This work constructed a recombinant lactic acid bacterium secreting β-galactosidase for GOS formation during milk fermentation. First, GalINF, a β-galactosidase derived from infant feces, was characterized to effectively produce GOS in milk, reaching a content of 10.03 g/L. To export GalINF in Lactococcus lactis, six signal peptide candidates were employed, resulting in extracellular activities as low as 52.83-85.65 U/L. Then, GalINF (114.6 kDa) was split into two complementary modules, M1-P723 and A724-I1023, which could be independently secreted and actively reconstituted with the help of the protein scaffold SpyCatcher/SpyTag. The resultant Lc. lactis B1RG exhibited an extracellular β-galactosidase activity of 544.22 U/L. Fermentation of pasteurized milk with Lc. lactis B1RG and the traditional yogurt starters reduced lactose to 19.67 g/L and yielded 7.17 g/L GOS. This work established an effective strategy to export large-sized proteins extracellularly and demonstrated the applicability of LAB secreting β-galactosidase for GOS-enriched fermented dairy products.

Lacticaseibacillus paracasei (L. paracasei), a probiotic bacterium commonly found in the intestinal tract and fermented products, has been utilized as a tool for generating various bioactive components. However, its potential as a host cell for protein secretion is constrained by the limited availability of secretion signal peptides. This study explores the potential of endogenous signal peptides from L. paracasei for improving heterologous protein secretion. Endogenous secretory proteins from L. paracasei BL23 were analyzed to characterize their signal peptides. Using reporter genes such as nuc (nuclease) and amy (amylase) under the control of the nisin-inducible promoter PnisA, signal peptide SP230 was shown to be the most potent endogenous signal peptide for heterologous protein secretion in L. paracasei BL23. Additionally, a tandem linkage strategy employing identical signal peptides increased the secretion levels of heterologous proteins. The secretion of heterologous proteins was found to be dependent on the utilization of their optimal signal peptides. Tandem linkage of proteins with preferred signal peptides proved to be critical for efficient secretion of two distinct heterologous proteins simultaneously. The identification of novel signal peptides and the development of tandem linkage strategies in this study offer valuable insights for improving heterologous protein expression and secretion in L. paracasei. These findings enhance the potential of L. paracasei as a host for biotechnological applications, facilitating the secretion of diverse proteins.

Menaquinone, also known as vitamin K2, has extensive nutritional and pharmaceutical applications, such as in bone health maintenance and cardiovascular protection. It consists of a naphthoquinone ring and a side chain with varying numbers of isoprenoid units; hence, it is abbreviated as MK-n, where n represents the number of isoprenoid units. Since long-chain MKs (n=5–13), which are produced only by bacteria, exhibit longer half-lives and higher bioavailability than short-chain MKs, the microbial production of long-chain MKs has attracted great interest in the fields of food microbiology and synthetic biology. Lactococcus lactis, a key starter strain for dairy processing, is an excellent candidate as it produces MK-8, MK-9, and MK-10. In recent years, research on MK biosynthesis in L. lactis has gained increasing attention, and improvements in MK production have been achieved through physiological exploration, fermentation optimization, and metabolic engineering. This review summarizes the MK biosynthetic pathway, its physiological functions, and regulation of the MK profile as well as the production titer in L. lactis. Moreover, it discusses the high-yield schemes of MK in Escherichia coli and Bacillus subtilis to provide insights for developing L. lactis as an MK-producing cell factory.

