Innovative Tools for Molecular and Cell Biology

Innovative Tools for Molecular and Cell Biology

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NICE® Expression System for Lactococcus lactis

The Effective Nisin Controlled Gene Expression System for L. lactis

Controlled gene expression in L. lactis – an emerging alternative to recombinant protein production

Small scale L. lactis culture

Small scale L. lactis culture

Features

  • The system is fully food grade
  • No endotoxins are produced
  • No inclusion bodies
  • No spores
  • No extracellular proteases
  • Tightly controlled gene expression allows production of toxic proteins
  • Simple fermentation, scale-up and downstream processing

Description

  • Next to the wealth of traditional food applications, L. lactis is increasingly used for modern biotechnological applications such as the production of recombinant proteins for food, feed, pharmacological and biocatalysis applications. Owing to the easy genetic accessibility of L. lactis the Nisin Controlled gene Expression (NICE®) system developed at NIZO has played a key role in this development.

Applications

  • Production of homologous and heterologous proteins for food, feed, pharmacological and biocatalysis applications
  • Production of prokaryotic and eukaryotic membrane proteins
  • Production of exopolysaccharides
  • Production of ingredients through metabolic engineering: e.g. alanine, folate, diacetyl
  • Preparation of L. lactis as a biocatalyst by expression of a suitable enzyme as, e.g., dehydrogenases and in-situ cofactor regeneration
  • High-throughput screening for enzyme evolution or enzyme comparison.

Plasmids for Intracellular Expression and Secretion

Vector map of pNZ9530
  1. enlarge

Vector map of pNZ9530

Plasmids for Intracellular Expression

  • The replicons of the vectors pNZ8008, pNZ8148, pNZ8149 and pNZ8150 are identical and came originally from the Lactococcus lactis plasmid pSH71. However, this replicon has a broad host range. Plasmids with this replicon can replicate in many Gram-positive bacteria, such as Lactobacillus plantarum and Streptococcus thermophilus.

  • pNZ8148 – In this vector the nisA promoter is followed by an NcoI site for translational fusions at the ATG. It contains a terminator after the MCS. Sequence adaptation for cloning in NcoI can result in a change of the second amino acid of a protein (Mierau and Kleerebezem, 2005).

  • pNZ8149 – This vector has the lacF gene as food grade selection marker. To enable selection of transformants, it needs a host strain with the lactose operon without the lacF gene like Lactococcus lactis NZ3900. The nisA promoter is followed by an NcoI site for translational fusions at the ATG. It contains a terminator after the MCS. Sequence adaptation for cloning in NcoI can result in a change of the second amino acid of a protein (no reference, example: (Mierau et al., 2005)).

  • pNZ8150 – The nisA promoter is followed by a ScaI site for translational fusions precisely at the ATG. Blunt end fragments are generated by PCR and cannot be cut out again after ligation to the ScaI site (Mierau and Kleerebezem, 2005).

  • pNZ8151 – This vector carries the lacF gene as food grade selection marker. The nisA promoter is followed by an ScaI site for translational fusions at the ATG start codon. It contains a terminator after the MCS (Mierau and Kleerebezem, 2005).

  • pNZ8152 – This vector contains the alr gene as food grade selection marker. The nisA promoter is followed by an ScaI site for translational fusion at the ATG start codon. It contains a terminator after the MCS (Mierau and Kleerebezem, 2005).

  • pNZ8008 – This is a reference plasmid for testing the nisin induction in Lactococcus and other lactic acid bacteria genera. A gusA gene without promoter was fused to the nisin A promoter (PnisA) (de Ruyter et al., 1996).

  • pNZ9530 – A low copy plasmid with pAMΒ1 origin of replication that carries the nisR and nisK genes. For cloning in Lactococcus strains and in strains of other lactic acid bacteria genera that do not have the regulatory genes integrated into the chromosome. In this case, a two plasmid system is used for nisin induced expression: e.g. pNZ9530 (nisRnisK) + pNZ8150 (+insert) (Kleerebezem et al., 1997).

