Characterization of a Panel of Constitutive Promoters from Lactococcus cremoris for Fine-Tuning Gene Expression.

Lactococcus cremoris (homotypic synonym: Lactococcus lactis) is receiving increasing attention as a prominent vehicle for the delivery of live vaccines. This can hardly be achieved without developing tools for the genetic manipulation of L. cremoris, and the paucity of studies on L. cremoris endogenous promoters has attracted our attention. Here, we report the discovery and characterization of 29 candidate promoters identified from L. cremoris subsp. cremoris NZ9000 by RNA sequencing analysis. Furthermore, 18 possible constitutive promoters were obtained by RT-qPCR screening from these 29 candidate promoters. Then, these 18 promoters were cloned and characterized by a reporter gene, gusA, encoding ß-glucuronidase. Eventually, eight endogenous constitutive promoters of L. cremoris were obtained, which can be applied to genetic manipulation of lactic acid bacteria.

The Arginine Repressor ArgR2 Controls Conjugated Linoleic Acid Biosynthesis by Activating the cla Operon in Lactiplantibacillus plantarum.

CLA (conjugated linoleic acid) has attracted substantial attention due to its physiological functions, including regulating immunity, reducing obesity, and contributing to cancer suppression. In Lactiplantibacillus plantarum, CLA oleate hydratase (CLA-HY), CLA short-chain dehydrogenase (CLA-DH), and CLA acetoacetate decarboxylase (CLA-DC) catalyze the biotransformation of linoleic acid (LA) to CLA. However, the underlying transcriptional regulation mechanism of this pathway remains largely unknown. In this study, the potential transcriptional regulators that might bind to the cla promoter of L. plantarum AR195 were investigated by DNA pulldown. Interestingly, ArgR2, the transcriptional regulator of arginine metabolism, was identified as a potential regulator involved in the regulation of CLA biotransformation. Electrophoretic mobility shift assay (EMSA) and molecular interaction results demonstrated the specific binding of ArgR2 to the regulatory region of the cla operon. The knockout of argR2 led to the downregulation of cla-dh and cla-dc by 91% and 34%, respectively, resulting in a decline in the CLA yield by 14%. A segmental EMSA revealed that ArgR2 bound to three distinct sites in the cla regulatory region, and these binding sites were highly conserved and rich in AT. The regulatory mechanism of ArgR2 on CLA biosynthesis further expanded our knowledge of the regulatory mechanism of CLA biosynthesis in L. plantarum and laid the theoretical foundation for the production and application of CLA. IMPORTANCE CLA (conjugated linoleic acid) has received extensive attention owing to its important physiological functions. CLA from natural sources is far from meeting people's demands. Lactic acid bacteria can efficiently synthesize cis-9,trans-11-CLA and trans-10,cis-12-CLA, which possess physiological activities. However, little is known about the regulatory mechanism. In this study, we identified that the biosynthesis of CLA in L. plantarum AR195 was transcriptionally regulated by the arginine biosynthesis regulatory protein ArgR2. The regulation mechanism of ArgR2 on CLA biosynthesis lays a theoretical foundation for the regulation of CLA synthesis and industrial production.

Determination of the regulatory network and function of the lysR-type transcriptional regulator of Lactiplantibacillus plantarum, LpLttR.

BACKGROUND: Lactiplantibacillus plantarum has various healthcare functions including the regulation of immunity and inflammation, reduction of serum cholesterol levels, anti-tumor activity, and maintenance of the balance of intestinal flora. However, the underlying metabolic and regulatory mechanisms of these processes remain unclear. Our previous studies have shown that the LysR type transcriptional regulator of L. plantarum (LpLttR) regulates the biotransformation of conjugated linoleic acids (CLAs) through the transcriptional activation of cla-dh (coding gene for CLA short-chain dehydrogenase) and cla-dc (coding gene for CLA acetoacetate decarboxylase). However, the regulatory network and function of LpLttR have not yet been characterized in L. plantarum. RESULTS: In this study, the regulatory role of LpLttR in various cellular processes was assessed using transcriptome analysis. The deletion of LpLttR had no evident influence on the bacterial growth. The transcriptome data showed that the expression of nine genes were positively regulated by LpLttR, and the expression of only two genes were negatively regulated. Through binding motif analysis and molecular interaction, we demonstrated that the regulatory region of the directly regulated genes contained a highly conserved sequence, consisting of a 15-base long box and rich in AT. CONCLUSION: This study revealed that LpLttR of L. plantarum did not play a global regulatory role similar to that of the other transcriptional regulators in this family. This study broadens our knowledge of LpLttR and provides a theoretical basis for the utilization of L. plantarum.

