wild-type RANTES [90]. Apart from the above-mentioned strategy, L. jensenii had been engineered to secrete the two-domain CD4 (2D CD4) proteins to inhibit HIV infection by blocking its entry into target cells. Single-cycle infection assay indicated that this engineered strain could inhibit the HIV-1HxB2 entry into HeLa cells by 95% and inhibit the HIV-1JR − FL entry by 55% [91]. To inhibit the transmission of HIV, L. rhamnosus GG and GR-1 were engineered to express carbohydrate-binding agent griffithsin (GRFT). The cytosolic protein fractions of two engineered strain were able to inhibit the T-tropic (X4) HIV-1 NL4.3 infection with an EC 50 value of 1/1710 and 1/3021 [92]. Besides, these two engineered strains showed significant activity against the M-tropic (R5) HIV-1 BaL strain, with a dilution factor of 1/605 and 1/1143 [92].

Genome integration-based secretion of functional element

To fight against HIV infection, L. jensenii 1153–1666 was engineered to secret the HIV inhibitor cyanovirin-N (CV-N). This engineered strain could colonize the vagina and prevent the repeated vaginal simian-HIV (SHIVSF162P3)-challenged Rhesus Macaque from HIV infection by remodeling the vaginal mucosal environment, e.g., lowering pH by an estimated 0.4 pH units and increasing the anti-inflammatory cytokine IL-1RA [93].

Future perspectives and challenges

Though probiotics inherently carry genetic signatures that promote the health of the host, the metabolically engineered Lactobacilli are further augmented with functional elements for the potential treatment of disease indications. The Lactobacilli have been engineered to secrete or surface display the functional elements to treat various diseases. As reported, the microbial cell surface display technology, which fused the heterologous protein to the anchor proteins, had been widely used in the application of biotechnology and biomedicine and vaccine delivery. Compared to the protein secretion system, the cell surface display strategy showed enhanced protein activity and stability [94]. However, there are several unavoidable challenges for this strategy, e.g., the size limitation of the displayed protein, the low display efficiency, the lack of proper carrier protein on cell surface, and the insertion site of the heterologous protein into the carrier (the insertion site affects the stability and activity of the displayed protein) [94, 95]. Based on these limitations, the smaller proteins, the functional fragment of big proteins, peptides, and vaccines can be displayed on the cell surface to exert specific functions. However, if the desired protein is too big to be displayed on surface, the protein secretion system should be used.

Given that both plasmid-based gene expression systems and genome integration systems have been widely utilized in metabolic engineering of host cells, we conducted a comprehensive comparison of their advantages and disadvantages across several key aspects: Firstly, the simplicity for metabolic engineering. Compared to genome integration systems, plasmid-based expression systems offer greater convenience and flexibility for metabolic engineering of host cells. Secondly, the gene expression level. In the genome integration strategy, a single copy of the functional element was inserted into the host cell’s genome, resulting in relatively low expression levels of the target element. In contrast, the use of high-copy-number plasmids enables significantly higher expression levels of the plasmid-encoded functional elements. Thirdly, the growth stress of host strain. Previous studies have reported that overexpression of heterologous proteins in bacterial systems can impose substantial metabolic burden on host cells, resulting in significantly reduced growth rates. Based on this conception, the plasmid-based high-level gene expression system may impose greater growth inhibition on host cells compared to the genome integration approach. Fourthly, the stable inheritance of the functional genes. For the plasmid-based gene expression system, the plasmids are unstable and can be lost after the long-term passage, especially under the antibiotic-free condition. In contrast, chromosomal integration of genes through genome-editing technologies ensures stable inheritance and maintenance of functional genes. Fifthly, the usage of antibiotic genes. The antibiotic genes on the plasmid can be diffused through transposition, transfer, or homologous recombination during operation, resulting the emergence of drug-resistant strains [96]. When functional elements were integrated into the host genome, the resulting strains did not require antibiotic resistance genes for maintenance. Lastly, the stability of genetic element. Replication of plasmids through the rolling circle mechanism generates unstable single-stranded DNA intermediates, which can result in deletions of genetic elements [97]. However, this disadvantage can be overcome by integrating the functional element in the genome of host cell.