Lactococcus lactis is a well-known workhorse for dairy products, whose important industrial traits are tightly associated with numerous cytoplasmic membrane proteins. However, roles of the signal recognition particle (SRP) pathway responsible for membrane protein targeting have not been studied in L. lactis. In this work, the putative genes ffh and ftsY encoding SRP pathway components were identified in the genome of L. lactis NZ9000. Experimental evidence showed that sequence mutation in either the ffh or ftsY was not lethal, but prolonged the lag phase of the resultant mutants Δffh and ΔftsY by 2 h and lowered their biomass to 85.7 % of the wild type under static conditions, as well as deprived the mutants of improved growth capacity under aerobic respiration conditions. Besides, the speeds of glucose consumption and lactate production were significantly decreased in the mutants. Then, the impact of the SPR components on acid resistance was detected, showing that the ffh and ftsY were transcriptionally upregulated by 3.02 ± 1.21 and 8.66 ± 1.01-fold in the wild type during acid challenge at pH 3.0, and cell survival of the Δffh and ΔftsY decreased by10- and 100-fold compared with the wild type. To explore the possible mechanism about the SRP pathway involved in the above physiological traits, proteomics analysis was performed and revealed that disruption of the Ffh or FtsY led to decrease in ribosomal proteins, but increase in DnaK, GroEL and heat shock protein GrpE, indicating that the SRP pathway was closely linked to protein synthesis and folding in L. lactis. Decrease in the fructose-bisphosphate aldolase, respiratory complexes NADH dehydrogenase, as well as glutamate decarboxylase was also detected in the Δffh and ΔftsY, which is consistent with the phenomena of impaired sugar metabolism and acid resistance. Our results demonstrated the dispensable SRP pathway could contribute to the maintenance of metabolism homeostasis and acid resistance of L. lactis.

A novel bivalent oral vaccine candidate against H5N1 and H9N2 avian influenza virus (AIV) was developed using Lactobacillus surface display technology without genetic modification. The hemagglutinin subunit 1 (HA1) antigens from both subtypes were fused to the surface layer-binding domain of Lactobacillus crispatus K313, expressed in Escherichia coli, and purified. Wild-type Lactobacillus johnsonii H31, isolated from chicken intestine, served as a delivery vehicle by adsorbing and stably displaying the HA1 proteins on its surface. This approach eliminates the need for bacterial engineering while utilizing lactobacilli’s natural capacity to protect surface-displayed antigens, as evidenced by HA1’s protease resistance. Mouse immunization studies demonstrated induction of strong systemic IgG and mucosal IgA responses against both H5N1 and H9N2 HA1. The system offers several advantages, including safety through non-GMO probiotics, potential for multivalent vaccine expansion, and intrinsic antigen protection by lactobacilli. These findings suggest this platform could enable development of cost-effective, multivalent AIV vaccines.

Limosilactobacillus reuteri is a well-recognized probiotic that improves gut homeostasis through the tryptophan metabolite indole-3-lactic acid (ILA), while the functional pathway underlying ILA biosynthesis remains unidentified. In this study, we identified two Lb. reuteri gut isolates, SDMCC050455 and SDMCC050493 exhibiting significant and negligible ILA production, respectively. Comparative genomic analysis and transcriptional determination suggested that six aromatic amino acid aminotransferases (AraT1 to AraT6) and two indole lactate dehydrogenases (FldH2 and FldH3) might participate in ILA biosynthesis. To identify the key aminotransferase, all six transcriptionally active AraTs were overexpressed, and AraT1 increased ILA production by 24.8% in SDMCC050455 and restored ILA production in SDMCC050493. Conversely, transcription suppression of AraT1 significantly reduced the ILA yield in SDMCC050455. Biochemical characterization confirmed AraT1 as a real aminotransferase with high affinity for l-tryptophan. Finally, an ILA biosynthesis pathway was reconstructed by coexpressing AraT1 with either FldH2 or FldH3, endowing Lactococcus lactis with ILA-producing ability. Homology searches using AraT1 and FldH2/3 identified sequences with over 90% identity in 720 of 1020 Lb. reuteri genomes, indicating prevalence of the ILA biosynthesis pathway. Our results demonstrated a novel AraT and functional FldHs forming a complete pathway for ILA biosynthesis in Lb. reuteri, providing insights for probiotic screening and customized ILA production.

Streptococcus thermophilus is traditionally used as a starter culture in the production of dairy fermented products. Recently this species has highlighted its potential benefits for skin health. This study evaluated the protective effects of S. thermophilus CGMCC 24468 on keratinocytes HaCaT. Co-incubation with the supernatant of sonicated fermented milk lysate (SFML) enhanced the expression of tight junction proteins (CLDN-1, OCLN, and JAM-1) and the transcription of antioxidative enzyme genes (CAT, SOD, and GPX), reducing intracellular ROS levels. Pre-treatment with SFML inhibited the activation of the MAPK pathway and accelerated wound closure. Collectively, these results demonstrated the potential application of S. thermophilus CGMCC 24468 fermented milk as a natural ingredient in cosmetic formulations for preventing ROS-induced skin photoaging.