Reasons for the differences in biotransformation of conjugated linoleic acid by Lactobacillus plantarum.

Conjugated linoleic acid (CLA) has attracted a great deal of attention for its functions in weight loss, regulation of metabolism, and antioxidant capabilities. Many microorganisms, including rumen bacteria, propionic acid bacilli, and Lactobacillus, have CLA biotransformation ability. The CLA production capability of different species is different, as are those different strains of the same species. However, the reasons for this discrepancy remain unclear. In this study, 14 strains of Lactobacillus plantarum were found, through gas chromatography-mass spectrometry analysis, to be capable of converting linoleic acid to CLA. The transcriptional levels of CLA-related genes in the high- (AR195, WCFS1, and AR488) and low-yield strains (AR176, AR269, and AR611) were analyzed using real-time quantitative PCR. The transcriptional levels of cla-hy, cla-dh, and cla-dc in AR195 were the lowest in the exponential phase, but it had the highest CLA yield. Correlation analysis showed no correlation between CLA yield and the transcription level of these genes in the exponential phase. The results showed that a high transcriptional level in the exponential phase of cla-hy, cla-dh, and cla-dc did not necessarily lead to high CLA production. Investigation of the transcription level in different growth phases showed that the CLA biotransformation abilities of Lactobacillus plantarum strains significantly depended on the transcriptional maintenance of cla-hy, cla-dh, and cla-dc. We observed a correlation between CLA production and increased levels of cla-hy transcription, but a prerequisite is needed: the transcription of cla-dh and cla-dc should be upregulated and maintained a high transcriptional level during the platform period. This study provides a new strategy for screening high CLA-producing strains. It also lays a theoretical foundation for regulating CLA biotransformation and increasing the yield of CLA.

High-efficiency transformation of Streptococcus thermophilus using electroporation.

BACKGROUND: Streptococcus thermophilus, one of the most important lactic acid bacteria, is widely used in food fermentation, which is beneficial to improve food quality. However, the current genetic transformation systems are inefficient for S. thermophilus S-3, which hinders its further study. RESULTS: We developed three electroporation transformation methods for S. thermophilus S-3, and optimized various parameters to enhance the transformation efficiency up to 1.3 × 106 CFU/µg DNA, which was 32-fold higher than that of unoptimized. Additionally, transcriptional analysis showed that a series of competence genes in S. thermophilus S-3 were remarkedly up-regulated after optimization, indicating that improvement of transformation efficiency was attributed to the expression level of competence genes. Furthermore, to prove their potential, expression of competence genes (comEA, cbpD and comX) were employed to increase transformation efficiency. The maximum transformation efficiency was obtained by overexpression of comEA, which was 14-fold higher than that of control. CONCLUSION: This is the first report of competence gene expression for enhancing transformability in S. thermophilus, which exerts a positive effect on the development of desirable characteristics strains. © 2021 Society of Chemical Industry.

Recent Research Advances in Small Regulatory RNAs in Streptococcus.

Small non-coding RNAs (sRNAs) are a class of regulatory RNAs 20-500 nucleotides in length, which have recently been discovered in prokaryotic organisms. sRNAs are key regulators in many biological processes, such as sensing various environmental changes and regulating intracellular gene expression through binding target mRNAs or proteins. Bacterial sRNAs have recently been rapidly mined, thus providing new insights into the regulatory network of biological functions in prokaryotes. Although most bacterial sRNAs have been discovered and studied in Escherichia coli and other Gram-negative bacteria, sRNAs have increasingly been predicted and verified in Gram-positive bacteria in the past decade. The genus Streptococcus includes many commensal and pathogenic Gram-positive bacteria. However, current understanding of sRNA-mediated regulation in Streptococcus is limited. Most known sRNAs in Streptococcus are associated with the regulation of virulence. In this review, we summarize recent advances in understanding of the functions and mechanisms of sRNAs in Streptococcus, and we discuss the RNA chaperone protein and synthetic sRNA-mediated gene regulation, with the aim of providing a reference for the study of microbial sRNAs.

