U.S. patent application number 09/759778 was filed with the patent office on 2002-09-19 for erythritol - producing moniliella strains.
Invention is credited to Chu, Wen-Shen, Huang, Chang-Cheng, Lin, Shie-Jea, Wen, Chiou-Yen.
Application Number | 20020132313 09/759778 |
Document ID | / |
Family ID | 25056915 |
Filed Date | 2002-09-19 |
United States Patent
Application |
20020132313 |
Kind Code |
A1 |
Lin, Shie-Jea ; et
al. |
September 19, 2002 |
Erythritol - producing moniliella strains
Abstract
An isolated strain of the Moniliella species that converts
glucose to erythritol with a conversion rate of at least about 45%
is disclosed, as is a method of producing erythritol from such a
strain.
Inventors: |
Lin, Shie-Jea; (Hsinchu,
TW) ; Wen, Chiou-Yen; (Hsinchu, TW) ; Huang,
Chang-Cheng; (Keelung, TW) ; Chu, Wen-Shen;
(Hsinchu, TW) |
Correspondence
Address: |
Y. ROCKY TSAO
FISH & RICHARDSON P.C.
225 Franklin Street
Boston
MA
02110-2804
US
|
Family ID: |
25056915 |
Appl. No.: |
09/759778 |
Filed: |
January 12, 2001 |
Current U.S.
Class: |
435/158 ;
435/254.1 |
Current CPC
Class: |
C12P 7/18 20130101; C12R
2001/645 20210501; Y10S 435/911 20130101; C12N 1/145 20210501 |
Class at
Publication: |
435/158 ;
435/254.1 |
International
Class: |
C12N 001/14; C12N
001/16; C12N 001/18 |
Claims
What is claimed is:
1. An isolated strain of the Moniliella species, wherein the strain
converts glucose to erythritol with a conversion rate of at least
about 45%.
2. The isolated strain of claim 1 wherein the strain converts
glucose to erythritol with a conversion rate of at least about
50%.
3. The isolated strain of claim 2 wherein the strain converts
glucose to erythritol with a conversion rate of at least about
60%.
4. The isolated strain of claim 1 wherein the strain is a mutant of
the Moniliella strain PTA-1227.
5. The isolated strain of claim 1 wherein the strain is a mutant of
the Moniliella strain PTA-1228.
6. The isolated strain of claim 1 wherein the strain is a mutant of
the Moniliella strain PTA-1229.
7. The isolated strain of claim 1 wherein the strain is a mutant of
the Moniliella strain PTA-1230.
8. The isolated strain of claim 1 wherein the strain is a mutant of
the Moniliella strain PTA-1232.
9. The isolated strain of claim 7 wherein the strain is N61188-12,
deposited with the American Type Culture Collection with the
accession number ______.
10. A method of producing erythritol, the method comprising:
growing the Moniliella strain of claim 1 in a culture; and
purifying erythritol from the culture.
11. A method of producing erythritol, the method comprising:
growing the Moniliella strain of claim 2 in a culture; and
purifying erythritol from the culture.
12. A method of producing erythritol, the method comprising:
growing the Moniliella strain of claim 3 in a culture; and
purifying erythritol from the culture.
13. A method of producing erythritol, the method comprising:
growing the Moniliella strain of claim 4 in a culture; and
purifying erythritol from the culture.
14. A method of producing erythritol, the method comprising:
growing the Moniliella strain of claim 5 in a culture; and
purifying erythritol from the culture.
15. A method of producing erythritol, the method comprising:
growing the Moniliella strain of claim 6 in a culture; and
purifying erythritol from the culture.
16. A method of producing erythritol, the method comprising:
growing the Moniliella strain of claim 7 in a culture; and
purifying erythritol from the culture.
17. A method of producing erythritol, the method comprising:
growing the Mon iliella strain of claim 8 in a culture; and
purifying erythritol from the culture.
18. A method of producing erythritol, the method comprising:
growing the Moniliella strain of claim 9 in a culture; and
purifying erythritol from the culture.
Description
BACKGROUND
[0001] 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, gastro-intestinal discomfort (Harald and Bruxelles
(1993) Starch/Starke 45:400-405).
