Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1999-05-11
2002-03-19
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
Reexamination Certificate
active
06358716
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the field of molecular biology and the use of recombinant organisms for the production of desired compounds. More specifically it describes the expression of cloned genes for glycerol-3-phosphate dehydrogenase (G3PDH) and glycerol-3-phosphatase (G3P phosphatase), either separately or together, for the enhanced production of glycerol.
BACKGROUND
Glycerol is a compound in great demand by industry for use in cosmetics, liquid soaps, food, pharmaceuticals, lubricants, anti-freeze solutions, and in numerous other applications. The esters of glycerol are important in the fat and oil industry.
Not all organisms have a natural capacity to synthesize glycerol. However, the biological production of glycerol is known for some species of bacteria, algae, and yeasts. The bacteria
Bacillus licheniformis
and
Lactobacillus lycopersica
synthesize glycerol. Glycerol production is found in the halotolerant algae Dunaliella sp. and
Asteronmonas gracilis
for protection against high external salt concentrations (Ben-Amotz et al., (1982)
Experientia
38:49-52). Similarly, various osmotolerant yeasts synthesize glycerol as a protective measure. Most strains of Saccharomyces produce some glycerol during alcoholic fermentation, and this can be increased physiologically by the application of osmotic stress (Albertyn et al., (1994)
Mol. Cell. Biol
. 14, 4135-4144). Earlier this century glycerol was produced commercially with Saccharomyces cultures to which steering reagents were added such as sulfites or alkalis. Through the formation of an inactive complex, the steering agents block or inhibit the conversion of acetaldehyde to ethanol, thus, excess reducing equivalents (NADH) are available to or “steered” towards dihydroxyacetone phosphate (DHAP) for reduction to produce glycerol. This method is limited by the partial inhibition of yeast growth that is due to the sulfites. This limitation can be partially overcome by the use of alkalis which create excess NADH equivalents by a different mechanism. In this practice, the alkalis initiated a Cannizarro disproportionation to yield ethanol and acetic acid from two equivalents of acetaldehyde.
The gene encoding glycerol-3-phosphate dehydrogenase (DAR1,GPD1) has been cloned and sequenced from
Sacchatromyces diastaticus
(Wang et al., (1994).
J. Bact
. 176:7091-7095). The DAR1 gene was cloned into a shuttle vector and used to transform
E. coli
where expression produced active enzyme. Wang et al., supra recognizes that DAR1 is regulated by the cellular osmotic environment but does not suggest how the gene might be used to enhance glycerol production in a recombinant organism.
Other glycerol-3-phosphate dehydrogenase enzymes have been isolated. For example, sn-glycerol-3-phosphate dehydrogenase has been cloned and sequenced from
S. cerevisiae
(Larason et al., (1993)
Mol. Microbiol
., 10:1101, (1993)). Albertyn et al., (1994)
Mol. Cell. Biol
., 14:4135) teach the cloning of GPD1 encoding a glycerol-3-phosphate dehydrogenase from
S. cerevisiae
. Like Wang et al., both Albertyn et al., and Larason et al. recognize the osmo-sensitivity of the regulation of this gene but do not suggest how the gene might be used in the production of glycerol in a recombinant organism.
As with G3DPH, glycerol-3-phosphatase has been isolated from
Saccharomyces cerevisiae
and the protein identified as being encoded by the GPP1 and GPP2 genes (Norbeck et al., (1996)
J. Biol. Chem
., 271:13875). Like the genes encoding G3DPH, it appears that GPP2 is osmotically-induced.
There is no known art that teaches glycerol production from recombinant organisms with G3PDH/G3P phosphatase expressed together or separately. Nor is there known art that teaches glycerol production from any wild-type organism with these two enzyme activities that does not require applying some stress (salt or an osmolyte) to the cell. Eustace ((1987),
Can. J. Microbiol
., 33:112-117)) teaches away from achieving glycerol production by recombinant DNA techniques. By selective breeding techniques, these investigators created a hybridized yeast strain that produced glycerol at greater levels than the parent strains: however, the G3PDH activity remained constant or slightly lower.
A microorganism capable of producing glycerol under physiological conditions is industrially desirable, especially when the glycerol itself will be used as a substrate in vivo as part of a more complex catabolic or biosynthetic pathway that could be perturbed by osmotic stress or the addition of steering agents.
The problem to be solved, therefore, is how to direct carbon flux towards glycerol production by the addition or enhancement of certain enzyme activities, especially G3PDH and G3P phosphatase which respectively catalyze the conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) and then to glycerol. This process has not previously been described for a recombinant organism and required the isolation of genes encoding the two enzymes and their subsequent expression. A surprising and unanticipated difficulty encountered was the toxicity of G3P phosphatase to the host which required careful control of its expression levels to avoid growth inhibition.
SUMMARY OF THE INVENTION
The present invention provides a method for the production of glycerol from a recombinant organism comprising: (i) transforming a suitable host cell with an expression cassette comprising either or both
(a) a gene encoding a glycerol-3-phosphate dehydrogenasc enzyme;
(b) a gene encoding a glycerol-3-phosphate phosphatase enzyme: (ii) culturing the transformed host cell in the presence of at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides. polysaccharides, and single-carbon substrates, or mixtures thereof whereby glycerol is produced; and (iii) recovering the glycerol. Glucose is the most preferred carbon source.
The invention further provides transformed host cells comprising expression cassettes capable of expressing glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase activities for the production of glycerol.
BRIEF DESCRIPTION OF BIOLOGICAL DEPOSITS AND SEQUENCE LISTING
Applicants have made the following biological deposits under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the Purposes of Patent Procedure:
Depositor Identification
Int'l. Depository
Reference
Designation
Date of Deposit
Escherichia coli
pAH21/DH5&agr;
ATCC 98187
26 September
(containing the GPP2 gene)
1996
Escherichia coli
(pDAR1A/AA200)
ATCC 98248
6 November
(containing the DAR1 gene)
1996
“ATCC” refers to the American Type Culture Collection international depository located at 12301 Parklawn Drive Rockville, Md. 20852 U.S.A. The designation is the accession number of the deposited material.
Applicants have provided 23 sequences in conformity with the Rules for the Standard Representation of Nucleotide and Amino Acid Sequences in Patent Applications (Annexes I and II to the Decision of the President of the EPO, published in Supplement No. 2 to OJ EPO. 12/1992) and with 37 C.F.R. 1.821-1.825 and Appendices A and B (Requirements for Application Disclosures Containing Nucleotides and/or Amino Acid Sequences).
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for the biological production of glycerol from a fermentable carbon source in a recombinant organism. The method provides a rapid inexpensive and environmentally-responsible source of glycerol useful in the cosmetics and pharmaceutical industries. The method uses a microorganism containing cloned homologous or heterologous genes encoding glycerol-3-phosphate dehydrogenase (G3PDH) and/or glycerol-3-phosphatase (G3P phosphatase). The microorganism is contacted with a carbon source and glycerol is isolated from the conditioned media. The genes may be incorporated into the host microorganism separately or together for the production of glycerol.
As used herein the following terms may be used for interpretation of t
Bulthuis Ben A.
Gatenby Anthony Arthur
Haynie Sharon Loretta
Hsu Amy Kuang-Hua
Lareau Richard D.
Achutamurthy Ponnathapu
E. I. du Ponte de Nemours and Company
Kerr Kathleen
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