LysR Family Regulator LttR Controls Production of Conjugated Linoleic Acid in Lactobacillus plantarum by Directly Activating the cla Operon.

Conjugated linoleic acids (CLAs) have attracted more attention as functional lipids due to their potential physiological activities, including anticancer, anti-inflammatory, anti-cardiovascular disease, and antidiabetes activities. Microbiological synthesis of CLA has become a compelling method due to its high isomer selectivity and convenient separation and purification processes. In Lactobacillus plantarum, the generation of CLA from linoleic acids (LAs) requires the combination of CLA oleate hydratase (CLA-HY), CLA short-chain dehydrogenase (CLA-DH), and CLA acetoacetate decarboxylase (CLA-DC), which are separately encoded by cla-hy, cla-dh, and cla-dc. However, the regulatory mechanisms of CLA synthesis remain unknown. In this study, we found that a LysR family transcriptional regulator, LTTR, directly bound to the promoter region of the cla operon and activated the transcription of cla-dh and cla-dc. The binding motif was also predicted by bioinformatics analysis and verified by electrophoretic mobility shift assays (EMSAs) and DNase I footprinting assays. The lttR overexpression strain showed a 5-fold increase in CLA production. Moreover, we uncovered that the transcription of lttR is activated by LA. These results indicate that LttR senses LA and promotes CLA production by activating the transcription of cla-dh and cla-dc. This study reveals a new regulatory mechanism in CLA biotransformation and provides a new potential metabolic engineering strategy to increase the yield of CLA.IMPORTANCE Our work has identified a novel transcriptional regulator, LTTR, that regulates the production of CLA by activating the transcription of cla-dh and cla-dc, essential genes participating in CLA synthesis in Lactobacillus plantarum This study provides insight into the regulatory mechanism of CLA synthesis and broadens our understanding of the synthesis and regulatory mechanisms of the biosynthesis of CLA.

The second messenger c-di-AMP mediates bacterial exopolysaccharide biosynthesis: a review.

Cyclic dimeric adenosine 3'-5'-monophosphate (c-di-AMP) is a recently discovered nucleotide messenger in bacteria. It plays an important role in signaling, transcription, and cell physiology, such as in bacterial growth, potassium transport, fatty acid synthesis, the metabolic balance of cell wall components, and biofilm formation. Exopolysaccharides (EPSs) have distinct physico-chemical properties and diverse bioactivities including antibacterial, hypolipidemic, and antioxidative activities, and they are widely used in the food, pharmaceutical, and cosmetic industries. Although c-di-AMP has been demonstrated to regulate the biosynthesis of bacterial EPSs, only a single c-di-AMP receptor, CabpA, has been identified in EPS synthesis. With the aim of describing current understanding of the regulation of microbial EPSs, this review summarizes c-di-AMP biosynthesis and degradation as well as the mechanism through which c-di-AMP regulates bacterial EPSs.

Short communication: Genome-wide identification of new reference genes for reverse-transcription quantitative PCR in Streptococcus thermophilus based on RNA-sequencing analysis.

Streptococcus thermophilus, one of the most important industrial lactic acid bacteria, is widely used for the production of fermented dairy products such as yogurt and cheese. The accuracy of gene expression-based analyses (e.g., reverse-transcription quantitative real-time PCR) relies heavily on the selection of reliable reference genes (RG), which provides the basis for correctly interpreting expression data. However, many traditional RG are not stably expressed in different systems. Here we used RNA-sequencing to systematically investigate gene expression variation at the genome scale and identify more stable RG in S. thermophilus. In total, 21 putative candidate RG were identified with variation coefficient values <10.0 based on the expression of all 1,911 genes under 4 different experimental conditions. We selected and validated 12 RG chosen from transcriptomes by using reverse-transcription quantitative real-time PCR, and ranked their expression stability by statistical algorithms geNorm and NormFinder. Compared with traditional RG 16S rRNA, genes encoding glycine-tRNA ligase subunit ß GlyS and fatty acid-binding protein DegV were more stable under all 4 treatments, which have never been used as RG in S. thermophilus. Our finding provides the foundation for more precise analysis of gene expression in S. thermophilus and other lactic acid bacteria species.

