Erythritol—producing Moniliella strains

Details Erythritol—producing Moniliella strains Erythritol—producing Moniliella strains

2001-01-12

2002-09-24

Lilling, Herbert J. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Fungi

C435S156000, C435S254110, C435S911000

Reexamination Certificate

active

06455301

ABSTRACT:

BACKGROUND
Erythritol is a sugar alcohol that can be found in lichens, hemp leaves, and mushrooms. It is also savored in fermented foods such as wine, soya sauce, or saki (Sasaki, T. (1989) Production technology of erythritol.
Nippon Nogeikagaku Kaishi
63: 1130-1132). Erythritol is a four-carbon polyol, which possesses several properties such as sweetness (about 70-80% of sucrose), tooth friendliness, very low calorific value (0.3 kcal/g, a tenth of sucrose), non-carcinogenicity and, unlike other polyols, causes little, if any, gastrointestinal discomfort (Harald and Bruxelles (1993)
Starch/Starke
45:400-405).
Traditional industrial erythritol production is carried out by adding catalysts such as hydrogen and nickel to the raw material sugars under the environment of high temperature and high pressure. Another process is performed by the chemo-reduction of raw materials such as meso-tartarate (Kent, P. W., and Wood, K. R. (1964)
J. Chem. Soc
. 2493-2497) or erythrose (Otey, F. H., and Sloan, J. W. (1961)
Ind Eng. Chem
. 53:267) to obtain erythritol. In addition, erythritol can be produced by a number of microorganisms. Such organisms include high osmophilic yeasts, e.g., Pichia, Candida, Torulopsis, Trigonopsis, Moniliella, Aureobasidium, and Trichosporon sp. (Onishi, H. (1967)
Hakko Kyokaish
25:495-506; Hajny et al. (1964)
Appl. Microbiol
. 12:240-246; Hattor, K., and Suziki, T. (1974)
Agric. Biol. Chem
. 38:1203-1208; Ishizuka, H., et al. (1989)
J. Ferment. Bioeng
. 68:310-314.)
SUMMARY
The invention features isolated strains of the Moniliella species with enhanced capacities for the conversion of glucose to erythritol. Such strains can produce erythritol from glucose with a conversion rate of at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or greater under optimal conditions.
Strains of the invention include isolates of Moniliella from a natural source; and the mutants of a Moniliella strains, e.g., a Moniliella strains assigned the American Type Culture Collection (ATCC) accession numbers of PTA-1227, PTA-1228, PTA-1229, PTA-1230, and PTA-1232. One particular mutant strain is the isolated strain, N61188-12, deposited with the American Type Culture Collection with the accession number PTA-2862.
As used herein, the term “mutant” refers to a strain whose genetic composition differs by at least one nucleotide, e.g., a substitution, insertion, or deletion, relative to a reference or parent strain. A mutant of the invention can be produced by a number of methods. One method is the selection of strains with increased erythritol conversion rates relative to a parent strain. The strains can be obtained by random mutagenesis of the parent strain, e.g., by means of a chemical mutagen, a transposon, or irradiation. In addition, a mutant strain of the invention can include a recombinant nucleic acid sequence. For example, a mutant may be a strain that harbors an additional nucleic acid sequence, e.g., a sequence transformed, transduced, or otherwise inserted into a cell of the parent strain. The additional nucleic acid sequence can encode a polypeptide that is generally or conditionally expressed. Alternatively, the additional nucleic acid sequence can encode a nucleic acid sequence capable of altering cell physiology, e.g., an anti-sense, a ribozyme, or other nucleic acid sequence. In another instance, the inserted nucleic acid is inserted into an endogenous gene, and alters (e.g., enhances or disrupts) its function. For example, the inserted nucleic acid can be a knockout construct that inactivates the endogenous gene; or an artificial enhancer or promoter that increases transcription of the endogenous gene. The mutation can disrupt the ability of the parental strain to import, assimilate, or consume erythritol or mannitol.
The invention also features a method of producing erythritol. The method includes growing a Moniliella strain of the invention, e.g., an enhanced mutant, in a culture; and purifying erythritol from the culture, e.g., from the supernatant or from the cell pellet.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects and advantages of the invention will be apparent from the description and drawings, and from the claims.