[0002] 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.,
etal. (1989) J. Ferment. Bioeng. 68:310-314.)
SUMMARY
[0003] 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.
[0004] 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-1 227, PTA-1 228, PTA-1 229, PTA-1
230, and PTA-1232. One particular mutant strain is the isolated
strain, N61 188-12, deposited with the American Type Culture
Collection with the accession number ______.
[0005] 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.
[0006] 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.
[0007] 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
[0008] 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.
[0009] Isolation of Enhanced Erythritol Producing Strains
[0010] Isolates of Moniliella can be obtained from a natural source
as described in U.S. patent application 09/585,926, filed Jun. 2,
2000. 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.multidot.10.sup.5 cells/ml) in a 50 ml flask in a rotary shaker
at 150 rpm and 30.degree. 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.degree. C. See
The Yeasts, A Taxonomic Study, Edited by Kurtzman et al., 4th Ed.,
page 785, Elsevier, Amsterdam (1998).
[0011] 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.degree. 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.degree. 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 .mu.g/ml NTG for 10
minutes.
[0012] 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.degree. C. for 6 days. Colonies are selected
randomly, inoculated into broth containing 30% glucose, and
incubated at 30.degree. 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.degree. 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 erythritol yield are subject
to further verification. For example, individual colonies are
obtained for the culture, re-grown as described above, and
reanalyzed. Selected colonies can be improved by additional rounds
of mutagenesis according to these procedures.
[0013] Measurement of Residual Glucose
[0014] 4-day-old culture broth is collected and centrifuged at
12,000 rpm for 10 min. The supernatant is diluted appropriately. 1
ml of each diluted solution is added to 0.5 ml of DNS
(dinitrosalicylic acid) reagent. DNS reagents (e.g., a. 1%
3,5-dinitrosalicylic acid (DNS). b. 0.2% phenol; c. 0.05%
NaHSO.sub.3 or 0.025% Na.sub.2S.sub.2O.sub.3; d. 1% NaOH; e. 0.5%
potassium sodium tartrate tetrahydrate) were prepared and used
according to method described in Miller, G. L. (1958) Anal. Chem.
31:426-428. The mixture is mixed well and incubated at 100.degree.
C. for 5 min. After cooling under room temperature, 9 ml water is
added and the absorbance at 540 nm (OD.sub.540 nm) is determined.
The absorbance at 540 nm is used to determine the concentration of
glucose by comparison with the standard curve, obtained by
measuring pure glucose at various concentrations.
[0015] Measurement of Erythritol Concentration
[0016] The amount of erythritol in a supernatant can be quantitated
by HPLC and TLC, e.g., to determine the erythritol-producing
capacity of a strain. HPLC analysis is performed by Hewlett Packard
H4033A analyzer on an Ion-300 chromatography column, using 0.1 N
sulfuric acid as the flowing phase with a flowing rate of 0.4
ml/min, the temperature being set at 75.degree. C. For TLC
analysis, the Neissner et al. procedure is followed. (Neissner, et
al. 1980. Herstellung, aanalyse und DC-trennung von fettsaure
erythritpartialestern. FETTE SEIFEN ANSTRICHMITTEL. 82:10-16.).
After rinsing Kieselgel 60F254 (Merck) with 4% boric acid, the gel
is heated in an incubator at 105.degree. C. for 20 minutes before
use. The spreading solvent is ethylmethylketone:acetone:water
(100:10:10 by vol.) and the color-developing agent is KMnO.sub.4 in
concentrated sulfuric acid.
[0017] Erythritol purified from a supernatant by HPLC or TLC can be
further purified by extraction and then dried under reduced
pressure. The further purified product and an erythritol standard
are acetylated according to the method of Shindou et al. (Shindou
et al. 1989. J. Agric. Food Chem. 37:1474-1476.). Erythritol
standards are commercially available, e.g., from Merck, Germany.
The resulting sample can be assayed by GC-MS to determine if the
re-purified product was identical to that of the standard
sample.
[0018] Large Scale Production of Erythritol
[0019] Following the specific examples provided below, a skilled
artisan can optimize erythritol yield of a mutant Moniliella strain
by identifying preferred pH, temperature, and carbon source for
growth and fermentation. Similar analysis can be used to optimize
aeration, stirring speed, culture volume, and culture time.