CRISPR-Cas-mediated gene editing in lactic acid bacteria.

The high efficiency, convenience and diversity of clustered regular interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are driving a technological revolution in the gene editing of lactic acid bacteria (LAB). Cas-RNA cassettes have been adopted as tools to perform gene deletion, insertion and point mutation in several species of LAB. In this article, we describe the basic mechanisms of the CRISPR-Cas system, and the current gene editing methods available, focusing on the CRISPR-Cas models developed for LAB. We also compare the different types of CRISPR-Cas-based genomic manipulations classified according to the different Cas proteins and the type of recombineering, and discuss the rapidly evolving landscape of CRISPR-Cas application in LAB.

Short communication: Dynamic changes in bacterial diversity during the production of powdered infant formula by PCR-DGGE and high-throughput sequencing.

Microorganisms such as thermophilic and psychrotrophic bacteria cause spoilage of milk and milk products [e.g., powdered infant formula (PIF)], mainly because they produce heat-stable extracellular enzymes. However, the dynamic changes in microbial diversity during PIF production are still not well understood. We used denaturing gradient gel electrophoresis (DGGE) and high-throughput sequencing (HTS) to investigate bacterial community structure and distribution during the major stages of PIF production: raw milk, pasteurization, mixing, evaporation, and spray-drying. Our PCR-DGGE analysis indicated that Lactobacillus and Pseudomonas spp. were the dominant bacteria at the raw milk and pasteurization stages; Lactococcus, Streptococcus, Enterococcus, and Lactobacillus spp. were abundant during mixing, evaporation, and spray-drying. Our HTS analysis showed that Pseudomonas had an abundance of 96.79% at the raw milk stage. Lactobacillus, Streptococcus, Thermus, Acinetobacter, and Bacteroides spp. were most common after pasteurization. The index of bacterial diversity was highest at the evaporation stage, suggesting a high potential risk of microbial contamination. The results from DGGE and HTS were consistent in reflecting changes in dominant flora, but different in reflecting the richness of bacterial communities present during PIF production: HTS revealed a much higher richness of bacterial species than DGGE. Our findings from DGGE and HTS showed that psychrophilic and thermophilic bacteria were the main flora present during PIF production: psychrophilic bacteria were mainly Pseudomonas spp. and thermophilic bacteria were mainly Lactobacillus, Streptococcus, and Bacillus spp. To our knowledge, this is the first study to report dynamic changes in microbial communities during PIF production. Our results provide insight into bacterial communities and identify potential contamination sources that could serve as a guide for reducing microbial risk.

Influence of freezing temperature before freeze-drying on the viability of various Lactobacillus plantarum strains.

Although freeze-drying is an excellent method for preserving microorganisms, it inevitably reduces cell activity and function. Moreover, probiotic strains differ in terms of their sensitivity to the freeze-drying process. Therefore, it is necessary to optimize the variables relevant to this process. The pre-freezing temperature is a critical parameter of the freeze-drying process, but it remains unclear whether the optimal pre-freezing temperature differs among strains and protectants. This study explored the effects of 4 different pre-freezing temperatures on the survival rates of different Lactobacillus plantarum strains after freeze-drying in the presence of different protectants. Using phosphate-buffered saline solution and sorbitol as protectants, pre-freezing at -196°C, -40°C, and -20°C ensured the highest survival rates after freeze-drying for AR113, AR307, and WCFS1, respectively. Using trehalose, pre-freezing at -20°C ensured the best survival rate for AR113, and -60°C was the best pre-freezing temperature for AR307 and WCFS1. These results indicate that the pre-freezing temperature can be changed to improve the survival rate of L. plantarum, and that this effect is strain-specific. Further studies have demonstrated that pre-freezing temperature affected viability via changes in cell membrane integrity, membrane permeability, and lactate dehydrogenase activity. In summary, pre-freezing temperature is a crucial factor in L. plantarum survival after freeze-drying, and the choice of pre-freezing temperature depends on the strain and the protectant.

