Biofortification as a solution for addressing nutrient deficiencies and malnutrition.

Malnutrition, defined as both undernutrition and overnutrition, is a major global health concern affecting millions of people. One possible way to address nutrient deficiency and combat malnutrition is through biofortification. A comprehensive review of the literature was conducted to explore the current state of biofortification research, including techniques, applications, effectiveness and challenges. Biofortification is a promising strategy for enhancing the nutritional condition of at-risk populations. Biofortified varieties of basic crops, including rice, wheat, maize and beans, with elevated amounts of vital micronutrients, such as iron, zinc, vitamin A and vitamin C, have been successfully developed using conventional and advanced technologies. Additionally, the ability to specifically modify crop genomes to improve their nutritional profiles has been made possible by recent developments in genetic engineering, such as CRISPR-Cas9 technology. The health conditions of people have been shown to improve and nutrient deficiencies were reduced when biofortified crops were grown. Particularly in environments with limited resources, biofortification showed considerable promise as a long-term and economical solution to nutrient shortages and malnutrition. To fully exploit the potential of biofortified crops to enhance public health and global nutrition, issues such as consumer acceptance, regulatory permitting and production and distribution scaling up need to be resolved. Collaboration among governments, researchers, non-governmental organizations and the private sector is essential to overcome these challenges and promote the widespread adoption of biofortification as a key part of global food security and nutrition strategies.

Antibiofilm Effect of Curcumin on Saccharomyces boulardii during Beer Fermentation and Bottle Aging.

In a prior study, we elucidated the biofilm formation of Saccharomyces boulardii on glass surfaces during beer bottle aging. Here, we supplemented brewing wort with curcumin at 25 µg/mL concentration to mitigate S. boulardii biofilm and enhance beer's functional and sensory attributes. An assessment encompassing biofilm growth and development, fermentation performance, FLO gene expression, yeast ultrastructure, bioactive content, and consumer acceptance of the beer was conducted throughout fermentation and aging. Crystal violet (CV) and XTT reduction assays unveiled a significant (p < 0.05) reduction in biofilm formation and development. Fluorescent staining (FITC-conA) and imaging with confocal laser scanning microscopy provided visual evidence regarding reduced exopolysaccharide content and biofilm thickness. Transcriptional analyses showed that key adhesins (FLO1, FLO5, FLO9, and FLO10) were downregulated, whereas FLO11 expression remained relatively stable. Although there were initial variations in terms of yeast population and fermentation performance, by day 6, the number of S. boulardii in the test group had almost reached the level of the control group (8.3 log CFU/mL) and remained stable thereafter. The supplementation of brewing wort with curcumin led to a significant (p < 0.05) increase in the beer's total phenolic and flavonoid content. In conclusion, curcumin shows promising potential for use as an additive in beer, offering potential antibiofilm and health benefits without compromising the beer's overall characteristics.

Identification, proteolytic activity quantification and biofilm-forming characterization of Gram-positive, proteolytic, psychrotrophic bacteria isolated from cold raw milk.

Psychrotrophic bacteria of raw milk face the dairy industry with significant spoilage and technological problems due to their ability to produce heat-resistant enzymes and biofilms. Despite extensive information about Gram-negative psychrotrophic bacteria in milk, little is known about Gram-positive psychrotrophic bacteria in milk, and their proteolytic activity and biofilm-forming characteristics. In the present study, Gram-positive, proteolytic, psychrotrophic bacteria of cold raw milk were identified, and their proteolytic activity and biofilm-forming capacity were quantified. In total, 12 genera and 22 species were represented among the bacterial isolates, however 50% belonged to three genera, namely Staphylococcus (19.4%), Bacillus (16.7%), and Enterococcus (13.9%). Different levels of proteolytic activity were detected in the identified isolates, even among the strains belonging to the same species. In addition, proteolytic activity was significantly higher at 25°C than at 7°C for all isolates. The crystal violet staining assay in polystyrene microtitre plates revealed a high level of variation in the biofilm-forming capacity at 7°C. After 72 hours of incubation, 11.1% of the strains did not produce a biofilm, while 27.8%, 52.8%, and 8.3% produced low, moderate, and high amounts of biofilm on polystyrene, respectively. The psychrotrophic bacteria were also able to produce biofilms on the surface of stainless steel coupons in ultra-high temperature milk after 72 h of incubation at 7°C; the number of attached cells ranged from 1.34 to 5.11 log cfu/cm2. These results expand the knowledge related to the proteolytic activity and biofilm-forming capacity of Gram-positive psychrotrophic milk bacteria.