Secretion Vectors

  • The secretion vectors pNZ8120, pNZ8121, pNZ8122, pNZ8123, and pNZ8124 are broad host range vectors with a chloramphenicol selection marker. They differ in their signal sequence and in their cloning sites.

  • pNZ8120 – A vector with the signal sequence of the lactococcal cell wall proteinase PrtP (Vos et al.). Cloning is possible via an NaeI site and the mature protein starts with its own first amino acid (results not published).

  • pNZ8121 – This vector has the signal sequence of PrtP also, but cloning is done via an EcoRV site, so that the mature protein starts with the first amino acid of PrtP after the signal cleavage site. This setting can contribute to a greater efficiency in removing the signal peptide (results not published).

  • pNZ8122 – A vector with the signal sequence of the Lactobacillus brevis SlpA protein (Novotny et al., 2005). Cloning is done via an NruI site, so the mature protein starts with the first amino acid of SlpA after the signal cleavage site. This setting can contribute to a greater efficiency in removing the signal peptide (results not published).

  • pNZ8123 – This vector provides the signal sequence of the lactococcal major secreted protein Usp45. Cloning is done via an NaeI site; mature protein starts with its own first amino acid (results not published).

  • pNZ8124 – Like pNZ8123 this vector has the signal sequence of Usp45, but cloning is done via an EcoRV site and the mature protein starts with the first amino acid of Usp45 (van Asseldong et al., 1993 & 1990) after the signal cleavage site. This setting can contribute to a greater efficiency in removing the signal peptide (results not published).

Overview on available Plasmids

Vector Order # Cloning Site Secretion Signal Peptide Food Grade Remarks
pNZ8148 VS-ELV00200-01 NcoI NO NO NO
pNZ8149 VS-ELV00300-01 NcoI NO NO YES
pNZ8150 VS-ELV00250-01 ScaI NO NO NO
pNZ8151 VS-ELV00310-01 ScaI NO NO YES
pNZ8152 VS-ELV00400-01 ScaI NO NO YES
pNZ8120 VS-ELV00600-01 NaeI YES PrtP NO
pNZ8121 VS-ELV00650-01 EcoRV YES PrtP NO
pNZ8122 VS-ELV00700-01 NruI YES SlpA NO
pNZ8123 VS-ELV00750-01 NaeI YES Usp45 NO
pNZ8124 VS-ELV00800-01 EcoRV YES Usp45 NO
pNZ8008 VS-ELV00100-01 n/a NO NO NO *
pNZ9530 VS-ELV00500-01 n/a NO NO NO **
  • * Reference plasmid carrying gusA gene
  • ** Carries nisR/nisK for expression in strains w/o nis regulating elements

Host Strains

Electron microscope image of Lactococcus lactis

Electron microscope image of Lactococcus lactis

Description

  • Lactococcus lactis NZ9000pepN::nisRnisK;
  • Standard host strain for nisin regulated gene expression (NICE®). The strain contains the regulatory genes nisR and nisK integrated into the pepN gene (broad range amino peptidase) (Kuipers et al., 1998; Mierau and Kleerebezem, 2005).

  • Lactococcus lactis NZ9100nisRnisK;
  • Standard host strain for nisin regulated gene expression (NICE®). The strain contains the regulatory genes nisR and nisK integrated in a neutral locus.

  • Lactoccoccus lactis NZ9130alr-, nisRnisK; This host strain is the same as N9100 used for nisin regulated gene expression. It carries an alr deletion (Δalr) that encodes for alanine racemase. Deletion of the alr gene results in auxotrophy for alanine suggesting that the strain is unable to grow on media without alanine unless alr is provided on a plasmid (Bron et al., 2002).

  • Lactococcus lactis NZ3900lacF-, pepN::nisRnisK;
  • Standard strain for food grade selection based upon the ability to grow on lactose. This strain is a progeny of NZ3000, a strain in which the lactose operon, that is generally present on plasmids, has been integrated into the chromosome and the lacF gene was deleted. Deletion of the lacF gene makes this strain unable to grow on lactose unless lacF is provided on a plasmid (de Ruyter et al., 1996a).