Lactococcus lactis and Streptococcus thermophilus are considered as ideal chassis of engineered probiotics, while food-grade genetic tools are limited in those strains. Here, a Zn2+-controlled gene expression (ZICE) system was identified in the genome of S. thermophilus CGMCC7.179, including a transcriptional regulator sczAst and a promoter region of cation transporter czcD (PczcDst). Specific binding of the SczAst to the palindromic sequences in PczcDst was demonstrated by EMSA analysis, suggesting the regulation role of SczAst on PczcDst. To evaluate their possibility to control gene expression in vivo, the sczAst-PczcDst was employed to drive the expression of green fluorescence protein (GFP) gene in L. lactis NZ9000 and S. thermophilus CGMCC7.179, respectively. Both of the transformants could express GFP under Zn2+ induction, while no fluorescence without Zn2+ addition. For optimal conditions, Zn2+ was used at a final concentration of 0.8 mM in L. lactis and 0.16 mM in S. thermophilus at OD600 close to 0.4, and omitting yeast extract powder in the medium unexpectedly improved GFP expression level by 2.2-fold. With the help of the ZICE system, engineered L. lactis and S. thermophilus strains were constructed to secret cytokine interleukin-10 (IL-10) with immunogenicity, and the IL-10 content in the supernatant of the engineered L. lactis was 59.37 % of that under the nisin controlled expression system. This study provided a tightly controlled expression system by the food-grade inducer Zn2+, having potential in development of engineered probiotics.

Bifidobacteria are the most prevalent members of the intestinal microbiota in mammals and other animals, and they play a significant role in promoting gut health through their probiotic effects. Recently, the potential applications of Bifidobacteria have been extended to skin health. However, the beneficial mechanism of Bifidobacteria on the skin barrier remains unclear. In this study, keratinocyte HaCaT cells were used as models to evaluate the protective effects of the cell-free supernatant (CFS), heat-inactivated bacteria, and bacterial lysate of Bifidobacterium animalis CGMCC25262 on the skin barrier and inflammatory cytokines. The results showed that all the tested samples were able to upregulate the transcription levels of biomarker genes associated with the skin barrier, such as hyaluronic acid synthetase (HAS) and aquaporins (AQPs). Notably, the transcription of the hyaluronic acid synthetase gene-2 (HAS-2) is upregulated by 3~4 times, and AQP3 increased by 2.5 times when the keratinocyte HaCaT cells were co-incubated with 0.8 to 1% CFS. In particular, the expression level of Filaggrin (FLG) in HaCaT cells increased by 1.7 to 2.7 times when incubated with Bifidobacterial samples, reaching its peak at a concentration of 0.8% CFS. Moreover, B. animalis CGMCC25262 also decreased the expression of the proinflammatory cytokine RANTES to one-tenth compared to the levels observed in HaCaT cells induced with tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). These results demonstrate the potential of B. animalis CGMCC25262 in protecting the skin barrier and reducing inflammatory response.

2-O-α-D-glucosyl glycerol (2-αGG) is a valuable ingredient in cosmetics, health-care and food fields. Sucrose phosphorylase (SPase) is a favorable choice for biosynthesis of 2-αGG, while its glucosyl-acceptor affinity and thermodynamic feature remain largely unknown, limiting 2-αGG manufacturing. Here, three SPases were obtained from lactobacilli and bifidobacteria, and the one encoded by Lb. reuteri SDMCC050455 (LrSP) had the best transglucosylation ability, with 2-αGG accounting for 86.01