Insight into phytase-producing microorganisms for phytate solubilization and soil sustainability.

The increasing demand for food has increased dependence on chemical fertilizers that promote rapid growth and yield as well as produce toxicity and negatively affect nutritional value. Therefore, researchers are focusing on alternatives that are safe for consumption, non-toxic, cost-effective production process, and high yielding, and that require readily available substrates for mass production. The potential industrial applications of microbial enzymes have grown significantly and are still rising in the 21st century to fulfill the needs of a population that is expanding quickly and to deal with the depletion of natural resources. Due to the high demand for such enzymes, phytases have undergone extensive research to lower the amount of phytate in human food and animal feed. They constitute efficient enzymatic groups that can solubilize phytate and thus provide plants with an enriched environment. Phytases can be extracted from a variety of sources such as plants, animals, and microorganisms. Compared to plant and animal-based phytases, microbial phytases have been identified as competent, stable, and promising bioinoculants. Many reports suggest that microbial phytase can undergo mass production procedures with the use of readily available substrates. Phytases neither involve the use of any toxic chemicals during the extraction nor release any such chemicals; thus, they qualify as bioinoculants and support soil sustainability. In addition, phytase genes are now inserted into new plants/crops to enhance transgenic plants reducing the need for supplemental inorganic phosphates and phosphate accumulation in the environment. The current review covers the significance of phytase in the agriculture system, emphasizing its source, action mechanism, and vast applications.

Finger Millet (Eleusine coracana) Plant-Endophyte Dynamics: Plant Growth, Nutrient Uptake, and Zinc Biofortification.

Endophytic fungi and bacteria were isolated from finger millet and their effects on finger millet growth parameters and zinc and NPK contents in grains were studied. Out of 70 fungal and 112 bacterial endophytes, the two best fungal and bacterial isolates were selected on the basis of zinc solubilization and plant-growth-promoting attributes. The fungal isolates identified were Aspergillus terreus and Lecanicillium sp., and the bacterial isolates were Pseudomonas bijieensis and Priestia megaterium. The endophytic zinc, NPK mobilization, and plant-growth-promoting efficacy were determined in a pot experiment with zinc carbonate as the zinc source. Endophytic-primed plants showed enhanced shoot and root lengths compared to the unprimed control. Endophytes increased the zinc content in grains by between 12.12% and 18.80% compared to control plants. Endophytes also augmented the NPK concentrations in seeds compared to control plants and exhibited stability in a diverse range of pHs, temperatures, and NaCl concentrations, and exhibited growth on various carbohydrate and nitrogen sources. This is the first study reporting the interaction of Aspergillus terreus, Lecanicillium sp., Pseudomonas bijieensis, and Priestia megaterium with finger millet for grain Zn biofortification and NPK concentration enhancement. This study indicated that zinc-dissolving endophytes possess the potential for enhancing the zinc and NPK content in grains in addition to the plant-growth-promoting attributes.

Genomics, Proteomics, and Metabolomics Approaches to Improve Abiotic Stress Tolerance in Tomato Plant.