Antrodin A from mycelium of Antrodia camphorata alleviates acute alcoholic liver injury and modulates intestinal flora dysbiosis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Antrodia camphorata (A. camphorata) is a rare functional fungus in Taiwan and contains a variety of biologically active ingredients. Antrodin A (AdA) is one of the main active ingredients in the solid-state fermented A. camphorata mycelium. It protects the liver from alcohol damage by improving the antioxidant and anti-inflammatory capacity of the liver and maintaining the stability of the intestinal flora. AIM OF THE STUDY: The aim of this study was to evaluate the hepatoprotective activities of ethyl acetate layer extract (EALE), AdA, and Antroquinonol (Aq) from mycelium of A. camphorata on alcoholic liver injury. MATERIALS AND METHODS: Mice were given with intragastrically vehicle (NC, 2% CMC-Na), alcohol (AL, 12 mL/kg bw), or different A. camphorata samples (EALE, AdA, Aq) at low (100 mg/kg bw) or high (200 mg/kg bw) dosages. The positive control (PC) group was given with silymarin (200 mg/kg bw). Except the NC group, each group of mice was fasted for 4 h after the last treatment and was intragastrically administrated with 50% alcohol (12 mL/kg bw). At the end of experiment, mouse serum was collected and the liver was excised. A portion of the liver was fixed in formalin and used for histopathological analysis, whereas the rest was used for biochemical analysis and real-time PCR analysis. The intestinal flora structure of feces was analyzed by determining the v3-v4 region sequence in 16S rDNA. RESULTS: The high-dose groups of the three samples (EALEH, AdAH, and AqH) significantly alleviated the alcohol-induced increases in liver index, serum ALT, AST, and AKP activities. Serum TG level was significantly reduced in all treatment groups. The increase of HDL-C content indicated that active ingredients of A. camphorata could reduce the lipid content in serum. Furthermore, MDA contents of the AdAH and AqH groups in liver were significantly reduced, accompanying with the levels of SOD, CAT, and GSH elevated to various extents. Antioxidant and anti-inflammatory capabilities in the liver were increased in the AdAH group, as evidenced by the mRNA expression levels of Nrf-2 and HO-1 were significantly increased; while those of CYP2e1, TNF-α, and TLR-4 were significantly decreased. Analysis of intestinal flora of feces showed that alcohol treatment significantly changed the composition of intestinal flora. Supplementation with AdA could mitigate dysbiosis of intestinal flora induced by alcohol. Flora of Faecalibaculum, Lactobacillus, and Coriobacteriaceae_UCG-002 showed significantly negative correlations with ALT, AST, AKP, and MDA levels. CONCLUSION: Antrodin A could improve the antioxidant and anti-inflammatory capacities of the liver and maintain the stability of intestinal flora. It is potentially a good candidate compound against acute alcoholic liver injury.

An increase in cell membrane permeability in the in situ extractive fermentation improves the production of antroquinonol from Antrodia camphorata S-29.

The goals of this study were to increase the production of antroquinonol (AQ) and to elucidate the response mechanism of the cell membrane during the in situ extractive fermentation (ISEF) of Antrodia camphorata S-29. Through ISEF, the concentration of AQ reached a maximum of 146.1 ± 2.8 mg/L, which was approximately (7.4 ± 0.1)-fold that of the control (coenzyme Q0-induced fermentation). Transcriptome sequencing showed that four genes (FAD2, fabG, SCD, and FAS1) related to fatty acid biosynthesis were upregulated. FAD2 and SCD may regulate the increase in oleic acid (C18:1) and linoleic acid (C18:2) in the cell membrane of A. camphorata S-29, resulting in an increase in cell membrane permeability. AQ was successfully transferred to the n-tetradecane phase through the cell membrane, reducing product feedback inhibition and improving the production of AQ from A. camphorata S-29.