CovRS two-component regulatory system involved in stress adaptation in Group A Streptococcus (GAS). This system has also been identified in the genome of Streptococcus thermophilus. To investigate its roles, covR (covRst) was knocked out, and transcriptome analysis was performed in S. thermophilus ST222. The deletion of the covRst resulted in the upregulation of the antioxidative enzymes, which possessed the conserved sequence WAAAAAGGAGV in their promoter regions. The transcriptional levels of the antioxidative enzyme genes of the peroxidase efeB and glutathione peroxidase gshF in the covRst defective mutant were increased by approximately 3.3-fold and 1.6-fold, respectively, as determined by qPCR analysis. EMSA analysis also confirmed the binding abilities of the CovRst protein to the promoter regions of gshF and efeB. Moreover, the glutathione (GSH) content and cell viability of the deficient mutant ST222 ΔcovR were higher than those of the wild-type ST222. Furthermore, the intestinal epithelium cells NCM 460 was used as a model to verify that the deficient mutant ST222 ΔcovR with strong cytoprotective roles against H2O2. Therefore, all experimental data demonstrated that CovRst served as a negative regulator to mediate the environmental adaptation of oxidative stress in S. thermophilus.

Streptococcus thermophilus is a traditional starter for yogurt making. Here, an eps gene cluster encoded for exopolysaccharides (EPS) was identified in the genome of S. thermophilus CGMCC 7.179. After purifying by DEAE-Sepharose Fast-Flow column chromatography, three main components ST-ESP1, ST-EPS2 and ST-EPS3, consisted of mannose, glucuronic acid, galacturonic acid, glucose and N-acetylglucosamine, were obtained. ST-EPS2 showed stronger antioxidant activity than the others in vitro. Moreover, ST-EPS2 could significantly upregulate the transcription levels of NF-E2-related factor-2 (Nrf2) and promote the expression of antioxidant-related proteins involved in Nrf2, suggesting ST-EPS2 protected the intestinal epithelial NCM 460 cells by attenuating the intracellular reactive oxygen species (ROS) level rising against oxidative stress. The potential binding mode of the mannose, galacturonic acid and N-acetylglucosamine with Keap1 simulated through molecular docking confirmed the action of ST-EPS2 with the protein Nrf2. Those results demonstrated that the ST-EPS2 provided opportunities for a natural antioxidant agent exploration in the food industry.

Lacticaseibacillus paracasei is an economically important bacterial species, used in the food industry and as a probiotic. Here, we investigate the roles of N6-methyladenine (6mA) modification in L. paracasei using multi-omics and high-throughput chromosome conformation capture (Hi-C) analyses. The distribution of 6mA-modified sites varies across the genomes of 28 strains, and appears to be enriched near genes involved in carbohydrate metabolism. A pglX mutant, defective in 6mA modification, shows transcriptomic alterations but only modest changes in growth and genomic spatial organization.

Lactobacillus plantarum is a catalase-negative species and distributes in human intestinal tracts. However, the cytoprotective effects of the catalase-activated L. plantarum strain have yet to be exploited against reactive oxygen species (ROS). Here, a catalase-activated L. plantarum CGMCC 6888 (CatA+) was obtained using exogenous added heme. The scavenging free radical abilities of this strain were obviously increased. Moreover, the activated catalase A in L. plantarum CGMCC 6888 endowed the intestinal epithelium NCM460 with lower ROS content after degrading H2O2. In addition, the transcription levels of Nrf2 and Nrf2-related antioxidant enzyme genes (HO-1, GCLC, NQO-1 and TXNRD1) and tight junction protein genes (ZO-1, OCLN, and JAM-1) were upregulated significantly when co-incubated with CGMCC 6888/CatA+. This work confirmed that the catalase A conferred L. plantarum with the strong protection effects in the intestinal epithelial cells against ROS.

