pNZ8122乳酸菌食品級表達載體

pNZ2122 plasmid

Food grade vector for constitutive gene expression under control of the lacA promoter for

L. lactis NZ3000. For transcriptional fusions. LacF-based selection. (Platteeuw et al.,1996)

Host strains

Lactococcus lactis subsp. cremoris MG1363

Plasmid-free progeny of the dairy strain NCDO712. Most widely used host strain for

cloning and gene expression in L. lactis (Gasson, 1983).

Lactococcus lactis NZ3000

Standard strain for food grade selection based upon the ability to grow on lactose. 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 (van Alen-Boerrigter, I. J.,

and W. M. de Vos, unpublished data; de Ruyter et al., 1996).

Strains Plasmids Medium

Lactococcus lactis MG1363 non-food grade M17 + 0.5% glucose + 10 μg/ml chloramphenicol

Lactococcus lactis NZ3000 food grade see 3.2 & 3.6

2.2 Plasmids

All plasmids are based on the pSH71 rolling circle replicon (de Vos, 1987).

pNZ124 General broad host range cloning vector with multiple cloning site for L. lactis and other

lactic acid bacteria. Chloramphenicol selection. (Platteeuw et al., 1994)

pNZ2105 General food grade cloning vector with multiple cloning site for L. lactis NZ3000. LacFbased

selection. (Platteeuw et al., 1996)

pNZ2103 Broad host range vector for constitutive gene expression under control of the lacA

promoter. For transcriptional fusion. Chloramphenicol selection. (Platteeuw et al., 1996)

pNZ7021 Broad host range vector for constitutive gene expression under control of the strong pepN

promoter. For transcriptional fusion. Chloramphenicol selection. (Wegkamp et al., 2007)

pNZ2122 Food grade vector for constitutive gene expression under control of the lacA promoter for

L. lactis NZ3000. For transcriptional fusions. LacF-based selection. (Platteeuw et al.,1996)

pNZ2123 Food grade vector. Identical to pNZ2122, but with inverted multiple cloning site (MCS).

LacF-based selection. (Platteeuw et al., 1996)

1 Introduction

The Constitutive Gene Expression System for Lactococcus lactis and Other Lactic

Acid Bacteria, developed at NIZO food research BV, The Netherlands, is easy-tooperate

and has advantages for the following applications:

? Overexpression of homologous and heterologous genes for functional studies and

to obtain large quantities of specific gene products

? Metabolic engineering

? Suitability for protein secretion (Novotny, R. et al. 2005; Ravn, P. et al. 2003; van

Asseldonk et al., 1990; Vos, P. et al. 1989) and anchoring in the cell envelope

? Large scale applications

Major advantages of this system over other expression systems are:

- Less endogenous and no exogenous proteases

- Endotoxin-free expression system

- Food grade protein expression possible

- No formation of inclusion bodies

- No formation of spores

- Simple fermentation, scale-up and downstream processing

- Suitable also for bacteria other than Lactococcus lactis

1.1 Lactococcus lactis

Lactococcus lactis is a homofermentative bacterium. Its primary function is rapid lactic

acid production from lactose. Functional characteristics that have extensively been

studied in lactococci include the carbon metabolism, the extracellular and intracellular

proteolytic system, the production of antibiotic substances, and their interaction with and

resistance to bacteriophages. The genome information of many L. lactis strains is publicly

available. This wealth of knowledge and experience has led to the use of lactococci in

several fields of biotechnology, e.g., the expression of bacterial and viral antigens for

safe vaccination via mucosal immunization, the production of human cytokines and other

therapeutic agents for in situ treatments, the use of lactococci as a cell factory for the

production of specific compounds, and the pilot production of pharmaceutical products

(Mierau, 2005).

1.2 Use of Lactococcus lactis plasmids in other Gram-positive bacteria

Plasmids with a replication origin from L. lactis can be used in all lactic acid bacteria and

in other Gram-positive bacteria such as Bacillus subtilis (Silke David, 1989, AEM

55:1483; Christ Platteeuw, 1994, AEM 60:587; Elisabeth Sorvig, 2005, Microbiology,

151:2439; Indranil Biswas, 2008, Microbiology, 154:2275).

1.3 Codon usage

Until very recently codon usage was an important issue in the possibility and efficiency to

express heterologous genes in L. lactis (GC content of the DNA of 35 - 37%). When a

gene donor organism is closely related to L. lactis, or the DNA GC content is similar to

that of L. lactis, the probability that a gene can successfully be expressed is high. With

the availability of cheap and reliable custom DNA synthesis, there are no longer

restrictions as to the origin of a specific target gene, since, from a known amino acid

sequence, a gene can be designed that fits the codon usage pattern of the host

organism. In addition to a general codon optimization, specific codon tables can be used,

such as the codon table for the highly expressed ribosomal protein genes, to further

increase product formation.

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