To explore changes in proteins and metabolites under stress circumstances, genomics, proteomics, and metabolomics methods are used. In-depth research over the previous ten years has gradually revealed the fundamental processes of plants' responses to environmental stress. Abiotic stresses, which include temperature extremes, water scarcity, and metal toxicity brought on by human activity and urbanization, are a major cause for concern, since they can result in unsustainable warming trends and drastically lower crop yields. Furthermore, there is an emerging reliance on agrochemicals. Stress is responsible for physiological transformations such as the formation of reactive oxygen, stomatal opening and closure, cytosolic calcium ion concentrations, metabolite profiles and their dynamic changes, expression of stress-responsive genes, activation of potassium channels, etc. Research regarding abiotic stresses is lacking because defense feedbacks to abiotic factors necessitate regulating the changes that activate multiple genes and pathways that are not properly explored. It is clear from the involvement of these genes that plant stress response and adaptation are complicated processes. Targeting the multigenicity of plant abiotic stress responses caused by genomic sequences, transcripts, protein organization and interactions, stress-specific and cellular transcriptome collections, and mutant screens can be the first step in an integrative approach. Therefore, in this review, we focused on the genomes, proteomics, and metabolomics of tomatoes under abiotic stress.

Antifungal metabolites, their novel sources, and targets to combat drug resistance.

Excessive antibiotic prescriptions as well as their misuse in agriculture are the main causes of antimicrobial resistance which poses a growing threat to public health. It necessitates the search for novel chemicals to combat drug resistance. Since ancient times, naturally occurring medicines have been employed and the enormous variety of bioactive chemicals found in nature has long served as an inspiration for researchers looking for possible therapeutics. Secondary metabolites from microorganisms, particularly those from actinomycetes, have made it incredibly easy to find new molecules. Different actinomycetes species account for more than 70% of naturally generated antibiotics currently used in medicine, and they also produce a variety of secondary metabolites, including pigments, enzymes, and anti-inflammatory compounds. They continue to be a crucial source of fresh chemical diversity and a crucial component of drug discovery. This review summarizes some uncommon sources of antifungal metabolites and highlights the importance of further research on these unusual habitats as a source of novel antimicrobial molecules.

Pseudomonas fluorescens group bacterial strains interact differently with pathogens during dual-species biofilm formation on stainless steel surfaces in milk.

In order to develop strategies for preventing biofilm formation in the dairy industry, a deeper understanding of the interaction between different species during biofilm formation is necessary. Bacterial strains of the P. fluorescens group are known as the most important biofilm-formers on the surface of dairy processing equipment that may attract and/or shelter other spoilage or pathogenic bacteria. The present study used different strains of the P. fluorescens group as background microbiota of milk, and evaluated their interaction with Staphylococcus aureus, Bacillus cereus, Escherichia coli O157:H7, and Salmonella Typhimurium during dual-species biofilm formation on stainless steel surfaces. Two separate scenarios for dual-species biofilms were considered: concurrent inoculation of Pseudomonas and pathogen (CI), and delayed inoculation of pathogen to the pre-formed Pseudomonas biofilm (DI). The gram-positive pathogens used in this study did not form dual-species biofilms with P. fluorescens strains unless they were simultaneously inoculated with Pseudomonas strains. E. coli O157:H7 was able to form dual-species biofilms with all seven P. fluorescens group strains, both in concurrent (CI) and delayed (DI) inoculation. However, the percentage of contribution varied depending on the P. fluorescens strains and the inoculation scenario. S. Typhimurium contributed to biofilm formation with all seven P. fluorescens group strains under the CI scenario, with varying degrees of contribution. However, under the DI scenario, S. Typhimurium did not contribute to the biofilm formed by three of the seven P. fluorescens group strains. Overall, these are the first results to illustrate that the strains within the P. fluorescens group have significant differences in the formation of mono-or dual-species biofilms with pathogenic bacteria. Furthermore, the possibility of forming dual-species biofilms with pathogens depends on whether the pathogens form the biofilm simultaneously with the P. fluorescens group strains or whether these strains have already formed a biofilm.

Comparative antioxidant potential of kefir and yogurt of bovine and non-bovine origins.