[0020] To produce erythritol on a larger scale, 0.2 ml of
Moniliella cells preserved in glycerol are added to 50 ml of broth
in a 500 ml flask, and incubated at 30.degree. C. on a rotary
shaker at 150 rpm for about 24 hours. From this culture, 2 ml are
used to inoculate a second 500 ml flask with 50 ml of broth. The
second culture is incubated at 30.degree. C. on a rotary shaker at
150 rpm for 48 hours. The second culture broth is used to inoculate
2 L of broth in a 5 L fermentor (NBS. Edison, N.J., USA). The
culture conditions are as follows. Aeration: 1 VVM; stirred speed:
500 rpm; temperature: 30.degree. C.; culture period: 5-7 days.
[0021] For these purposes, the broth can consist of 30%, 35%, 40%,
45%, or 50% glucose, together and 1% yeast extract. In addition,
KM72 and KM72F (Shin Etsu, Shin-Etsu Chemical Co., Ltd. 6- 1,
Ohtemachi 2-chome, Chiyoda-ku, Tokyo, Japan) can be used as a
defoamer.
[0022] Purification of Erythritol
[0023] Media from the fermentor is centrifuged to separate the
culture supernatant from pelleted cells. The supernatant is
decolored by passage over active carbon (e.g., powdered carbon as
can be obtained from a local supplier). The decolored supernatant
is desalted and de-proteinated by consecutive passage of over a
cation exchange resin, DIAION, WA30 (Mitsubishi) and an anion
exchange resin, AMBERLITE IR120 NA (Rohm and Haas Company). The
resulting solution is concentrated with the following apparati:
EYELA Rotary Vacuum Evaporator N-N Series; EYELA Waterbath SB-450;
and EYELA, Aspirator A-3 (Tokyo Rikakikai Co. LTD). The
concentrated solution is crystallized at room temperature. Crystals
are optionally washed with or re-crystallized in hydrous alcohol
and water (e.g., at 4.degree. C.) to remove the trace
impurities.
[0024] Verification of Erythritol Purification
[0025] To confirm the chemical identity of the purified product,
the NMR spectra of the purified product is compared to the NMR
spectra of a standard, e.g., erythritol purchased from Merck Co.
(NJ, USA), or another commercial supplier. The samples are
dissolved in 100% D.sub.2O and placed in an NMR spectrometer
(Bruker AM-500, Germany). The following conditions are used for
.sup.1H NMR spectra: 400.135 MHz; pulse length: 4.0 .mu.s;
acquisition time: 1.245 sec; pulse delay: 1 sec; chemical shifts:
D.sub.2O as 0 ppm. The following conditions are used for .sup.3C
NMR spectra: 100.536 MHz; pulse length: 5.0 .mu.s; acquisition
time: 0.623 sec; pulse delay: 2 sec; chemical shifts: 10 mM DSS as
0 ppm.
[0026] A skilled artisan can obtain a fungal mutant of the
invention and utilize it to the fullest extent to produce
erythritol based on the guidance of the following specific example,
which is merely illustrative, and not limitative of the scope of
the invention. All publications cited herein are incorporated in
their entirety by reference.
EXAMPLE
[0027] Moniliella Mutant Isolation
[0028] The erythritol-producing fungi Moniliella PTA-1230 was
mutagenized with NTG by the method described above. The procedure
was repeated such that an improved erythritol producer isolated in
one round is used as the parent strain for the subsequent round.
The N61188-12 mutant strain (ATCC deposit ______) was isolated
after six rounds of mutagenesis.
[0029] The N61188-12 mutant strain and the parental PTA-1230 were
cultured in broth containing 35% glucose and 1% yeast extract on
rotary shaker at 150 rpm for 6 days at the temperature of
25.degree. C., 30.degree. C., 34.degree. C., and 37.degree. C. At
each of these temperatures, the glucose-to-erythritol conversion
rates were respectively: 43.9%, 61.4%, 17.8%, and 2.2%, for the
N61188-12 mutant strain; and 18.9%, 30.5%, 17.9%, and 7.7% for the
parental PTA-1230. At 25.degree. C. and 30.degree. C., the
erythritol yields of the N61188-12 strain were at least twice as
great as that of the PTA-1230. The 61.4% yield observed for the
N61188-12 strain was unexpected, as it is remarkably close to the
theoretical upper limit for complete conversion of glucose to
erythritol--68%.
