Browsing by Subject "Lactococcus lactis"

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  • Fu, Y; Qiao, W; Zhu, D; Wang, X; Liu, F; Xu, H; Saris, Per Erik Joakim; Kuipers, Osacar; Qiao, Mingqiang (2018)
    Nisin, an important bacteriocin from Lactococcus lactis subsp., is primarily active against various Gram-positive bacteria. Leucocin C, produced by Leuconostoc carnosum 4010, is a class IIa bacteriocin used to inhibit the growth of Listeria monocytogenes. Because two bacteriocins have different modes of action, the combined use of them could be a potential strategy for effective inhibition of foodborne pathogens. In this study, L. lactis N8-r-lecCI (N8 harboring lecCI gene) coexpressing nisin–leucocin C was constructed based on the food-grade carrier L. lactis N8. Production of both bacteriocins was stably maintained. Antimicrobial measurements showed that the recombinant strain is effectively against Listeria monocytogenes and Staphylococcus aureus and moderately against Salmonella enterica serovar Enteritidis and Escherichia coli because of its stronger antibacterial activity than the parental strain, this result first demonstrated that the co-expression of nisin and leucocin C results in highly efficient antimicrobial activity. The checkerboard assay showed that the antibacterial activity of L. lactis N8-r-lecCI supernatant was enhanced in the presence of low concentration of EDTA. Analysis of the scanning electron microscope image showed the biggest cellular morphology change in L. monocytogenes treated with a mixture of EDTA and L. lactis N8-r-lecCI supernatant. The practical effect was verified in pasteurized milk through time-kill assay. The L. lactis N8-r-lecCI strain expressing both nisin and leucocin C has a promising application prospect in pasteurized milk processing and preservation because of its strong antibacterial activity.
  • Qiao, Wanjin; Qiao, Yu; Liu, Fulu; Zhang, Yating; Li, Ran; Wu, Zhenzhou; Xu, Haijin; Saris, Per Erik Joakim; Qiao, Mingqiang (2020)
    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 celcius, 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.
  • Qiao, Wanjin; Qiao, Yu; Liu, Fulu; Zhang, Yating; Li, Ran; Wu, Zhenzhou; Xu, Haijin; Saris, Per E J; Qiao, Mingqiang (BioMed Central, 2020)
    Abstract 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 ℃, 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.
  • Zhu, Duolong; Fu, Yuxin; Liu, Fulu; Xu, Haijin; Saris, Per E J; Qiao, Mingqiang (BioMed Central, 2017)
    Abstract 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.
  • Zhu, Duolong; Fu, Yuxin; Liu, Fulu; Xu, Haijin; Saris, Per Erik Joakim; Qiao, Mingqiang (2017)
    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.
  • Sorokina, Dina (Helsingfors universitet, 2015)
    Lactic acid bacteria (LAB) are generally recognized as safe micro-organisms and used in food preservation and as health promoting probiotics. Beside lactic acid, LAB produce several antimicrobial compounds of which especially bacteriocins provide new potential applications for food and pharmaceutical industries. Bacteriocins are ribosomally synthetized proteins or peptides with antimicrobial activity usually against closely related species. Whole genome sequencing project of lactic acid bacterium Lactococcus lactis N8 has revealed a new bacteriocin operon which consists of a bacteriocin gene and ABC transporter genes. Similar operon has been also found in several other L.lactis strains including IL1403. Peptides expressed by these bacteriocin genes belong to lactococcin 972 protein family according to their amino acid sequences. In this master’s thesis, these novel bacteriocin genes from L. lactis N8 and IL1403 were cloned into Escherichia coli with plasmid vectors. New bacteriocins were named encacin A and B. Strong inducible promoters were chosen to achieve high bacteriocin production. Encacins were expressed in cytosolic and periplasmic spaces to compare the effect of localization on antimicrobial activity of peptides. The prevalence of encacin genes among different L. lactis strains was also studied. Four of ten E. coli recombinant strains constructed during this study were shown to produce bacteriocins. Two of them, which produced encacins into periplasmic space, also appeared to be weakly active against L. lactis MG1614 strain. Therefore it seems that localization of encacins in E. coli bacterial cell has an impact on the bioactivity of peptides. Screening of bacteriocins genes showed that over 90 % of L. lactis stains bear encacin genes, from which encacin B is the more frequent form. More precise characterization of encacin genes and peptides may help to gain new information about qualities and mode of action of these novel potential bacteriocins.