The aim of this study was to compare the antioxidant potential of the yogurt and kefir produced from ewe, camel, goat, and cow milk. The antioxidant activity of the samples was assessed by measuring total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, ferric reducing antioxidant power (FRAP) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical reducing capacity during 20-day storage at 4 ºC. Kefir and yogurt prepared from ewe and camel milk had significantly higher antioxidative potential than samples made from goat and cow milk (P < 0.05). Ewe kefir (74.55-80.11 mg GAE 100 mL-1) showed the highest TPC followed by cow kefir (65-73.15 mg GAE 100 mL-1), camel kefir (61.2-69.91 mg GAE 100 mL-1) and goat kefir (58.31-73.5 mg GAE 100 mL-1) (P < 0.05). Camel yogurt possesses the highest TPC (56.5-68.25 mg GAE 100 mL-1) followed by ewe (40.32-46.5 mg GAE 100 mL-1), cow (29.5-35.5 mg GAE 100 mL-1) and goat (20.03-26.85 mg GAE 100 mL-1) yogurt (P < 0.05). According to DPPH, FRAP, and ABTS results, the antioxidant activity of samples was as follows in descending order: ewe kefir, camel kefir, ewe yogurt, camel yogurt, cow kefir, goat kefir, goat yogurt, cow yogurt. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13197-021-05139-9.

Lacticaseibacillus rhamnosus GG Survival and Quality Parameters in Kefir Produced from Kefir Grains and Natural Kefir Starter Culture.

The study aimed to determine the effect of starter cultures (kefir grains and natural kefir starter culture without grains) on Lacticaseibacillus rhamnosus GG (LGG) survival and on the quality characteristics of kefir. To this end, the viability of probiotic L. rhamnosus GG strain and the rheological properties and quality parameters of kefir beverages were tested during storage over 21 days at 4 °C. The final LGG counts were 7.71 and 7.55 log cfu/mL in natural kefir starter culture and kefir grain, respectively. When prepared with probiotic bacteria, the syneresis values of kefir prepared using natural kefir starter culture was significantly lower (p < 0.05) than that of kefir made using grains. However, the viscosity indices, hysteresis loop, and dynamic moduli were similar between kefir made with natural kefir starter culture and other kefir formulations (p > 0.05). Moreover, all samples showed shear-thinning behavior. The flavor scores for kefir prepared using natural kefir starter culture were significantly higher than for the other samples (p < 0.05), but overall acceptability was similar at the 10-day assessment across both starters (with and without grain) after the addition of probiotic bacteria (p > 0.05). Overall, the results indicate that natural kefir starter culture could be a potential probiotic carrier.

A novel co-cultivation strategy to generate low-crystallinity bacterial cellulose and increase nisin yields.

In this study, a co-culturing Enterobacter sp. and Lactococcus lactis strategy was developed to alter bacterial cellulose (BC) properties and increase nisin yields. We generated high nisin yields (6260 IU/mL) by altering inoculum ratios and inoculation times in a novel co-culture system. Critically, these were 85% higher than L. lactis monocultures. By monitoring fermentation broth pH and lactic acid yields, the pH was higher and lactic acid yields lower during co-culture conditions when compared with L. lactis monocultures, suggesting that co-culturing was more suitable for L. lactis nisin production. We also determined BC film yields and properties (BC, BC-N, and BC-N after nisin release). BC yields produced by co-culturing were not very different from Enterobacter sp. monocultures, but crystallinity was significantly altered. Collectively, our co-culture system adequately and economically modified BC fibers by interfering with self-assembly and crystallization processes during BC synthesis, with significantly improved nisin yields.

Evaluation of the probiotic and postbiotic potential of lactic acid bacteria from artisanal dairy products against pathogens.