DETAILED DESCRIPTION
The fungus Moniliella is capable of fermenting simple sugars to produce erythritol, a well-relished component of many cuisines. Screening and mutagenesis are used to identify improved strains of Moniliella that are capable of highly efficient erythritol production yields. Such strains are ideal for large-scale erythritol production, as can be achieved by the exemplary methods described herein.
Isolation of Enhanced Erythritol Producing Strains
Isolates of Moniliella can be obtained from a natural source as described in U.S. patent application Ser. No. 09/585,926, filed Jun. 2, 2000, now U.S. Pat. No. 6,300,107. For example, isolates of Moniliella can be obtained natural sources having high sugar content include honey, preserved fruit, and pollen. Each strain is identified based on its capability to convert glucose to erythritol and its various morphological and physiological traits. As used herein, the “glucose-to-erythritol conversion rate” is defined as the amount of erythritol produced divided by the amount of glucose consumed. The resulting ratio can be expressed as a percentage. The glucose-to-erythritol conversion rate of a fungal strain can be calculated by the following method. The strain is first cultured in a 10-ml broth containing 30% glucose and 1% yeast extract (initial cell density 1·10
5
cells/ml) in a 50 ml flask in a rotary shaker at 150 rpm and 30° C. for 6 days. Then, both the concentration of erythritol in the medium and the concentration of glucose in the medium are determined. The conversion of 1 g of glucose into 0.3 g of erythritol is termed a 30% conversion rate. The morphological traits are determined following growth on 4% malt extract, 0.5% yeast extract agar for 10 days at 20° C. See The Yeasts, A Taxonomic Study, Edited by Kurtzman et al., 4th Ed., page 785, Elsevier, Amsterdam (1998)
A mutant of a Moniliella strain can be obtained by the mutagenesis method described in Ishizuka, et al. (1989)
J. Ferment. Bioeng
. 68:310-314, or a variation thereof (see also U.S. Pat. No. 5,036,011). One variation for the mutagenesis of Moniliella cells with N-methyl-N-nitrosoguanidine (NTG) is described as follows. Moniliella cells are inoculated in broth with 30% glucose and 1% yeast extract, and cultured overnight at 30° C. on a rotary shaker at 150 rpm. This culture is diluted 1:100 into 10 ml of broth with 30% glucose, and incubated at 30° C. on a rotary shaker at 150 rpm for 1 day. The culture broth is centrifuged at 3,000 rpm for 15 min to form a cell pellet and the supernatant is discarded. The cell pellet is washed with 10 ml of sterile 0.1 M pH 7.0 phosphate buffered saline (PBS). The suspension is centrifuged (3,000 rpm, 15 min) and the supernatant is again discarded. The cells are resuspended in PBS, with 150 &mgr;g/ml NTG for 10 minutes.
After treatment with NTG, the Moniliella cells are grown in a glucose solution for 3 hours. The culture is then diluted appropriately and spread onto the medium containing 65% glucose and incubated at 30° C. for 6 days. Colonies are selected randomly, inoculated into broth containing 30% glucose, and incubated at 30° C. on a rotary shaker at 150 rpm overnight. A 1:100 dilution of the overnight culture is used to inoculate into a 30% glucose solution (10 ml) that is incubated at 30° C. on a rotary shaker at 150 rpm for 4 days. The medium from this culture is then centrifuged at 12,000 rpm for 10 min. The supernatant is diluted appropriately and the amount of residual glucose is measured using the DNS method (see below). Cultures with higher glucose consumption (i.e., lower residual glucose) are further analyzed to determine erythritol yield. The HPLC method described below can be used to quantitate erythritol yield. Cultures with indications of elevated erythrito

Affiliated with

Also associated with

Rating

Erythritol—producing Moniliella strains does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Erythritol—producing Moniliella strains, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Erythritol—producing Moniliella strains will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2881734