Isolation of biogenic amine-negative lactic acid bacteria for Chinese rice wine fermentation based on molecular marker reverse screening.

BACKGROUND: Chinese rice wine (CRW; a traditional alcoholic beverage in China with unique flavor and high nutritional value) containing high level of biogenic amines (BAs) may be deleterious to human health. The processes of rice soaking, primary fermentation and secondary fermentation were found to be the major factors for accumulation of BAs during industrial CRW production. RESULTS: To reduce the risk of the formation of BAs in CRW production, Enterococcus durans AR315, a BA-negative lactic acid bacterium, was isolated from CRW samples by PCR-based molecular marker reverse screening in this work. With addition of AR315 during steeping rice phase, the level of total BAs was significantly decreased by 45.1% in comparison with the control. Moreover, the final BA concentration with the addition of AR315 was 27.6% lower than that of the control during fermentation phase. CONCLUSIONS: To our knowledge, this is the first report of decreased accumulation of BAs in CRW production using a BA-negative lactic acid bacterium. Hence, using a BA-negative lactic acid bacterium as a starter culture could be an efficient strategy for significantly reducing the formation of BAs, which has the potential for industrial application in CRW production. © 2020 Society of Chemical Industry.

Characterization of a Panel of Strong Constitutive Promoters from Streptococcus thermophilus for Fine-Tuning Gene Expression.

Streptococcus thermophilus, one of the most important probiotic lactic acid bacteria (LAB), is widely used in the dairy industry and attracts a lot of attention in metabolic engineering and synthetic biology. However, the available well-characterized constitutive promoters are rather limited to modulate gene expression in S. thermophilus. Here, a pool of constitutive promoters was screened by RNA-seq analysis and characterized in S. thermophilus, Lactobacillius casei, and Escherichia coli using the reporter red fluorescent protein. To assess their application potential, six constitutive promoters were selected for the expression of superoxide dismutase and significantly improved enzyme activity in the above three bacteria. Moreover, two strong constitutive promoters were used to construct a dual-expression vector for overexpressing epsA and epsE, key proteins of exopolysaccharide (EPS) biosynthesis, resulting in the change of molecular weight and the titer of EPS. Taken together, this is the first well-characterized constitutive promoter library from S. thermophilus, which could be used as a basic toolbox for various applications in LAB and other bacteria.

Genomic and phenotypic analyses of exopolysaccharide biosynthesis in Streptococcus thermophilus S-3.

Streptococcus thermophilus, one of the most important industrial lactic acid bacteria, is widely used as a starter culture in the dairy industry. Streptococcus thermophilus S-3 isolated from Chinese traditional dairy products has shown great potential for the production of larger amounts of exopolysaccharides (EPS), which significantly affect the organoleptic properties of fermented milk products. To understand the relationship between the genotype and phenotype of S. thermophilus S-3 in terms of EPS biosynthesis, its genome of strain S-3 was sequenced and the genes related to carbohydrate utilization, nucleotide sugars synthesis, and EPS biosynthesis were investigated. The genomic analysis revealed that S. thermophilus S-3 can use sucrose, mannose, glucose, galactose, and lactose. Phenotypic analysis showed that S-3 prefers fermenting lactose to fermenting glucose or galactose. The genetic analysis of nucleotide sugars and EPS biosynthesis revealed that S-3 can synthesize uridine diphosphate (UDP)-glucose, deoxythymidine diphosphate-glucose, deoxythymidine diphosphate-rhamnose, UDP-galactose, UDP-N-acetylgalactosamine, and UDP-N-acetylglucosamine. A high yield of EPS from S-3 cultivated with lactose rather than glucose as the carbon source was correlated with high transcriptional levels of the genes associated with metabolism of these nucleotide sugars and EPS biosynthesis. Our results provide a better understanding of EPS biosynthesis in S. thermophilus and can facilitate enhanced EPS production by lactic acid bacteria fermentation via genetic and metabolic engineering approaches.

Comparison of gal-lac operons in wild-type galactose-positive and -negative Streptococcus thermophilus by genomics and transcription analysis.