INTRODUCTION: Probiotic and postbiotic potential of thirty-two strains of lactic acid bacteria (LAB), obtained earlier from artisanal dairy sources in Pakistan, have been investigated against major multi-drug resistant (MDR) and food borne pathogenic bacteria. METHODOLOGY: LAB strains were identified by 16S rRNA gene sequencing and their antibacterial activity was assessed by the microdilution method. Four LAB isolates, Weissella confusa PL6, Enterococcus faecium PL7, and Lactobacillus delbrueckii PL11 and PL13 were shortlisted. Their ability to degrade lactose and safety for human consumption in terms of hemolysis and antibiotic susceptibility were assessed in vitro. The antibacterial components in the cell-free supernatants (CFSs) of isolate cultures were characterized biochemically by HPLC. RESULTS: Acid neutralization but not protease treatment abolished the antibacterial activity of CFSs. Lactic, acetic and propionic acids were the main acids in the CFSs, and acid production peaked in the stationary phase of growth. The antibacterial activity of the LAB cultures resulted from secretion of organic acids that lowered the pH. The strains exhibited variable ability to degrade lactose and were non-hemolytic and susceptible to the most common antibiotics. CONCLUSIONS: These LAB strains are probiotic candidates for further investigation of their postbiotic role in naturally preserving processed foods and for attenuation of lactose intolerance.

Genomic Features and Construction of Streamlined Genome Chassis of Nisin Z Producer Lactococcus lactis N8.

Lactococcus lactis is a commonly used fermenting bacteria in cheese, beverages and meat products. Due to the lack of simplified chassis strains, it has not been widely used in the fields of synthetic biology. Thus, the construction of lactic acid bacteria chassis strains becomes more and more important. In this study, we performed whole genome sequencing, annotation and analysis of L. lactis N8. Based on the genome analysis, we found that L. lactis N8 contains two large plasmids, and the function prediction of the plasmids shows that some regions are related to carbohydrate transport/metabolism, multi-stress resistance and amino acid uptake. L. lactis N8 contains a total of seven prophage-related fragments and twelve genomic islands. A gene cluster encoding a hybrid NRPS-PKS system that was found in L. lactis N8 reveals that the strain has the potential to synthesize novel secondary metabolites. Furthermore, we have constructed a simplified genome chassis of L. lactis N8 and achieved the largest amount of deletion of L. lactis so far. Taken together, the present study offers further insights into the function and potential role of L. lactis N8 as a model strain of lactic acid bacteria and lays the foundation for its application in the field of synthetic biology.

Engineering Lactococcus lactis as a multi-stress tolerant biosynthetic chassis by deleting the prophage-related fragment.

BACKGROUND: In bioengineering, growth of microorganisms is limited because of environmental and industrial stresses during fermentation. This study aimed to construct a nisin-producing chassis Lactococcus lactis strain with genome-streamlined, low metabolic burden, and multi-stress tolerance characteristics. RESULTS: The Cre-loxP recombination system was applied to reduce the genome and obtain the target chassis strain. A prophage-related fragment (PRF; 19,739 bp) in the L. lactis N8 genome was deleted, and the mutant strain L. lactis N8-1 was chosen for multi-stress tolerance studies. Nisin immunity of L. lactis N8-1 was increased to 6500 IU/mL, which was 44.44% higher than that of the wild-type L. lactis N8 (4500 IU/mL). The survival rates of L. lactis N8-1 treated with lysozyme for 2 h and lactic acid for 1 h were 1000- and 10,000-fold higher than that of the wild-type strain, respectively. At 39 â„ƒ, the L. lactis N8-1 could still maintain its growth, whereas the growth of the wild-type strain dramatically dropped. Scanning electron microscopy showed that the cell wall integrity of L. lactis N8-1 was well maintained after lysozyme treatment. Tandem mass tags labeled quantitative proteomics revealed that 33 and 9 proteins were significantly upregulated and downregulated, respectively, in L. lactis N8-1. These differential proteins were involved in carbohydrate and energy transport/metabolism, biosynthesis of cell wall and cell surface proteins. CONCLUSIONS: PRF deletion was proven to be an efficient strategy to achieve multi-stress tolerance and nisin immunity in L. lactis, thereby providing a new perspective for industrially obtaining engineered strains with multi-stress tolerance and expanding the application of lactic acid bacteria in biotechnology and synthetic biology. Besides, the importance of PRF, which can confer vital phenotypes to bacteria, was established.

