Increasing production of proteins in gram-positive...

Details Increasing production of proteins in gram-positive... Increasing production of proteins in gram-positive...

2000-03-22

2003-01-14

Achutamurthy, Ponnathapu (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...

C435S320100, C435S252300, C435S252310

Reexamination Certificate

active

06506579

ABSTRACT:

FIELD OF THE INVENTION
The present invention generally relates to expression of proteins in gram-positive microorganisms and specifically to the gram positive microorganism secretion factor, SecG. The present invention also provides expression vectors, methods and systems for the production of proteins in gram-positive microorganisms.
BACKGROUND OF THE INVENTION
Gram-positive microorganisms, such as members of the group Bacillus, have been used for large-scale industrial fermentation due, in part, to their ability to secrete their fermentation products into the culture media. In gram-positive bacteria, secreted proteins are exported across a cell membrane and a cell wall, and then are subsequently released into the external media usually obtaining their native conformation.
Secretion factors from Gram-positive microorganisms which have been identified and reported in the literature include SecA (Sadaie Y., Takamatsu h., Nakamura k., Yamane k.; Gene 98:101-105, 1991)., SecY (Suh J.-W., Boylan S. A., Thomas S. M., Dolan K. M., Oliver D. B., Price C. W.; Mol. Microbiol. 4:305-314, 1990)., SecE (Jeong S., Yoshikawa H., Takahashi H.; Mol. Microbiol. 10:133-142, 1993), FtsY an FfH (PCT/NL 96/00278), and PrsA (WO 94/19471).
By contrast, in the gram-negative microorganism,
E.coli
, protein is transported to the periplasm rather than across the cell membrane and cell wall and into the culture media.
E.coli
has at least two types of components of the secretory mechanism, soluble cytoplasmic proteins and membrane associated proteins. Reported
E.coli
secretion factors include the soluble cytoplasmic proteins, SecB and heat shock proteins; the peripheral membrane-associated protein SecA; and the integral membrane proteins SecY, SecE, SecD and SecF.
In spite of advances in understanding portions of the protein secretion machinery in procaryotic cells, the complete mechanism of protein secretion, especially for gram-positive microorganisms, such as Bacillus, has yet to be fully elucidated.
SUMMARY OF THE INVENTION
The capacity of the secretion machinery of a Gram-positive microorganism may become a limiting factor or bottleneck to protein secretion and the production of proteins in secreted form, in particular when the proteins are recombinantly introduced and overexpressed by the host cell. The present invention provides a means for alleviating that bottle neck.
The present invention is based, in part, upon the discovery of a
Bacillus subtilis
SecG secretion factor (also referred to herein as YVAL) identified in heretofore uncharacterised translated genomic DNA by its homology with a consensus sequence for SecG (based upon SecG sequences for Escherichia, Haemophilus, and Mycoplasma organisms) and the demonstration that
B. subtilis
SecG is a functional homolog of
E.coli
Sec G. The present invention is also based, in part, upon the discovery that
B.subtilis
SecG in combination with other
B.subtilis
secretion factors forms a functional preprotein translocase.
The present invention provides isolated nucleic acid and deduced amino acid sequences for
B. subtilis
SecG. The amino acid sequence for
B. subtilis
SecG is shown in
FIG. 1
(SEQ ID No:1). The nucleic acid sequence encoding
B. subtilis
SecG is shown in
FIG. 1
(SEQ ID No:2).
The present invention also provides improved methods for secreting proteins from gram-positive microorganisms. Accordingly, the present invention provides an improved method for secreting a desired protein in a gram-positive microorganism comprising the steps of obtaining a gram-positive microorganism host cell comprising nucleic acid encoding SecG wherein said nucleic acid is under the control of expression signals capable of expressing SecG in a gram-positive microorganism said microorganism further comprising nucleic acid encoding said protein; and culturing said microorganism under conditions suitable for expression of SecG and expression and secretion of said protein. in one embodiment of the present invention, the desired protein is homologous or naturally occurring in the gram-positive microorganism. In another embodiment of the present invention, the desired protein is heterologous to the gram-positive microorganism.
In one aspect of the present invention, a microorganism is genetically engineered to produce a desired protein, such as an enzyme, growth factor or hormone. The enzyme is selected from the group consisting of proteases, carbohydrases including amylases, cellulases, xylanases, and lipases; isomerases such as racemases, epimerases, tautomerases, or mutases; transferases, kinases and phophatases acylases, amidases, esterases, reductases, oxidases. In a further embodiment the expression of the secretion factor SecG is coordinated with the expression of other components of the secretion machinery. Preferably other components of the secretion machinary, i.e., translocase, SecA, SecY, SecE and/or other secretion known to those of skill in the art are modulated in expression at an optimal ratio to SecG. For example, it may be desired to overexpress multiple secretion factors in addition to SecG for optimum enhancement of the secretion machinary. In one particular embodiment disclosed herein,
B.subtilis
SecG is expressed along with
B.subtilis
SecYE and SecA to form a functional preprotein translocase.
The present invention also provides a method of identifying homologous gram positive microorganism SecG that comprises hybridizing part or all of
B. subtilis
SecG nucleic acid shown in
FIG. 1
(SEQ ID No:1) with nucleic acid derived from gram-positive microorganisms. In one embodiment, the nucleic acid is of genomic origin. In another embodiment, the nucleic acid is a cDNA. The present invention encompasses novel gram-positive microorganism secretion factors identified by this method.


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