Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2000-03-24
2001-07-31
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S190000, C435S252300, C435S252330, C435S254110, C435S254210, C536S023200, C536S023700
Reexamination Certificate
active
06268189
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the isolation of a fungal lactate dehydrogenase (Ldh) from
Rhizopus oryzae,
to the gene encoding the Ldh, to nucleic acid constructs containing the gene, and to the expression of the gene in host transformants for the purpose of lactic acid production.
Lactic acid is commonly used as a food additive for preservation, flavor, acidity and for the manufacture of the biodegradable plastic, polylactic acid (PLA). The global lactic acid market is estimated to be in excess of 100,000 tons per year and is expected to increase substantially in next few years as new PLA facilities become operational. Another demand that may grow substantially is the biodegradable solvent ethyl lactate. This ester is considered non-toxic and has many applications that include electronic manufacturing, paints and coatings, textiles, cleaners and degreasers, adhesives, printing, and de-inking. It has been estimated that lactate esters could potentially replace as much as 80% of the 3.8 million tons of solvents used each year in the U.S. However, fermentation efficiency must be improved to ensure the economic feasibility of these anticipated market expansions.
2. Description of the Prior Art
Fermentative methods for production of lactic acid are often preferred over chemical synthesis which results in a mixture of both D and L isomers. The products of microbiological fermentations are dependent on the organism used. They may yield a mixture of the two isomers or optically pure acid in a stereospecific form. The desired stereospecificity of the product depends on the intended use. However, L-(+)-lactic acid is the most desired form for the majority of applications.
Bacterial fermentations with Lactobacilli are common for industrial production of lactic acid, but these fermentations rarely yield optically pure product. Additionally, the fastidious nature of these bacteria requires that considerable amounts of supplemental nutrients be added to the growth medium, adding additional cost and making purification more difficult. Yeast are not capable of producing appreciable levels of lactic acid, although recombinant
Saccharomyces cerevisiae
strains have been described that contain the ldh gene from either Lactobacillus or bovine origins [Patent WO 99/14335 and Adachi et al. J. Ferment. Bioeng. 86:284-289(1998)]. While capable of producing up to 2-4% (w/v) lactic acid, these strains exhibit poor productivity and a significant portion of the glucose is converted to ethanol.
The filamentous fungus
Rhizopus oryzae
(syn.
R. arrhizus
) is also used for industrial production of lactic acid. It was in 1936 that
R. oryzae
was first described as being able to aerobically convert glucose, in a chemically defined medium, to large amounts of optically pure L-(+)-lactic acid. Research on lactic acid production by Rhizopus has continued primarily because of the ease of product purification in a minimal growth medium and the ability of the fungus to utilize both complex carbohydrates and pentose sugars (Hang et al., U.S. Pat. No. 4,963,486). This allows the fungus to be utilized for conversion of low value agricultural biomass to lactic acid.
It is extraordinary to find a filamentous fungus, like
R. oryzae,
that converts such a high percentage of the available carbon source to a fermentative by-product such as lactic acid. Most eukaryotic organisms rely primarily on oxidative phosphorylation when oxygen is available and use fermentation as a means for regenerating NAD
+
only when necessary. Fermentation is much less energy efficient than oxidative phosphorylation, but is often necessary in the absence of oxygen to ensure the availability NAD
+
for continued glycolysis and ATP production. However, it has been suggested that there might be a selective advantage for an organism to convert available sugars to another compound that can still be utilized as an energy source. This is especially true if the fermentation by-product is not as desirable for other organisms that might be competing for the same starting sugars. Rhizopus is very acid tolerant, while most bacteria are inhibited by lactic acid. It may be less efficient for the fungus to ferment sugars to lactic acid, but it is a way to minimize competition by other microorganisms. Similar, theories have also been proposed for ethanologenic yeast that ferment most of the available sugar to ethanol instead relying primarily on oxidative phosphorylation.
Other metabolic products made by Rhizopus include, ethanol, fumaric acid, and glycerol. Production levels for the different metabolites vary tremendously among the Rhizopus species, with some producing predominantly lactic acid and others accumulating only fumaric acid. An ideal lactic acid producing strain of Rhizopus would accumulate little or none of these metabolites, since their production depletes sugar that could be used for conversion to lactate.
Ethanol is believed to be produced by most Rhizopus species primarily as a result of low oxygen conditions. While Rhizopus is not typically considered an organism that grows under anaerobic conditions, it does possess ethanol fermentative enzymes that allow the fungus to grow for short periods in the absence of O
2
. These enzymes have not been purified to homogeneity, but the alcohol dehydrogenase proteins have been partially characterized.
Fumaric acid production has been well-studied in Rhizopus (U.S. Pat. No. 4,877,731) and the fumarase gene has also been isolated. Synthesis is believed to occur primarily through the conversion of pyruvate to oxaloacetate, by pyruvate dehydrogenase. Conditions leading to increased fumaric acid are usually associated with aerobic growth in high glucose levels and low available nitrogen. Accumulation of fumarate is often a problem with lactic acid production, because its low solubility can lead to detrimental precipitations that compromise the fermentation efficiency.
Glycerol is a byproduct that is often produced by Rhizopus grown in high glucose containing medium. There has not been much written specifically about this metabolite accumulation in Rhizopus, but it is likely that regulation is similar to that found in Saccharomyces. There are at least two genes that encode glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) in these organisms. It appears that one of these genes is expressed primarily during anoxic conditions, so that glycerol may act as a redox sink for excess cytosolic NADH. The other gene is involved in osmoregulation and is turned on during osmotic stress. Accumulation of the glycerol is presumably to allow the cell to maintain turgor pressure in the presence of high sugars or salts. Mutants deleted for both activities do not produce detectable glycerol, are highly osmosensitive, and are unable to grow under reduced oxygen conditions.
The ability to modify lactic acid production by genetic modification in Rhizopus and other fungi has been limited. Efforts in this area have been hampered by the lack of cloned ldh genes, encoding functional NAD
+
dependent L-lactate dehydrogenase, of fungal origin. Such a gene would have distinct advantages for expression in filamentous fungi and yeast.
SUMMARY OF THE INVENTION
I have now, for the first time, isolated a fungal Ldh protein and a gene encoding that protein. I have also constructed expression vectors with these genes, transformed host organisms with the vectors, and observed enhanced levels of optically pure L-(+)-lactic acid when the transformants were cultivated on a fermentable medium.
In accordance with this discovery, it is an object of this invention to provide a novel Ldh protein and ldh gene for use in lactic acid production.
It is another object of this invention to make constructs for use in transforming any of numerous host organisms for expressing the ldh gene when the host organism is cultivated in a fermentable medium.
It is also an object of the invention to enhance the L-(+)-lactic acid production of lactic acid-producing organisms
Fado John D.
Pak Yong
Ribando Curtis P.
Silverstein M. Howard
Slobodyansky Elizabeth
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