Restructured Lactococcus lactis strains with emergent properties constructed by a novel highly efficient screening system.

BACKGROUND: After 2.83% genome reduction in Lactococcus lactis NZ9000, a good candidate host for proteins production was obtained in our previous work. However, the gene deletion process was time consuming and laborious. Here, we proposed a convenient gene deletion method suitable for large-scale genome reduction in L. lactis NZ9000. RESULTS: Plasmid pNZ5417 containing a visually selectable marker PnisZ-lacZ was constructed, which allowed more efficient and convenient screening of gene deletion mutants. Using this plasmid, two large nonessential DNA regions, L-4A and L-5A, accounting for 1.25% of the chromosome were deleted stepwise in L. lactis 9k-3. When compared with the parent strain, the mutant L. lactis 9k-5A showed better growth characteristics, transformability, carbon metabolic capacity, and amino acids biosynthesis. CONCLUSIONS: Thus, this study provides a convenient and efficient system for large-scale genome deletion in L. lactis through application of visually selectable marker, which could be helpful for rapid genome streamlining and generation of restructured L. lactis strains that can be used as cell factories.

Contribution of YthA, a PspC Family Transcriptional Regulator of Lactococcus lactis F44 Acid Tolerance and Nisin Yield: a Transcriptomic Approach.

To overcome the adverse impacts of environmental stresses during growth, different adaptive regulation mechanisms can be activated in Lactococcus lactis In this study, the transcription levels of eight transcriptional regulators of L. lactis subsp. lactis F44 under acid stress were analyzed using quantitative reverse transcription-PCR. Eight gene-overexpressing strains were then constructed to examine their influences on acid-resistant capability. Overexpressing ythA, a PspC family transcriptional regulator, increased the survival rate by 3.2-fold compared to the control at the lethal pH 3.0 acid shock. Moreover, the nisin yield was increased by 45.50%. The ythA-overexpressing strain FythA appeared to have higher intracellular pH stability and nisin-resistant ability. Subsequently, transcriptome analysis revealed that the vast majority of genes associated with amino acid biosynthesis, including arginine, serine, phenylalanine, and tyrosine, were predominantly upregulated in FythA. Arginine biosynthesis (argG and argH), arginine deiminase pathway, and polar amino acid transport (ysfE and ysfF) were proposed to be the main regulation mechanisms of YthA. Furthermore, the transcription of genes associated with pyrimidine and exopolysaccharide biosynthesis were upregulated. The transcriptional levels of nisIPRKFEG genes were substantially higher in FythA, which directly contributed to the yield and resistance of nisin. Three potential DNA-binding sequences were predicted by computer analysis using the upstream regions of genes with prominent changes. This study showed that YthA could increase acid resistance and nisin yield and revealed a putative regulation mechanism of YthA.IMPORTANCE Nisin, produced by Lactococcus lactis subsp. lactis, is widely used as a safe food preservative. Acid stress becomes the primary restrictive factor of cell growth and nisin yield. In this research, we found that the transcriptional regulator YthA was conducive to enhancing the acid resistance of L. lactis F44. Overexpressing ythA could significantly improve the survival rate and nisin yield. The stability of intracellular pH and nisin resistance were also increased. Transcriptome analysis showed that nisin immunity and the biosynthesis of some amino acids, pyrimidine, and exopolysaccharides were enhanced in the engineered strain. This study elucidates the regulation mechanism of YthA and provides a novel strategy for constructing robust industrial L. lactis strains.

Enhanced heterologous protein productivity by genome reduction in Lactococcus lactis NZ9000.