Based on the abovementioned advantages of genome integration system, more and more researchers had focused on constructing the synthetic probiotics by editing the genome of Lactobacilli. Until now, several genome-editing technologies had been developed to facilitate the genome editing of Lactobacilli, e.g., the methods based on the insertion sequence (IS) elements [98], Cre-lox-based systems [99], and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) systems [100]. However, compared with the high copy number of functional genes in the plasmid-based expression system, the genes that integrated into the genome has only one copy, which will limit the expression of antigens or functional genes. Thus, the expression of the genome-integrated element should be enhanced by optimizing the expression related elements, e.g., the optimization of promoters or ribosomal binding sites. Until now, the number of genome-edited Lactobacilli are much lower than that of the plasmid-containing microbes, thus, more efforts should be done to generate the genome engineered Lactobacilli.

Taking the biosafety into consideration, the leakage of engineered microbes to environment will bring the problem of bio-contamination. To provide biocontainment for engineered microbes, several useful strategies had been applied during the construction of engineered probiotics: (1) Generation of auxotrophs to prevent the engineered probiotics from escaping to the environment [101]; (2) Construction of orthogonalized genetic central dogma by using artificial elements or coding principles [102, 103]. Based on this strategy, the biocontainment can be achieved by inhibiting the survival of engineered probiotics under the condition without non-natural synthetic substances; (3) Application of suicide circuit. For the passive suicide circuit, the killing module is coupled with a sensing module which can detect environmental signals. The engineered bacteria that harbours the suicide circuit commit suicide when the living environment is changed [104].

Collectively, with the assistance of the efficient genome-editing technologies and the biocontainment strategies, further studies should focus on constructing the engineered Lactobacilli with stable and safe characteristics for disease treatment.

Conclusion

Engineered probiotics are promising avenues for the treatment of various diseases by expressing functional elements or delivering intestine-directed therapeutics. Among these probiotics, Lactobacilli is widely used as functional chassis. In this review, the research progresses on engineering Lactobacilli for disease treatment are summarized according to the different engineering strategies. By discussing the shortcomings of the plasmid-based strategies that are used for the construction of synthetic microbes, our study stresses the importance of constructing synthetic probiotics with stable characteristics. Moreover, our study provides biocontainment strategies to improve the biosafety of the engineered microbes for the potential treatment of disease indications.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

This work was supported by grants from the National Science and Technology Innovation 2030 Major Program (2021ZD0200900), the National Key Research and Development Program of China (2022YFF0710901), the National Natural Science Foundation of China Grant (82021001 and 31825018), and the Biological Resources Program of the Chinese Academy of Sciences (KFJ-BRP-005). This work was also supported by the 111 Project D18007 and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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  1. Yunpeng Yang and Peijun Yu contributed equally to this work.

Authors and Affiliations

  1. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China

    Yunpeng Yang, Yufei Huang & Wanying Zhang

  2. Institute of Comparative Medicine, Yangzhou University, Yangzhou, 225009, China

    Yunpeng Yang, Yufei Huang & Wanying Zhang

  3. Shanghai Center for Brain Science and Brain-Inspired Technology, Shanghai, 201602, China

    Yunpeng Yang, Yanhong Nie & Changshan Gao

  4. Institute of Neuroscience, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, 200031, China

    Yunpeng Yang, Peijun Yu, Yanhong Nie & Changshan Gao

  5. University of Chinese Academy of Sciences, Beijing, 100049, China

    Peijun Yu

  6. Key Laboratory of Genetic Evolution & Animal Models, Chinese Academy of Sciences, Kunming, 650201, China

    Peijun Yu, Yanhong Nie & Changshan Gao

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Contributions

“Y.P.Y. conceived the project. Y.P.Y., P.J.Y., Y.F.H., W.Y.Z., Y.H.N. and C.S.G. wrote the main manuscript text. Y.P.Y. prepared Figs. 1-2. Y.P.Y. and P.J.Y. prepared the Tables 1-5. All authors reviewed the manuscript.”

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Correspondence to Yunpeng Yang.

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Yang, Y., Yu, P., Huang, Y. et al. Metabolic engineering of Lactobacilli spp. for disease treatment. Microb Cell Fact 24, 53 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12934-025-02682-4

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Keywords

Microbial Cell Factories

ISSN: 1475-2859

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