  • Lactococcus lactis NZ3910lacF-, nisRnisK;
  • Same as NZ3900 for food grade lacF selection, but with nisRnisK integrated in a neutral locus.

Overview on L. lactis Host Strains

Strain Order # Species Food Grade Remarks
NZ9000 VS-ELS09000-01 L. lactis NO pepN::nisRnisK
NZ9100 VS-ELS09100 L. lactis NO nisRnisK in neutral locus
NZ9130 VS-ELS09130-01 L. lactis YES nisRnisK in neutral locus, alr-
NZ3900 VS-ELS03900-01 L. lactis YES pepN::nisRnisK, lacF-
NZ3910 VS-ELS03910-01 L. lactis YES nisRnisK in neutral locus, lacF-

ORDER INFORMATION

  • For shipping and storage information please click on Order#.
Order# Description Amount Price Data Sheet
VS-ELV00100-01 NICE pNZ8008 Reference plasmid with gusA gene 10 µg 482,00 PDF
VS-ELV00200-01 NICE pNZ8148 Lactococcus lactis expression vector, NcoI site 10 µg 482,00 PDF
VS-ELV00250-01 NICE pNZ8150 Lactococcus lactis expression vector, ScaI site 10 µg 482,00 PDF
VS-ELV00300-01 NICE pNZ8149 Lactococcus lactis expression vector, food grade (lacF), NcoI site 10 µg 482,00 PDF
VS-ELV00310-01 NICE pNZ8151 Lactococcus lactis expression vector, food grade (lacF), Scal site 10 µg 482,00 PDF
VS-ELV00400-01 NICE pNZ8152 Lactococcus lactis expression vector, food grade (alr), Scal site 10 µg 482,00 PDF
VS-ELV00500-01 NICE pNZ9530 Lactococcus lactis nisRnisK vector, in Strain NZ9000 1 ml 294,00 PDF
VS-ELV00600-01 NICE pNZ8120 Lactococcus lactis secretion vector (SP PrtP/NaeI) 10 µg 612,00 PDF
VS-ELV00650-01 NICE pNZ8121 Lactococcus lactis secretion vector (SP PrtP/EcoRV) 10 µg 612,00 PDF
VS-ELV00700-01 NICE pNZ8122 Lactococcus lactis secretion vector (SP SlpA/NruI) 10 µg 612,00 PDF
VS-ELV00750-01 NICE pNZ8123 Lactococcus lactis secretion vector (SP Usp45/NaeI) 10 µg 612,00 PDF
VS-ELV00800-01 NICE pNZ8124 Lactococcus lactis secretion vector (SP Usp45/EcoRV) 10 µg 612,00 PDF
VS-ELS09000-01 NICE Lactococcus lactis expression Strain NZ9000 1 ml 294,00 PDF
VS-ELS09100 NICE Lactococcus lactis expression Strain NZ9100 1 ml 318,00 PDF
VS-ELS09130-01 NICE Lactococcus lactis expression Strain NZ9130, food grade, (alr) 1 ml 383,00 PDF
VS-ELS03900-01 NICE Lactococcus lactis expression Strain NZ3900, food grade, (lacF) 1 ml 294,00 PDF
VS-ELS03910-01 NICE Lactococcus lactis expression Strain NZ3910, food grade, (lacF) 1 ml 294,00 PDF
VS-ELS-10610-01 NICE E. coli host Strain MC1061 1 ml 305,00 PDF
VS-ELS10710-01 MC1061 Chemically Competent Escherichia coli 5 x 100 µl 438,00 PDF
VS-ELK01000-02 NICE Nisin kit, 1 g nisin (concentration 2.5\% (balance sodium chloride and denatured milk solids)), 1 ml 5\% acetic acid Kit 122,00 PDF
  • All prices are in EURO excl. VAT and shipping. For further pricing and order information please ask your local distributor.

