Expression system for altered expression levels

Details Expression system for altered expression levels Expression system for altered expression levels

2000-01-06

2001-05-01

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S252340, C435S320100, C536S023200

Reexamination Certificate

active

06225106

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the discovery of the lipase regulation cascade of
Pseudomonas alcaligenes
. Specifically, the present invention provides the nucleic acid and amino acid sequences of various components of the lipase regulation cascade which may be used in expression methods and systems designed for the production of heterologous proteins.
BACKGROUND OF THE INVENTION
The isolation and identification of a microorganism that can naturally secrete a product of potential industrial production is one of, if not the most, vital steps in the process of fermentation biotechnology. The ability to secrete the protein of interest usually leads to easier downstream processing. The next critical stage is the mutagenesis of a naturally occurring strain to a hyper-producing strain. Over a number of years, scientists have developed screening strategies from which a number of exo-protein producing bacteria have been isolated. Following isolation, a large number of rounds of mutagenesis can be used to continuously select higher producing strains. However, classical strain improvement cannot be used indefinitely to further increase production levels. Therefore, a more direct method of characterization and molecular genetic manipulation is needed to achieve higher production levels.
Several patents and publications have claimed or described a lipase modulator gene (WO 94/02617; EP 331,376; Nakanishi et al. (1991)
Lipases
-
Struct. Mech. Genet. Eng.
GBF Monographs 16:263-266). However, later research has shown that the product of the gene, now called lif, is concerned with folding of the lipase rather than regulating the expression of the lipase. A review of various lipase expression systems that use the lif gene product can be found in Jaeger et al. (1994)
FEMS Microbiol. Rev.
15:29-63.
Another publication discusses the sigma 54 promoter and the types of genes that have been described to be under control of this type of promoter. Morrett and Segovia (1993)
J. Bacter.
175:6067-6074.
The search has continued for an expression system that can efficiently express a heterologous protein, particularly a lipase in
Pseudomonas
, in particular
Pseudomonas alcaligenes
. Pseudomonas expression of lipase is very difficult and often is at lower levels than industry would like to see.
The present invention solves the problem of low levels of expression of proteins in Pseudomonas as well as other microbial hosts.
SUMMARY OF THE INVENTION
The present invention relates to the discovery of a Pseudomonas lipase regulation cascade and provides individual components of the regulation cascade that can be used in expression systems for the production and secretion of proteins in host cells. The regulation cascade comprises, surprisingly, a two-component part that includes a kinase and a DNA binding regulator. The two components work in concert with a promoter and an upstream binding sequence to efficiently express a protein. The regulation cascade also comprises secretion factors that can be used in host cells to enhance the secretion of produced proteins.
The present invention provides nucleic acid and amino acid sequences for the various components of the
Pseudomonas alcaligenes
lipase regulation cascade. The present invention also provides new, efficient expression systems, i.e., expression vectors, and host cells that can be used to express proteins at increased levels. The new expression systems allow for increased expression of a protein whose gene is functionally linked to components of the expression system, i.e., components of the lipase regulation cascade. A hyper-producing strain can thus be developed and used in a commercial setting.
In one embodiment of the invention, an isolated nucleic acid encoding a kinase that can regulate the expression of a protein, preferably a lipase, is provided. The nucleic acid encoding a kinase is preferably derived from a Gram-negative bacteria such as a pseudomonad, preferably from
Pseudomonas alcaligenes
and is most preferably lipQ. Further, nucleic acid encoding the kinase preferably has the sequence as shown in
FIGS. 1A-1B
(SEQ ID NO: 1) and/or has at least 50% homology with that sequence. The kinase protein is also provided and it is preferably derived from a bacteria, preferably from a Gram-negative bacteria such as a pseudomonad, most preferably, the kinase is from
Pseudomonas alcaligenes
. In a preferred embodiment, the kinase is LipQ. The kinase preferably has the sequence shown in
FIGS. 1A-1B
, (SEQ ID NO: 2) and/or has at least 50% homology with that sequence.
In another embodiment, the present invention provides a nucleic acid encoding a kinase that can regulate the expression of a lipase in
Pseudomonas alcaligenes
. In another embodiment, the present invention provides a kinase capable of regulating the expression of a lipase in
Pseudomonas alcaligenes.
In a further embodiment of the invention, an isolated nucleic acid encoding a DNA binding regulator that can regulate the expression of a protein, preferably a lipase, is provided. The DNA binding regulator nucleic acid is preferably lipR. Further, it preferably has the sequence as shown in
FIGS. 2A-2B
(SEQ ID NO: 3) and/or has at least 50% homology with that sequence. The DNA binding regulator protein is also provided and it is preferably LipR. The DNA binding regulator preferably has the sequence shown in
FIGS. 2A-2B
(SEQ ID NO: 4) and/or has at least 50% homology with that sequence. Preferably, the DNA binding regulator is from bacteria. More preferably, the DNA binding regulator is from a Gram-negative bacteria such as a pseudomonad. Most preferably, the DNA binding regulator is from
Pseudomonas alcaligenes.
In yet a further embodiment, the present invention provides an isolated nucleic acid that encodes a DNA binding regulator that can regulate the expression of a lipase in
Pseudomonas alcaligenes
. In another embodiment, the present invention provides the DNA binding regulator itself.
In yet another embodiment of the invention, nucleic acid encoding a portion of a polymerase that can regulate the expression of a protein, preferably a lipase, is provided. The polymerase nucleic acid is preferable orfZ. Further, it preferably has the sequence as shown in
FIGS. 9A-9B
(SEQ ID NO: 36) and/or has at least 75% homology with that sequence. A portion of the polymerase protein is also provided and it is preferable OrfZ. The polymerase protein preferable has the sequence shown in
FIGS. 9A-9B
(SEQ ID NO: 37) and/or at least 75% homology with the sequence. Preferably, the polymerase is from Gram-negative bacteria such as pseudomonad. Most preferably, the polymerase is from
Pseudomonas alcaligenes.
In another embodiment, the kinase, the DNA binding regulator and a portion of the polymerase are present in one nucleic acid. In another embodiment, the kinase, the DNA binding regulator and the polymerase have the nucleic acid sequence shown in
FIGS. 4A-4G
(SEQ ID NO: 28).
In another embodiment of the invention, an isolated nucleic acid encoding a
Pseudomonas alcaligenes
sigma 54 promoter is provided.
In a further embodiment of the invention, an isolated nucleic acid encoding a
Pseudomonas alcaligenes
upstream activating sequence is provided. The upstream activating sequence is preferably UAS. Further, it preferably has the sequence as shown in SEQ ID NO: 5 and/or has at least 50% homology with that sequence. Preferably, the upstream activating sequence is from bacteria. More preferably, the upstream activating sequence is from a Gram-negative bacteria such as a pseudomonad. Most preferably, the upstream activating sequence is from
Pseudomonas alcaligenes.
In yet another embodiment of the invention, isolated nucleic acids encoding secretion factors are provided. The secretion factors are preferably XcpP, XcpQ, OrfV, OrfX, XcpR, XcpS, XcpT, XcpU, XcpV, XcpW, XcpX, XcpY, XcpZ and another protein, OrfY, having the C-terminal amino acid sequence shown in SEQ ID NO: 35. Further, they preferably have the nucleic acid sequence as shown in SEQ ID NOS: 12,

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