ABC transporter gene cluster in pseudomonas fluorescens for...

Details ABC transporter gene cluster in pseudomonas fluorescens for... ABC transporter gene cluster in pseudomonas fluorescens for...

2000-01-26

2001-12-11

Carlson, Karen Cochrane (Department: 1653)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C536S023100, C536S023710, C435S006120, C435S069100, C435S320100, C544S361000

Reexamination Certificate

active

06329172

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to ABC transporter gene cluster, tliDEF, in Pseudomonas fluorescens(“
P. fluorescens”
), more specifically, to ABC transporter gene of
P. fluorescens
encoding proteins which enhance the secretion of foreign proteins extracellularly, a recombinant expression vector containing the transporter gene, a microorganism transformed therewith and a process for preparing foreign proteins which comprises the steps of incubating the transformant, inducing expression of the proteins and leading the proteins to be secreted extracellularly.
BACKGROUND OF THE INVENTION
For the last scores of years, various genes from microorganism to human have been isolated and sequenced. In line with the development of biotechnology, foreign proteins have been produced by expressing these genes in host organism such as animal, plant, microorganism, etc. and used it various fields of medicaments, enzymes for biochemical synthesis, food additives and agriculture.
Escherichia coli
(“
E. coli”
), a representative host organism used for introducing gene, has been employed as a research tool for a long time, since it has been throughly investigated to use conveniently. Further many
E. coli
strains for various purposes have been developed so far, which accelerate the use of
E. coli
for the production of useful proteins necessary for industrial purposes and research purposes as well.
E coli
has an advantage of excessive production of desired protein, since it accumulates foreign gene-derived proteins in a level of 20-50% of total proteins. However, there are many cases that proteins are produced not as an active form but as an inactive inclusion body. Production of inclusion body occurred when proteins can not be secreted extracellularly and then excess amount of proteins accumulated in the cell. These proteins are accumulated as an inclusion body before they were refolded to have a biological activity(see: Mitraki et al., Bio/Technology, 7:693(1989)). Many attempts have been made to solve these problems, for examples, change of fermentation condition(see: Schein, Bio/Technology, 7.1141(1989)) or insertion of chaperon preventing inclusion body from being produced(see: Hockney, Trends Biotechnol., 12:456(1994)).
As an alternative approach, a method for secreting intracellular protein to extracellular medium has been investigated. If tale expressed proteins are secreted extracellularly before the accumulation in the cell, inclusion body is not formed any more. Expressed proteins can be ideally manufactured from microorganism by secreting the proteins in the cell, removing the microorganism from culture media, and obtaining the desired proteins from the media. If the expressed proteins are not secreted to extracellular media, inactive inclusion body is formed, or if the inclusion body is not formed, mechanical, chemical and biochemical methods are required to lyse cells and desired proteins should be purified from many other proteins contained in the cell. This accompanies great loss of expense and loss of proteins. Accordingly, it is of great importance to improve is method for secreting proteins expressed in microorganism to extracellular medium.
In an association with protein secretion, general export pathway(GEP) has been extensively studied, by which N-terminal sequence of protein is recognized by sec protein in the cell to secrete the protein extracellularly(see: Pugsley, Microbiol. Rev., 57:50(1993)) The industrial use of GEP has been considered, since GEP can lead intracellular proteins to attach to cell surface or to be secreted extracellularly(see: U.S. Pat. Nos. 5,156,959; 5,541,087; 5,658,755; and, 5,665,590). By way of GEP, protein is secreted extracellularly in case of Gram-positive bacteria having only one cell membrane, however, it should pass inner membrane, stay at periplasm and bypass outer membrane in case of Gram-negative bacteria like
E. coli
having two cell membranes. Many proteins(≧14) are involved in the export through only outer membrane, which in turn, reveals a serious problem that secretion efficiency is relatively low(see: Akrim et al., Mol. Microbiol., 10:431(1993); Pugsley and Possot, Mol. Microbiol., 10:665(1993)).
Another pathway, mediated by ABC transporter(ATP binding cassette transporter), has been also known in the art, where ATP is hydrolyzed to secrete protein(see: Fath and Kolter, Microbiol. Rev., 57:995(1993)). Protein thus secreted has a C-terminal targeting signal, which is recognized by ABC transporter, to secret the protein extracellularly. Protein secretion is carried out by the aid of ABC transporter protein located at cell membrane and proteins of both Gram-positive and Gram-negative. bacteria can pass cell membrane at once. That is, in case of Gram-negative bacteria, secretory protein makes inner membrane fuse partly with outer membrane, which allows the protein to pass 2 membranes at the same time. Accordingly, the secretion pathway based on ABC transporter is more efficient in Gram-negative bacteria such as
E. coli.
Until now, many ABC transporters have been successively known in the art, those associated with cyclolysin(see: Glaser et al., EMBO J., 7:3997(1988)), leucotoxin(see: Highlander et al., DNA, 8:15(1989)), protease and lipase, since a secretion system associated with &agr;-hemolysin(see: Felmlee et al., J. Bacteriol., 163:94(1985)) was discovered in
E. coli
. Especially, ABC transporter secreting &agr;-hemolysin has been extensively studied and it is proposed as a universal secretory system, based on the results that other proteins can be secreted by C-terminal targeting signal(see: Blight and Holland, Trends Biotechnol., 12:450(1994) In this connection, ABC transporters cloned from Pseudomonas sp., Erwinia sp. and Serratia sp., are practically applied for the secretion of protease extracellularly(see: Duong et al., Gene, 121:47(1992); Letoffe et al., EMBO J., 9:1375(1990); Letoffe et al., T. Bacterol., 173:2160(1991)). However, little attraction has been, paid to ABC transporter for secreting lipase.
So far, two kinds of ABC transporters secreting, lipase extracellularly have been reported in the art: First, a gene secreting lipase was cloned from
Serratia marcescens
(see: Akatsuka et al., J. Bacteriol., 177:6381(1995)); second, ABC transporter secreting protease from
P. aeruginosa
was revealed to secrete lipase of
P. fluorescens
(see: Duong et al., Mol. Microbiol., 11; 1117(1994)). Naturally, ABC transporter has not been practically applied in industry since only the ABC transporters from
Serratia marcescens
and protease from
P. fluorescens
are available to secrete lipase. In addition, ABC transporter for lipase secretion has not been found in
P. fluorescens
, though the lipase from the microorganism was cloned in many research groups(see: Lee et al, Biochim. Biophys. Acta., 1169:156(1993); Tan et al., Appl. Environ. Microbiol., 58:1402(1992); John et al., Appl. Environ Microbiol., 58:1776(1992)).
Accordinqly, there are strong reasons for exploring ABC transporter gene from
P. fluorescens
, and to develop a novel process for preparing foreign proteins by ABC transporter in a simple and efficient manner.
SUMMARY OF THE INVENTION
The present inventors first cloned a gene encoding ABC transporter from
P. fluorescens
, and constructed an expression vector containing the said gene and lipase or protease coding gene which can be expressed in Gram-negative bacteria such as
E. coli
and Pseudomonas sp., to secrete the lipase or protease proteins. As a result, it was found that these proteins can be produced and accumulated in extracellular medium with a high efficiency. Since the ABC transporter enhancing the secretion of foreign proteins functions at a low temperature of below 25° C., it can prevent inclusion body formation, which allows universal use of the ABC transporter for industrial purposes.
The first object of the invention is, therefore, to provide ABC transporter gene from
P. fluoresens
which enhances the secretion of foreign proteins to extracellular medium.
The second

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