  • Juuti, Noora (Helsingin yliopisto, 2020)
    Usp45 is the major secreted protein in Lactococcus lactis. Protein’s 27-aa signal peptide (SPUsp45) is widely used for increased secretion and improved yields of heterologous proteins. L. lactis, previously used mainly in food production, has gained increasing popularity in gene technology and, thanks to extensive research, became a Gram-positive model organism alongside Bacillus subtilis. Despite the widespread use of its signal peptide, the biological role of Usp45 protein remains largely a mystery. The aim of this study was to test whether decreased translation of the naturally highly secreted Usp45 protein would lead to improved secretion of desired heterologous proteins. The hypothesis was that high levels of secreted Usp45 cause the Sec translocon becoming a bottleneck and by reducing this strain on the secretion route, the capacity to secrete other proteins could increase. Based on literature, usp45 is not part of the L. lactis core genome and was assumed to be non-vital. To study this, the L. lactis strain NZ9000 was transformed with the plasmid pLEB805 which contains a nisin inducible antisense-usp45 gene (ASusp45) resulting in strain LAC455. The effect of antisense-RNA mediated silencing on growth and morphology of the cells was observed as well as the changes in quantity and quality of secreted proteins in ASusp45 induced cells. The secretion of heterologous proteins was tested with bacteriocins leucocin A and C that were introduced to the cells in expression vectors. This study brought new information on the function of the usp45 gene in L. lactis. The results show that the silencing of the usp45 gene leads to retarded growth rate, multifold ingrowth of the cell wall, aggregation of the cells and the leakage of cytoplasmic proteins leading to loss of viability of the cells. These results demonstrate that Usp45 is vital for the structure of the cell wall, cell separation and normal chain formation, and it probably acts as a vital peptidoglycan hydrolase.
  • Ahonen, Susanna (Helsingin yliopisto, 2018)
    Bacteriocins are ribosomally synthesized antimicrobial proteins or peptides that inhibit growth of other bacteria. Bacteriocins can be divided into three classes based on their characteristics. Bacteriocins from the genus Lactococcus have only been found from the classes I and II. These two classes include small heat resistant peptides. Class III includes larger heat sensitive proteins. Bacteriocins have different ways to inhibit bacteria. They can either kill the bacteria or prevent them from multiplying. Due to their safe properties, bacteriocins can be used in food production to prevent the growth of harmful bacteria. In this Master’s thesis, antimicrobial compound produced by Lactococcus lactis LAC460 isolated from idli batter, was studied. Based on inhibition tests it was found that the LAC460 prevented the growth of some lactic acid bacteria by forming an inhibition zone on indicator plate. Uncommon to bacteriocins, the zone expanded during several weeks of incubation. This indicates that the antimicrobial compound can also kill inactive non-growing target cells. Based on the enzymatic tests the antimicrobial compound turned out to be a protein, probably a bacteriocin. The LAC460 bacteriocin lost its activity already at 53 °C, as well as both in low and high pH. This indicates that the compound was not a peptide but rather a bigger protein. The strain LAC460 produced more bacteriocin when the growth media was strongly buffered at least to pH 7. The bacteriocin was produced only at later growth phase. Based on the results from the ribosomal 16S rDNA sequencing, API-test and whole genome sequencing, LAC460 turned out to be a mixed culture of two bacteria, L. lactis and Enterococcus faecium. These bacteria were separated from each other with selective media and it was found out that Lactococcus was the actual producer of the bacteriocin. No obvious bacteriocin gene was found from the genome sequence, but the results suggest that the LAC460 bacteriocin belongs to class III bacteriolysins. As Lactococcus strains have not been found to produce class III bacteriocins, the antimicrobial protein characterized in this study seems to be a new bacteriocin.
  • Liu, Fulu; Zhang, Yating; Qiao, Wanjin; Zhu, Duolong; Xu, Haijin; Saris, Per Erik Joakim; Qiao, Mingqiang (2019)
    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 P-nisZ-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.
  • Liu, Fulu; Zhang, Yating; Qiao, Wanjin; Zhu, Duolong; Xu, Haijin; Saris, Per E J; Qiao, Mingqiang (BioMed Central, 2019)
    Abstract 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.
  • Väistö, Anne (Helsingfors universitet, 2010)
    The properties and evaluation methods of viili, the actions and interactions of viili starters in milk and bacteriophages of the viili starters were reviewed. The aim of the experimental study was to explore whether it was possible to make viili with single strain starters and combine them just before adding the starter to the milk. A new, second viili starter was made from the new strains. The success of the new starters was evaluated by sensory evaluation and by analysing the texture and chemical properties of viili. The starter strains were cultivated in a bioreactor, concentrated by a centrifuge and frozen at –75 °C. The starter strains were combined approx. 1 day before the viili production. The sensory evaluation of the viili was performed by groups of 3 to 6 persons. The texture (consistency, firmness and cohesiveness) of the viili and chemical analysis were made. The results of the sensory analysis were analysed statistically and new strain combinations were formulated based results. The viilis made by the traditional viili starter strains were evaluated by the triangle test (n = 10–11) and the second viili starter was evaluated by descriptive analysis (n = 8). The texture measurements and chemical analyses were also performed. The viili produced by the second starter was infected by the factory phage samples and the pH was measured. After infecting the viili with phage samples, the viili produced by second starter was acidified to pH 4.5 from 0 to 10 hours later compared to the viili without the phage sample. The viili produced by traditional starter did not acidify when the phage was added. The aroma producers did not grow properly in viili when the starter was made by single strains. The viilis made by the present viili starter strains were not distinguished by the triangle test which meant that the starters are possible to make from single strains. The viilis produced by the second viili starter differed from the viili made by traditional starter by appearance and texture characteristics. There was no difference in taste characteristics between the traditional and new starter.