Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process...
Reexamination Certificate
1998-11-19
2002-10-01
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
C435S069100, C435S183000, C435S198000, C435S201000, C435S219000
Reexamination Certificate
active
06458557
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of molecular biology and in particular to the identification of molecules involved in formate transport and utilization in Bacillus. The present invention also provides methods for increasing the yields of polypeptides produced in Bacillus.
INTRODUCTION
Gram-positive microorganisms, such as Bacillus, have been used for large-scale industrial fermentation due, in part, to their ability to secrete their fermentation products into the culture media. Secreted proteins are exported across a cell membrane and a cell wall, and then are subsequently released into the external media. It is advantageous to produce proteins of interest in gram-positive microorganisms since exported proteins usually maintain their native conformation.
Suppmann et al. (1994, Molecular Microbiology vol. 11(5), pg. 965-982) describe a putative formate transporter in a gram-negative microorganism,
E. coli
. Nagy et al. (1995, Journal of Bacteriology, vol: 177, pg. 1292) describe a formyltetrahydrofolate hydrolase in
E. coli
. Mazel et al (1997, J. Mol. Biol. 266:939-949) describe a polypeptide deformylase function in Eubacterial lineage. Little is known, however, about the uptake and utilization of formate in gram-positive microorganisms used in large scale fermentation methods for the production of heterologous proteins.
Gene products which may be associated with formate utilization have been identified in Bacillus. An operon for the production of the co-enzyme tetrahydrofolate (THF) was disclosed by de Siazieu (1997, Microbiology 143:979-989). It is also known that a 10-formyltetrahydrofolate synthetase (ligase) activity and a 5,10-methylenetetrahydrofolate dehydrogenase have been shown to exist in
B. subtilis
(Whitehead et al., 1988, Bacteriology 170:995-997) and Saxild et al. (1994, Mol. Gen. Genet. 242:415-420) have identified a 5′-phosphoribosyl-1-glycinamide (GAR) transforylase which catalyses a one carbon transfer reaction in purine biosynthesis. This enzyme, the product of the purT locus, was found to be dependent on formate added-either to the growth medium or to in vitro assays using cell-free extracts.
There remains a need in the art to optimize grapositive expression systems so that production of products in these systems can be increased.
SUMMARY OF THE INVENTION
Prior to the present invention, very little was known about formate transport, utilization or cycling in gram-positive microorganisms. While studying the effect of different additives on the growth of a gram-positive microorganism, Bacillus, in shake flask, a growth enhancement phenomenon was observed when sodium formate was added to the medium. Also, in the absence of exogenous formate, the phenomenon of endogenous formic acid production during gram-.positive microorganism fermentation was observed.
The present invention is therefore based, in part, upon the modification(s) in gram-positive microorganism growth observed in the presence of endogenous or exogenous sodium formate. The present invention is also based upon the evidence presented herein that formate is transported into Bacillus by a symport transport mechanism. Accordingly, the present invention provides a method for modifying the growth of gram-positive microorganisms comprising modifying formate transport in the gram-positive microorganism.
The present invention is also based, in part, upon the identification and characterization of four Bacillus proteins found encoded by genomic nucleic acid sequences of
Bacillus subtilis
which appear to be associated with the formate transport, utilization and cycling: formate transport associated protein 1 (FTAP1) and formate transport associated protein 2 (and FTAP2) which have about 35% and 30% identity, respectively, with the
E. coli
protein. FocA, a formate channel protein;
Bacillus subtilis
PurU, which has about 48% identity at the amino acid level with PurU of
E. coli
, a N10-formytetrahydrofolate hydrolase which is involved in the cycling of tetrahydrofolate and formyl tetrahydrofolate, and a formylmethionine deformylase (FMD), which has about 40% similarity to a formylmethionine deformylase (YkrB).
The present invention is further based upon data which shows that in the presence of exogenous formate, a Bacillus cell cultured in shake flask and having an interruption of the gene encoding FTAP1 exhibits about a 50% decrease in the growth enhancement normally seen in the presence of exogenous formate. In the presence of exogenous formate, a Bacillus cell cultured in shake flask and having an interruption of the gene encoding FTAP 2 grows more slowly and the density of the culture declines over time. Thus, it appears that FTAP 1 and FTAP 2 are associated with formate transport and utilization in Bacillus.
Therefore, modulating the expression of molecules involved in formate transport, utilization and cycling, e.g., FTAP 1, FTAP 2, PurU, and FMD either individually or in combination with each other or other associated molecules, provides a means for regulating the levels of formate production in gram-positive microorganisms. It may be desirable to increase the expression of such molecules, decrease the expression of such molecules, or regulate the expression of such molecules, i.e., provide a means for expressing such molecules during a defined time in cell growth, depending upon the type of gram-positive microorganism and culture conditions desired.
Accordingly, the present invention provides a method for increasing the production of a product in a gram-positive microorganism comprising the steps of obtaining a microorganism capable of expressing the product and comprising nucleic acid encoding either one or both of i) formate transport associated protein 1 (FTAP 1) and ii) formate transport associated protein 2 (FTAP 2); and culturing said microorganism in the presence of formate and under conditions suitable for expression of said product. The product includes naturally occurring products obtainable from a gram-positive microorganism, such as anti-microbial compounds, antibiotics, antigens, antibodies, surfactant, chemical products and enzymes, as well as products, such as proteins and polypeptides, which are encoded by recombinantly introduced nucleic acid.
In one aspect, the product is a recombinant protein. In one embodiment, the recombinant protein is homologous to said gram-positive microorganism and in another embodiment, the recombinant protein is heterologous to said gram-positive microorganism. In one aspect of the present invention, the gram-positive organism is a Bacillus and in yet another embodiment, the Bacillus includes
B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus
and
Bacillus thunringiensis.
In one aspect of the present invention, the recombinant protein includes hormones, enzymes, growth factor and cytokine and in another, the enzyme includes protease, lipase, amylase, pullulanase, cellulase, glucose isomerase, laccase and a protein disulfide isomerase.
Under large scale Bacillus fermentation conditions performed in the absence of exogenous formate, an excess of endogenous formate or formate “spillover” has been observed in the culture media. Therefor, it may be desirable to delete, mutate or otherwise interrupt the genes encoding FTAP 1 and 2 in order to maintain appropriate endogenous formate levels. Accordingly, the present invention provides a method for producing a product in a gram-positive microorganism comprising the steps of obtaining a gram-positive microorganism capable of expressing said product said microorganism having a mutation in the nucleic acid encoding either one or both of FTAP 1 and FTAP 2 said mutation resulting in inhibition of production by said microorganism of the FTAP 1 and/or FTAP 2 activity; and b) culturing said microorganism under conditions suitable for expression of said product.
Furthermore, based upon the overall amino acid sequence homology of Bacillus PurU with
E. coli
PurU, it appe
Diaz-Torres Maria
Miller Brian
Genencor International Inc.
Genencor International, Inc
Kerr Kathleen M.
Saidha Tekchand
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