Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications (original) (raw)
Related papers
Microbial cell factories, 2005
The NIsin-Controlled gene Expression system NICE of Lactococcus lactis is one of the most widespread used expression systems of Gram-positive bacteria. It is used in more than 100 laboratories for laboratory-scale gene expression experiments. However, L. lactis is also a micro-organism with a large biotechnological potential. Therefore, the aim of this study was to test whether protein production in L. lactis using the NICE system can also effectively be performed at the industrial-scale of fermentation. Lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus) from S. simulans biovar. Staphylolyticus, was used as a model system. Food-grade lysostaphin expression constructs in L. lactis were grown at 1L-, 300-L and 3000-L scale and induced with nisin for lysostaphin production. The induction process was equally effective at all scales and yields of about 100 mg/L were obtained. Up-scaling was easy and required no specific effort. Furthermore, we describe a simple ...
Plasmid, 2011
Over the last 10 years, the NIsin Controlled Expression (NICE) system has been extensively used in the food-grade bacterium Lactococcus lactis subsp. cremoris to produce homologous and heterologous proteins for academic and biotechnological purposes. Although various L. lactis molecular tools have been developed, no expression vectors harboring the popular Gateway recombination system are currently available for this widely used cloning host. In this study, we constructed two expression vectors that combine the NICE and the Gateway recombination systems and we tested their applicability by recombining and overexpressing genes encoding structural proteins of lactococcal phages Tuc2009 and TP901-1. Over-expressed phage proteins were analyzed by immunoblotting and purified by His-tag affinity chromatography with protein productions yielding 2.8-3.7 mg/l of culture. This therefore is the first description of L. lactis NICE expression vectors which integrate the Gateway cloning technology and which are suitable for the production of sufficient amounts of proteins to facilitate subsequent structural and functional analyses.
Applied and Environmental Microbiology, 2000
The potential of lactic acid bacteria as live vehicles for the production and delivery of therapeutic molecules is being actively investigated today. For future applications it is essential to be able to establish dose-response curves for the targeted biological effect and thus to control the production of a heterologous biopeptide by a live lactobacillus. We therefore implemented in Lactobacillus plantarum NCIMB8826 the powerful nisin-controlled expression (NICE) system based on the autoregulatory properties of the bacteriocin nisin, which is produced by Lactococcus lactis. The original two-plasmid NICE system turned out to be poorly suited to L. plantarum. In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein (-glucuronidase) or a model antigen (the C subunit of the tetanus toxin, TTFC), the lactococcal nisRK regulatory genes were integrated into the chromosome of L. plantarum NCIMB8826. Moreover, recombinant L. plantarum producing increasing amounts of TTFC was used to establish a dose-response curve after subcutaneous administration to mice. The induced serum immunoglobulin G response was correlated with the dose of antigen delivered by the live lactobacilli.
Bioreactor Strategies to Increase the Engineered Protein Production in Lactococcus Lactis
2020
Lactococcus lactis is a lactic acid bacterium widely used in various food and fermentation processes. L. lactis is one of the organism which is treated as generally regarded as safe (GRAS) organisms, whichch is used for as genetic machinery for the genetic and chromosomal engineering and vectors to facilitate cloning and gene expression and produces a number of important proteins. However, the production of large quantities of biologically active recombinant proteins in this organism is restricted due to a number of factorsand biological constraints. One of the constraints with this system is that, in glucose growing culture, L. lactis develops a stress response and cessation of growth due to the acidification of medium. Prior work in our laboratory has shown that suppression of this response increases the streptokinase productivity in L.lactis. It was shown that buffering the medium suppresses development. In this work, the correlation between ATR suppression and increase in recomb...
Nisin inducible production of listeriolysin O in Lactococcus lactis NZ9000
Microbial Cell Factories, 2008
Background: Listeria monocytogenes is a well-characterized food-borne pathogen that infects pregnant women and immunocompromised individuals. Listeriolysin O (LLO) is the major virulence factor of the pathogen and is often used as a diagnostic marker for detection of L. monocytogenes. In addition, LLO represents a potent antigen driving T cell-mediated immunity during infection. In the present work, Lactococcus lactis NZ9000 was used as an expression host to hyper-produce LLO under inducible conditions using the NICE (NIsin Controlled Expression) system. We created a modified pNZ8048 vector encoding a six-His-tagged LLO downstream of the strong inducible PnisA promoter.
Applied and environmental microbiology, 1997
A transferable dual-plasmid inducible gene expression system for use in lactic acid bacteria that is based on the autoregulatory properties of the antimicrobial peptide nisin produced by Lactococcus lactis was developed. Introduction of the two plasmids allowed nisin-inducible gene expression in Lactococcus lactis MG1363, Leuconostoc lactis NZ6091, and Lactobacillus helveticus CNRZ32. Typically, the beta-glucuronidase activity (used as a reporter in this study) remained below the detection limits under noninducing conditions and could be raised to high levels, by addition of subinhibitory amounts of nisin to the growth medium, while exhibiting a linear dose-response relationship. These results demonstrate that the nisin-inducible system can be functionally implemented in lactic acid bacteria other than Lactococcus lactis.