DOWNLOADS

  • NICE® Expression System for Lactococcus lactis Handbook (PDF)

Vector maps and Sequences

  • Vector map of NICE® pNZ8008 Reference plasmid (pic)
  • Vector map of NICE® pNZ8148 (pic)
  • Vector map of NICE® pNZ8149 (pic)
  • Vector map of NICE® pNZ8150 (pic)
  • Vector map of NICE® pNZ8151 (pic)
  • Vector map of NICE® pNZ8152 (pic)
  • Vector map of NICE® pNZ9530 (pic)
  • Vector map of NICE® pNZ8120 (pic)
  • Vector map of NICE® pNZ8121 (pic)
  • Vector map of NICE® pNZ8122 (pic)
  • Vector map of NICE® pNZ8123 (pic)
  • Vector map of NICE® pNZ8124 (pic)
  • Sequence of NICE® pNZ8008 Reference plasmid (txt)
  • Sequence of NICE® pNZ8148 (txt)
  • Sequence of NICE® pNZ8149 (txt)
  • Sequence of NICE® pNZ8150 (txt)
  • Sequence of NICE® pNZ8151 (txt)
  • Sequence of NICE® pNZ8152 (txt)
  • Sequence of NICE® pNZ9530 (txt)
  • Sequence of NICE® pNZ8120 (txt)
  • Sequence of NICE® pNZ8121 (txt)
  • Sequence of NICE® pNZ8122 (txt)
  • Sequence of NICE® pNZ8123 (txt)
  • Sequence of NICE® pNZ8124 (txt)

Literature

  • Bernaudat, F. Frelet-Barrand, A., et al. (2011), “Heterologous expression of membrane proteins: choosing the appropriate host” PLoS One. 2011;6(12):e29191. PubMed
  • de Ruyter, P. G., O. P. Kuipers, et al. (1996). "Functional analysis of promoters in the nisin gene cluster of Lactococcus lactis." Journal of Bacteriology 178(12): 3434-3439. de Vos, W. M. (1987). "Gene cloning and expression in lactic streptococci." FEMS Microbiology Letters 46: 281-295. Pubmed
  • Dolatabadi, S. et al. (2015). "Lactococcus lactis as an oral vector for cloning of heat shock protein A from Helicobacter pylori."
    International Journal of Biosciences, Vol. 6, No. 3, p. 410-415, 2015. Full Text
  • Frelet-Barrand, A., Boutigny, S., Kunji, E. R. S., und Rolland, N. (2010). "Membrane protein expression in Lactococcus lactis."
    Methods Mol. Biol 601, 67-85.
  • Gasson, M. J. (1983). "Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing."
    Journal of Bacteriology 154(1): 1-9. Pubmed
  • Kleerebezem, M., M. M. Beerthuyzen, et al. (1997). "Controlled gene expression systems for lactic acid bacteria: transferable nisin-inducible expression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp."
    Applied and Environmental Microbiology 63(11): 4581-4584. Pubmed
  • Kuipers, O. P., M. M. Beerthuyzen, et al. (1993). "Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis. Requirement of expression of the nisA and nisI genes for development of immunity."
    European Journal of Biochemistry 216(1): 281-291. Pubmed
  • Mierau, I. and M. Kleerebezem (2005). "10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis."
    Applied Microbiology and Biotechnology 9: 1-13. Pubmed
  • Mierau, I., P. Leij, et al. (2005). "Industrial-scale production and purification of a heterologous protein in Lactococcus lactis using the nisin-controlled gene expression system NICE: the case of lysostaphin."
    Microbial Cell Factories 4: 15. Pubmed
  • Simon, D. and A. Chopin (1988). "Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis."
    Biochimie 70(4): 559-566. Pubmed
  • Villatoro-Hernandez, J. et al. (2012). "Heterologous protein expression by Lactococcus lactis."
    In A. Lorence (ed.), Recombinant Gene Expression: Reviews and Protocols, Third Edition, Methods in Molecular Biology, vol. 824. Chapter 8, p. 155 - 165.