[0030] For the purposes of verification, pure erythritol was
obtained from a fermentor culture of the N61188-12 strain using the
above-described methods. The pure erythritol from N61188-12 was
analyzed by nuclear magnetic resonance as described above. Its
spectra were identical to the spectra of an erythritol standard
indicating that the product recovered, purified, and crystallized
was, indeed, erythritol.
[0031] Optimization of Erythritol Production Conditions.
[0032] The erythritol yields were determined in parallel for the
parental PTA-1230 and the N61188-12 strain under conditions of
varying pH, temperature (see above), and carbon source.
[0033] pH. The parental PTA-1230 and the N61188-12 strains were
cultured in 35% glucose and 1% yeast extract broth adjusted to
various pH's at 30.degree. C. on a rotary shaker at 150 rpm for 6
days. For the pH's 3.0, 4.0, 5.0, 6.0, and 7.0, the erythritol
yield of the PTA-1230 was 31.2%, 39.3%, 38.4%, 34.4%, and 34.2%
respectively, whereas the erythritol yield of the N61188-12 strain
was 56.6%, 59.4%, 58.5%, 60.3%, and 57.3%, respectively.
[0034] Glucose concentration. Culture broths containing 20%, 30%,
35%, 40%, and 50% glucose together with 1% yeast extract were
prepared. Both PTA-1230 and N61188-12 strains were cultured in the
above broths at 30.degree. C. on a rotary shaker at 150 rpm for 6
days. At each of these glucose concentrations, the erythritol yield
of the PTA-1230 strain was 40.6%, 37.1%, 34.5%, 29.4%, and 19.2%,
respectively, whereas the erythritol yield of the N61188-12 strain
was 56.3%, 57.5%, 62.8%, 55.6%, and 35.8%, respectively (Table 10).
The optimal yield of the PTA-1230 strain was with the 20% glucose
solution, the yield decreasing with the increasing glucose
concentration. The optimal yield of the N61188- 12 strain was with
the 35% glucose broth. The yields obtained from other glucose
concentrations, such as 20%, 30%, and 40% glucose solution, were
similar to each other, while that obtained from 50% glucose
solution was reduced to 35.8%. At high glucose concentrations,
e.g., 40% and 50%, the erythritol yield of the N61188-12 strain was
nearly twice that of the PTA-1230 strain. These results indicate
the unexpectedly improved erythritol production capacity of the
N61188-12 strain in comparison to the wild type PTA-1230
strain.
[0035] Carbon source. The culture broths containing 35% of either
glucose, maltodextrin, maltose, sucrose, fructose, or lactose as
carbon source, and 1% yeast extract as nitrogen source were
prepared. Both PTA-1230 and N61188-12 strains were cultured in
above broths at 30.degree. C. on a rotary shaker at 150 rpm for 6
days. Respectively for glucose, maltodextrin, maltose, sucrose,
fructose, or lactose, strain PTA-1 230 produced 120.8, 44.5, 0,
154.0, 111.0, and 0 g/L of erythritol, whereas the N61188-12 strain
produced 220.0, 15.1, 22.8, 239.4, 211.4, and 0 g/L. These results
indicated that sucrose has best conversion capacity for both fungal
strains, and the next being glucose, and then fructose. Notably,
strain PTA-1230 cannot utilize maltose and lactose for erythritol
production, whereas the N61188-12 strain can utilize maltose, but
not lactose for erythritol production. For the PTA-1230 strain, the
erythritol yield using sucrose as the carbon source was 27.5%
higher than that using glucose, whereas the yield was only 9%
higher under the same conditions for the N61188-12 strain.