Nisin expression production fromLactococcus lactis in milk whey medium
Journal of Chemical Technology & Biotechnology, 2008
BACKGROUND: Nisin is a commercially available bacteriocin produced by Lactococcus lactis ATCC 11454 and used as a natural agent in the biopreservation of food. In the current investigation, milk whey, a byproduct from dairy industries was used as a fermentation substrate for the production of nisin. Lactococcus lactis ATCC 11454 was developed in a rotary shaker (30 • C/36 h/100 rpm) using two different media with milk whey (i) without filtration, pH 6.8, adjusted with NaOH 2 mol L −1 and without pH adjustment, both autoclaved at 121 • C for 30 min, and (ii) filtrated (1.20 µm and 0.22 µm membrane filter). These cultures were transferred five times using 5 mL aliquots of broth culture for every new volume of the respective media.
Enhancement of nisin production by Lactococcus lactis in periodically re-alkalized cultures
Biotechnology and Applied Biochemistry, 2003
One of the biggest problems when producing nisin from lactic acid bacteria on an industrial scale is the high cost of the complex peptide sources of the specific commercial media. Improving nisin production through optimization of fermentation parameters would make nisin more cost-effective for various applications. In this work the possibility of using soya permeate as a substitute for commercial peptide sources was studied. The growth and nisin production by Lactococcus lactis subsp. lactis MTCC 440 was investigated in fixed volume batch fermentation on soya permeate, and MRS medium. Kinetic parameters and nisin production was higher to those obtained with bactopeptone and commercial media. Soya permeate was also supplemented with growth stimulating amino acids. Nisin biosynthesis is strongly dependent on the presence of a sulphur source, either an inorganic salt (magnesium sulphate or sodium thiosulphate) or the amino acids methionine, or cysteine. The amino acids serine, threonine and cysteine highly stimulate nisin production without affecting the final cell yield, indicating their precursor role during nisin biosynthesis.
Journal of Food Science, 2010
Lactococcus lactis UQ2 is a nisin A-producing native strain. In the present study, the production of nisin by L. lactis UQ2 in a bioreactor using supplemented sweet whey (SW) was optimized by a statistical design of experiments and response surface methodology (RSM). In a 1st approach, a fractional factorial design (FFD) of the order 2(5-1) with 3 central points was used. The effect on nisin production of air flow, SW, soybean peptone (SP), MgSO(4)/MnSO(4) mixture, and Tween 80 was evaluated. From FFD, the most significant factors affecting nisin production were SP (P = 0.011), and SW (P = 0.037). To find optimum conditions, a central composite design (CCD) with 2 central points was used. Three factors were considered, SW (7 to 10 g/L), SP (7 to10 g/L), and small amounts of added nisin as self-inducer (NI 34.4 to 74.4 IU/L). Nisin production was expressed as international units (IU). From RSM, an optimum nisin activity of 180 IU/mL was predicted at 74.4 IU/L NI, 13.8 g/L SP, and 14.9 or 5.11 g/L SW, while confirmatory experiments showed a maximum activity of 178 +/- 5.2 IU/mL, verifying the validity of the model. The 2nd-order model showed a coefficient of determination (R(2)) of 0.828. Optimized conditions were used for constant pH fermentations, where a maximum activity of 575 +/- 17 IU/mL was achieved at pH 6.5 after 12 h. The adsorption-desorption technique was used to partially purify nisin, followed by drying. The resulting powder showed an activity of 102150 IU/g. Practical Application: Nisin production was optimized using supplemented whey as alternative culture medium, using a native L. lactis UQ2 strain. Soybean peptone, SW, and subinhibitory amounts of nisin were successfully employed to optimize nisin production by L. lactis UQ2. Dried semipurified nisin showed an activity of 102150 IU/g.
Brazilian Archives of Biology and Technology, 2010
The influence of various physiochemical parameters on the growth of Lactococcus lactis sub sp. lactis MTCC 440 was studied at shake flask level for 20 h. Media optimization (MRS broth) was studied to achieve enhanced growth of the organism and also nisin production. Bioassay of nisin was done with agar diffusion method using Streptococcus agalactae NCIM 2401 as indicator strain. MRS broth (6%, w/v) with 0.15µg/ml of nisin supplemented with 0.5% (v/v) skimmed milk was found to be the best for nisin production as well as for growth of L lactis. The production of nisin was strongly influenced by the presence of skimmed milk and nisin in MRS broth. The production of nisin was affected by the physical parameters and maximum nisin production was at 30 0 C while the optimal temperature for biomass production was 37 0 C.