Streptococcus thermophilus is one of the most important homo-fermentative thermophilic bacteria, which is widely used as a starter culture in dairy industry. Both wild-type galactose-negative (Gal-) S. thermophilus AR333 and galactose-positive (Gal+) S. thermophilus S-3 in this study were isolated from Chinese traditional dairy products. Here, to access the mechanism of the difference of galactose utilization between strains AR333 and S-3, the expression of gal-lac operons was examined using real-time qPCR in the presence of different sugars, and the gene organization of gal-lac operons was characterized using comparative genomics analysis. As compared with medium containing glucose, the expression of gal-lac operons in AR333 and S-3 was significantly activated (> 5-fold) in the presence of galactose or lactose in the medium. More importantly, the expression of gal operon in S-3 was higher than that of AR333, suggesting that the strength of gal promoter in AR333 and S-3 may be different. The genomes of AR333 and S-3 were the first time sequenced to provide insight into the difference of gal-lac operons in these two strains. Comparative genomics analysis showed that gene order and individual gene size of gal-lac operons are conserved in AR333 and S-3. The DNA sequence of gal operon responsible for galactose utilization between AR333 and S-3 is almost identical except that galK promoter of S-3 possesses single base pair mutation (G to A substitution) at -9 box galK region. Moreover, the expression of red fluorescent protein can be activated by galK promoter of S-3, but cannot by galK promoter of AR333 in galactose medium, suggesting that gal operon is silent in AR333 and active in S-3 under galactose-containing medium. Overall, our results indicated that single point mutation at -9 box in the galK promoter can significantly affect the expression of gal operon and is largely responsible for the Gal+ phenotype of S. thermophilus.

Functional analysis and heterologous expression of bifunctional glutathione synthetase from Lactobacillus.

Bifunctional glutathione synthetase (GshF) has recently been reported to simultaneously catalyze the 2-step ATP-dependent biosynthesis of reduced glutathione (GSH). In this work, 19 putative gshF were mined from the complete sequenced genome of 20 representative Lactobacillus species. To functionally analyze these putative GshF, GshF from Lactobacillus plantarum and Lactobacillus casei were selected and successfully expressed in Escherichia coli. Compared with the control without expressing GshF, GSH titers were enhanced significantly in E. coli with overexpression of GshF, demonstrating that putative GshF from Lactobacillus have functional activities on GSH biosynthesis. Moreover, with the expression of GshF from L. plantarum in E. coli as a paradigm, GSH yield (286.5 µM) was strongly improved by 177.9% with optimized induced conditions and precursor concentration compared with the control under unoptimized conditions. Transcriptional analysis showed that key genes of endogenous GSH metabolism and precursor biosynthesis were remarkably suppressed by GshF expression, indicating that the increase of GSH titer was attributed to heterologous expression of GshF. Overall, our results suggested that gshF is enriched in Lactobacillus and that heterologous expression of GshF is an efficient strategy for improving GSH biosynthesis.

Short communication: Improving the activity of bile salt hydrolases in Lactobacillus casei based on in silico molecular docking and heterologous expression.

Bile salt hydrolase (BSH) plays an essential role in the cholesterol-removing effect of lactic acid bacteria, which hydrolyze conjugated bile salts to amino acid and deconjugated bile salts. However, Lactobacillus casei lacks the bsh gene, which may make it highly sensitive to bile salt stress. We wanted to improve the BSH activity of L. casei for various food-industry applications (e.g., milk fermentation). Plate assay testing indicated that Lactobacillus plantarum AR113 has the highest BSH activity. We cloned and sequenced 4 bsh genes from the genome of L. plantarum AR113. Structure modeling and molecular docking of BSH indicated that BSH1 and BSH3 could react efficiently with bile salts, so we selected BSH1 and BSH3 for heterologous expression in L. casei. Compared with single expression of BSH1 or BSH3, co-expression of both protein sequences showed the highest hydrolysis activity by HPLC analysis. Our results suggested that heterologous expression of BSH in L. casei can significantly improve host activity against bile salts, and in silico molecular docking could be an efficient method of rapid screening for BSH with high activity.