β-Galactosidase is one of the most important enzymes used in dairy processing. It converts lactose into glucose and galactose, and also catalyzes galactose to form galactooligosaccharides (GOS), so-called prebiotics. However, most of the β-galactosidases from the starter cultures have low transgalactosylation activities, the process that results in galactose accumulation in yogurt. Here, a site-directed mutation strategy was attempted, to genetically modify β-galactosidase from Streptococcus thermophilus. Out of 28 Strep. thermophilus strains, a β-galactosidase gene named bgaQ, encoded for high β-galactosidase hydrolysis activity (BgaQ), was cloned from the strain Strep. thermophilus SDMCC050237. It was 3,081 bp in size, with 1,027 deduced amino acid residuals, which belonged to the GH2 family. After replacing the Tyr801 and Pro802 around the active sites of BgaQ with His801 and Gly802, the GOS synthesis of the generated mutant protein BgaQ-8012 increased from 20.5% to 26.7% at 5% lactose, and no hydrolysis activity altered obviously. Subsequently, the purified BgaQ or BgaQ-8012 was added to sterilized milk inoculated with 2 starters from Strep. thermophilus SDMCC050237 and Lactobacillus delbrueckii ssp. bulgaricus ATCC11842. The GOS yields with added BgaQ or BgaQ-8012 increased to 5.8 and 8.3 g/L, respectively, compared with a yield of 3.7 g/L without enzymes added. Meanwhile, the addition of the BgaQ or BgaQ-8012 reduced the lactose content by 49.3% and 54.4% in the fermented yogurt and shortened the curd time. Therefore, this study provided a site-directed mutation strategy for improvement of the transgalactosylation activity of β-galactosidase from Strep. thermophilus for GOS-enriched yogurt making.

Most Streptococcus thermophilus strains possess the conserved gadBC gene cluster for gamma-aminobutyric acid (GABA) synthesis, endowing yogurt with special beneficial characteristics. Here, we found that GABA yields of S. thermophilus were always positively correlated with fast-acidification. To explore the regulation mechanism of acid production to GABA synthesis, the activities of β-galactosidase (LacZ), lactate dehydrogenase (Ldh) and glutamate decarboxylase (GAD) were determined in strains SDMCC050243 and SDMCC050242. The results showed the higher enzyme activities of LacZ and Ldh at the exponential growth phases, the higher GAD activity. Furthermore, the activity of PgadB promoter responding to the transcription of gadB gene increased with the decrease of pH, suggesting that acid production activated the expression of GAD to synthesize GABA. Meanwhile, the viability of S. thermophilus increased with the accumulation of GABA, confirming the protection role of GABA synthesis against acid stress. In milk fermentation using ten GABA-producing strains, the more GABA yields were always accompanied with the shorter curd time, further illustrating that fast-acidification was beneficial to GABA synthesis. Therefore, this study revealed that fast-acidification promoted GABA biosynthesis in S. thermophilus, subsequently protected strains against acid stress, providing theoretical foundations for making the GABA-enriched yogurt.

Streptococcus thermophilus is a common yogurt starter that consumes lactose as its primary carbon source. The enzyme β-galactosidase is essential for the lactose metabolism and the growth of this species. Streptococcus thermophilus appears to be a promising cell factory. Food-grade vectors have advantages in heterologous protein expression. This study aimed to determine whether the β-galactosidase of S. thermophilus has the α-complementary characteristic and to develop a novel food-grade vector based on this phenomenon. The N-terminal 7 to 36 AA residues of the β-galactosidase in S. thermophilus were deleted. The obtained mutant S. thermophilus Δα lost β-galactosidase activity and growth ability in the lactose medium. Subsequently, plasmids expressing α-fragments with different lengths of 1 to 36 (Sα1), 1 to 53 (Sα2), and 1 to 88 (Sα3) AA were constructed and transformed into S. thermophilus Δα. Recombinant S. thermophilus Δα expressing Sα2 or Sα3 recovered the ability to grow in the lactose medium, and their β-galactosidase activity accounted for 24.5% or 11.5% of the wild strain, respectively. These results indicated that the α-complementation system of β-galactosidase existed in S. thermophilus. Based on the characteristic, a food-grade vector pSEα was constructed. Except for Sα2, vector pSEα expressed the α-donor derived from E. coli β-galactosidase. This facilitated the construction of recombinant plasmids in E. coli DH5α and thus improved the transformation efficiency of S. thermophilus. Green fluorescent protein as a reporter protein could be highly expressed in S. thermophilus using this vector. As a result, pSEα is an efficient and safe vector for S. thermophilus with potential food applications.