[0036] Byproduct accumulation. The concentrations of the metabolic
byproducts--glycerol, pentitol, and alcohol--were monitored in the
aforementioned carbon sources. For example, when glucose was used
as the carbon source, the concentration of glycerol and pentitol in
the PTA-1230 strain culture broth was 36.4 and 17.2 g/L,
respectively, whereas there was no glycerol present, and the
content of pentitol was only 3.8 g/L in the N61188-12 strain
culture broth. Results for additional carbon sources are
illustrated in Table 1.
[0037] No alcohol was producing during the fermentation of the
PTA-1230 strain with glucose as the carbon source. However, in
other carbon sources, both the PTA-1230 and N61188-12 strains
produced alcohol for the initial five days after inoculation.
However, the alcohol was exhausted on the 6.sub.th day. The only
exception was some residual alcohol (0.7 g/L) on the 6.sub.th day
when fructose was used for the N61188-12 culture. In sum, these
results indicate that the use of sucrose for culturing the
N61188-12 strain results in a high conversion capacity to
erythritol without the accumulation of byproducts.
1TABLE 1 Production of erythritol and byproducts of PTA-1230 and
N61188-12 strains Byproducts from Byproducts from PTA-1230 strain
(g/L) N61188-12 strain (g/L) Carbon Source Erythritol Glycerol
Pentitol Alcohol Erythritol Glycerol Pentitol Alcohol glucose 120.8
36.4 17.2 0 220 0 3.8 0 maltodextrin 45.5 0 0 0 15.1 0 0 0 maltose
0 0 0 0 22.8 0 0 0 sucrose 154 23.4 0 0 239.4 0 0 0 fructose 111
26.3 7.6 0 211.4 13.6 4.2 0.7
[0038] Each carbon source was present at 35%; nitrogen source 1%
yeast extract; and incubation at 150 rpm, 6 days.
[0039] Gross properties of mutant strain N61188-12. The parental
PTA-1230 strain and the mutant N61188-12 strain were grown under
various conditions and compared. Their cell morphologies were
substantially the same. However, on plates, the mutant strain grew
to only a quarter the size of the PTA-1230 strain. Notably, the two
strains have different physiological properties. These differences
are reflected in their abilities to ferment and assimilate
different sugars. The mutant N61188-12 strain can ferment galactose
(Table 2), whereas the PTA-1230 strain cannot. In addition, the
N61188-12 strain is unable to assimilate erythritol and mannitol
(Table 3) in contrast to the PTA-1230 strain.
2TABLE 2 Fermentation of various carbon sources PTA-1230 N61188-12
Carbon source strain strain glucose + + galactose - + maltose + +
sucrose + + lactose - -
[0040]
3TABLE 3 Assimilation study of PTA-1230 and N61188-12 strains on
various carbon sources PTA-1230 PTA-1230 Carbon source strain
N61188-12 strain arbon source strain N61188-12 strain glucose + +
ribitol - - galactose - - xylitol - - sorbose - - arabinitol - -
glucosamine - - glucitol - - ribose - - mannitol + - xylose - -
galactitol - - L-arabinose - - myo-inositol - - D-arabinose - -
glucono-1,5-lactone + - rhamose - - 2-keto-gluconate - - sucrose +
+ gluconate - - maltose + + glucuronate - - trehalose - -
galacturona - - methyl-D-glucoside - - lactate - - cellobiose + +
succinate + + salicin - - citrate - - arbutin + + methanol + +
melibiose - - ethanol - - lactose - - propane - - raffinose - -
butane - - melezitose - - quinate - - inulin - - saccarate - -
glycerol + + galactonate - - erythritol + W* W* refers to weak
growth and meager assimilation of the carbon source.
[0041] Cell density. Under various conditions such as temperature,
pH, carbon source, and glucose concentration, the turbidity
(A.sub.660) of the culture broth for the N61188-12 strain was less
than that of the PTA-1230 strain. Overall (except for use of
maltose and lactose as the carbon source), the turbidity of the
culture broth for the N61188-12 strain was between 31% and 77% of
that for the PTA-1230 strain. In most cases the turbidity of the
N61188-12 strain was less than 50% of the PTA-1230 strain. Thus, it
is inferred that the N61188-12 strain reduced the proportion of
carbon source applied to cell growth, and instead converted a
greater proportion of the carbon source into erythritol.
OTHER EMBODIMENTS
[0042] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
* * * * *