BACKGROUND: The implementation of novel chassis organisms to be used as microbial cell factories in industrial applications is an intensive research field. Lactococcus lactis, which is one of the most extensively studied model organisms, exhibits superior ability to be used as engineered host for fermentation of desirable products. However, few studies have reported about genome reduction of L. lactis as a clean background for functional genomic studies and a model chassis for desirable product fermentation. RESULTS: Four large nonessential DNA regions accounting for 2.83% in L. lactis NZ9000 (L. lactis 9 k) genome (2,530,294 bp) were deleted using the Cre-loxP deletion system as the first steps toward a minimized genome in this study. The mutants were compared with the parental strain in several physiological traits and evaluated as microbial cell factories for heterologous protein production (intracellular and secretory expression) with the red fluorescent protein (RFP) and the bacteriocin leucocin C (LecC) as reporters. The four mutants grew faster, yielded enhanced biomass, achieved increased adenosine triphosphate content, and diminished maintenance demands compared with the wild strain in the two media tested. In particular, L. lactis 9 k-4 with the largest deletion was identified as the optimum candidate host for recombinant protein production. With nisin induction, not only the transcriptional efficiency but also the production levels of the expressed reporters were approximately three- to fourfold improved compared with the wild strain. The expression of lecC gene controlled with strong constitutive promoters P5 and P8 in L. lactis 9 k-4 was also improved significantly. CONCLUSIONS: The genome-streamlined L. lactis 9 k-4 outcompeted the parental strain in several physiological traits assessed. Moreover, L. lactis 9 k-4 exhibited good properties as platform organism for protein production. In future works, the genome of L. lactis will be maximally reduced by using our specific design to provide an even more clean background for functional genomics studies than L. lactis 9 k-4 constructed in this study. Furthermore, an improved background will be potentially available for use in biotechology.

Isolation of strong constitutive promoters from Lactococcus lactis subsp. lactis N8.

The synthesis of heterologous proteins in Lactococcus lactis is strongly influenced by the promoter selected for the expression. The nisin A promoter is commonly used for induced expression of proteins in L. lactis, whereas few constitutive promoters (P45 and the weaker P32) have been used for protein expression studies. In this study, eight different putative strong constitutive promoters were identified through transcriptional analysis of L. lactis N8 and were investigated for their capability to drive nisZ gene expression with promoters P45 and P32 as control. Four strong promoters (P8, P5, P3 and P2) were identified as having a transcriptional activity that was higher than that of P45 through RT-qPCR and agar-diffusion experiments. In addition, these four promoters were fused to the erythromycin resistant gene (ermC) with promoter P45 as control and inserted into the backbone of the pNZ8048 vector. The transcriptional efficiencies of promoters P8, P5, P2 and P3 were all higher than promoter P45 based on the obtained MIC50 values and they all showed different activity levels. In conclusion, four strong constitutive promoters with a wide range of promoter activities were identified and are suitable for protein production in L. lactis.

Loss of IrpT function in Lactococcus lactis subsp. lactis N8 results in increased nisin resistance.

The antibiotic nisin, produced by Lactococcus lactis subsp. lactis N8, offers an extensive commercial prospect as natural food preservatives. The nisin immunity of the L. lactis strains is regulated by a variety of mechanisms. In this study, we isolated a L. lactis L31 strain with increased nisin resistance from a mini-Mu transposon mutant pool of strain N8. The single Mu insertion in strain L31 was in the irpT gene with unknown function. By comparing the proteomic profiles of L. lactis L31 and its parental strain, we found that changes occurred in the synthesis of a protein involved in cell wall biosynthesis (RmlD). Strain L31 had 13.7% higher content of rhamnose in the cell wall than the N8 strain. Overexpression of RmlD involved in the synthesis of dTDP-L: -rhamnose in the nisin-sensitive MG1363 strain increased nisin resistance of the strain. The results indicate that these cellular proteins effected nisin resistance in L. lactis N8.