A simple generic method for enhancing extracellular protein yields in engineered bacteria is still lacking. Here, we demonstrated that phage-encoded holin can be used to export proteins to the extracellular medium in both Gram-negative Escherichia coli and -positive Lactococcus lactis. When a putative holin gene LLNZ_RS10380 annotated in the genome of L. lactis NZ9000 (hol380) was recombinantly expressed in E. coli BL21(DE3), the Hol380 oligomerized up to hexamer in the cytoplasmic membrane, yielding membrane pore to allow the passage of cytosolic β-galatosidase (116 kDa), whose extracellular production reached 54.59 U/μl, accounting for 76.37% of the total activity. However, the overexpressed Hol380 could not release cytosolic proteins across the membrane in L. lactis NZ9000, but increased the secretory production of staphylococcal nuclease to 2.55-fold and fimbrial adhesin FaeG to 2.40-fold compared with those guided by signal peptide Usp45 alone. By using a combination of proteomics and transcriptional level analysis, we found that overexpression of the Hol380 raised the accumulation of Ffh and YidC involved in the signal recognition particle pathway in L. lactis, suggesting an alternative road participating in protein secretion. This study proposed a new approach by expressing holin in bacterial cell factories to export target proteins of economic or medical interest.

Enteric diseases caused by Salmonella are prevalent in poultry farming. With the forbiddance of antibiotics in feedstuff industry, Bacillus subtilis (B. subtilis) preparation as antibiotic alternatives against Salmonella infection has gained increasing attention recently. However, the protection modes of B. subtilis against Salmonella infection in broilers are strain-specific. In this study, probiotic B. subtilis LF11 significantly reduced diarrhea and mortality of broilers caused by Salmonella braenderup (S. braenderup) in spite of no inhibition effect on it in vitro. Here, the intestinal epithelial cells NCM460 were incubated to explore the protection of B. subtilis LF11 on intestinal epithelium against Salmonella. The results revealed that B. subtilis LF11 showed obvious exclusion activity with the decrease of adhesion and invasion of S. braenderup to NCM460 cells, accordingly with the increase of NCM460 cell survival compared with S. braenderup challenge alone. Meanwhile, RT-PCR and Western blot proved that the gene transcription and expression levels of four tight junction proteins in NCM 460 cells were upregulated, which was further confirmed by immunofluorescence observation. Besides, B. subtilis LF11 downregulated the gene transcription levels of the proinflammatory cytokines IL-6, IL-8, and TNF-α induced by S. braenderup H9812. ELISA analysis also verified that B. subtilis LF11 reduced the IL-8 production significantly. In general, B. subtilis LF11 has the ability to protect the intestinal epithelium against Salmonella infection by reducing the Salmonella adhesion and invasion, enhancing the intestinal barrier and attenuating the enterocyte inflammatory responses, and has the potential as probiotics to prevent enteric diseases in broilers.

Lactococcus lactis is a food-grade chassis for delivery of bioactive molecules to the intestinal mucosa in situ, while its ability to produce lycopene for detoxification of reactive oxidative species (ROS) is not realized yet. Here, L. lactis NZ9000 was engineered to synthesize lycopene by heterologous expression of a gene cluster crtEBI in plasmids or chromosomes, yielding the recombinant strains NZ4 and NZ5 with 0.59 and 0.54 mg/L lycopene production, respectively. To reroute the pyruvate flux to lycopene, the main lactate dehydrogenase and α-acetolactate synthase pathways were sequentially disrupted. The resultant strains NZΔldh-1 and NZΔldhΔals-1 increased lycopene accumulation to 0.70 and 0.73 mg/L, respectively, while their biomasses were reduced by 12.42% and the intracellular NADH/NAD+ ratios increased by 3.05- and 2.10-fold. To increase the biomasses of these engineered strains, aerobic respiration was activated and tuned by the addition of exogenous heme and oxygen. As a result, the engineered L. lactis strains partly recovered the growth and redox balance, yielding the lycopene levels of 0.91-1.09 mg/L. The engineered L. lactis strain protected the intestinal epithelial cells NCM460 against H2O2 challenge, with a 30.09% increase of cell survival and a 29.2% decrease of the intracellular ROS level compared with strain NZ9000 treatment. In summary, this work established the use of the engineered probiotic L. lactis for lycopene production and prospected its potential in the prevention of intestinal oxidative damage.