Note: Descriptions are shown in the official language in which they were submitted.
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1
PRODUCTION OF LYSINE USING
SALT TOLERANT, METHANOL UTILIZING BACILLUS
Background of the Invention
This invention relates to production of lysine using salt tolerant, methanol
utilizing Bacillus.
Microorganisms that utilize one-carbon compounds more reduced than
carbon dioxide (methylotrophs) are diverse and ubiquitous. Anthony, The
Biochemistry of Methylotrophs, page 3 (Academic Press, London 1982); Hanson,
Adv. Appl. Microbiol., 26:3 ( 1980). Those methylotrophic bacteria reported to
utilize methane are all gram-negative and nearly all have an obligate
requirement for
one-carbon compounds as energy sources. Anthony, supra; Whittenburg et al. J.
Gen. Microbiol., 61:219-226 ( 1970). Bacteria that grow on methanol and
methylamines but not methane include several facultative as well as obligate
methylotrophs. Anthony, supra; (-ianson, supra. All the obligate methylotrophs
unable to utilize methane are gram-negative aerobic bacteria. Anthony, supra.;
Whittenburg, supra. Of the facultative methylotrophs isolated that utilize
methanol,
methylamine or both, only a few were gram positive and were assigned to the
genera
Bacillus, Corynebacterium, Arthrobacter, or Nocardia. Akiba et al, J. Ferment.
Technol., 48:323-328 ( 1970); Clement et al. Abstracts of the Fifth
International
Symposium Microbiol. Growth on C, Compounds. p. 69 (Free Univ. Press,
Amsterdam 1986); Hazen et al, Arch. Microbiol.. 135:205-210 (1983); Mimura et
al., J. Ferment. Technol., 56:243-252 ( 1978).
Some species of facultative gram positive methylotrophs that utilize
methanol, methylamine or both have now been classified together and named
Bacillus methanolicus. Adman et al., Int. J. System. Bact., 42:438 (1992).
Characteristics of Bacillus methanolicus are identified in Arfman et al.,
cited supra.
The industrial advantages of a thermophilic methanol utilizing fermentation
process at elevated temperatures have been described, Snedecor and Cooney,
Appl.
Microbiol., 27:112-1117 (1974). For example, use of elevated temperatures can
significantly reduce cooling costs. Use of methanol as a carbon and energy
source is
cost efficient because of its wide availability and low cost. A methanol
utilizing,
thermophilic mixed culture that included an endospore-forming species was
selected
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by Snedccor acrd Cooney; however, Snedecor aad Cooney, were unable to isolate
a
pure culture capable of growth on methanol. It is exticmely difficult or
impossible
to isolate appropriate salt tolerant strains from mixed or impure cultures.
-~ Large scale production of lysine is desired for many commercial
applications.
For example, lysine is used in the supplementation of animal feeds low in this
amino
acid. The market for lysine has been estimated as 200,000 tons per year. To
date no
production of amino acids, such as lysine, using an isolated salt tolerant
Bacillus
species capable of rapid growth on rncthsnoI at temperatures above 50°C
has
occurred.
Accordingly, there is a need for a ~rrethod of producing lysine using a salt
tolerant. aciltus which exhibits sustained growth on methanol at a
tcanparatura of at
50°C. There i s also a need for an inexpensive method of producing
lysine on an
industrial scale.
Summa
roof the Inyention
The invention provides using microorganisms in a method far producing
lysine. The method involves culturing salt tolerant, methanol utilizing
Bacillus
t~ in media with methanol as a..arbon source and recovering lysine from
the nutrient media. In one embodiment, salt tolerant, methanol utilizing
methanolieus culture is in mcdiucn including tnethionine untU lysine is
produced at a
concentration of at Least about 3 slL, preferably more than about 30 g/L. In
another
embodiment, the lysine producing methanol utili~ng flacillus methanolicus is
an
auxotrophic mutant. The_f3aeillus~nethanolieus can also be an amino acid
analog
resistant isolate or mutant of a salt tolerant ~acillua methanolicus culture.
The
ZS method is especially useful to produce lysine on an industrial scale from
an
inexpensive a~td readily available substrate such as methanol.
Strains of salt tolerant, methanol utili:dng bacillus methanntj~u used is the
invention have the following characteristics: (1) gram positive; (2) sport
forming
with sports present at a subtermina,l to central position; and (3) growth is
obligately
aerobic and occurs at temperatures 35-65°C, preferably 45-60 °C,
with optimum
growth at about 55°C.
According to the invention, the salt tolerant strain Ha~illus methanol3ee~ or
amino acid resistant isolate or mutant thereof exhibits sustained growth at
50°C in
Atv"ENaED SEE'(
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3
nutrient media comprising methanol as a carbon source and produces lysine at a
concentration of at least about 3 g/l. More preferably the salt tolerant
strain or
amino acid resistant isolate or mutant therefrom produces about 25 to about
150 g/1
lysine, and most preferably about 50 to about 110 g/1 lysine.
In the preferred version, lysine is produced by growth of a salt tolerant
strain
of Bacillus or amino acid resistant isolate or mutant therefrom under fed-
batch or
semi-continuous culture conditions.
Description of the Drawings
FIGURE 1 is a phase contrast photomicrograph of a strain of methanol
utilizing Bacillus methanolicus grown on MV medium at 53°C. The bar
indicates
10 ~tm.
FIGURE 2 is a phase contrast photomicrograph of a strain of methanol
utilizing Bacillus methanolicus grown on SM medium at 53°C and shifted
to 37°C.
I S The bar represents 10 Vim.
FIGURE 3 shows the amino acid biosynthetic pathways employed by strains
of methanol utilizing Bacillus methanolicus.
Detailed Descrietion of the Invention
A. Isolation and Characteristics of Methanol Utilizing Bacillus Strains
Although certain characteristics, such as fermentation substrates can vary
among strains, there are several characteristics that identify a bacterium as
a
methanol utilizing Bacillus. These characteristics include: ( 1 ) the bacteria
are gram
positive; (2) the bacteria form spores at a subterminai to central position;
and
(3) growth is obligately aerobic and occurs at temperatures 35-65°C,
with optimum
growth at about 55°C.
Characteristics of a preferred methanol utilizing Bacillus strain are that it
is a
gram positive, spore-forming rod that can grow at 50°C in an aqueous
nutrient media
that includes methanol as a sole carbon and energy source, and that is salt
tolerant.
As used herein, salt tolerance refers to the ability of the strain of methanol
utilizing
Bacillus to grow at higher salt concentrations than other strains of methanol
utilizing
Bacillus. Salt tolerant strains can be selected using nutrient medium
including one
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4
or more of several salts. For example, a salt tolerant strain of methanol
utilizing
Bacillus can be selected by culture in nutrient medium including greater than
1%,
preferably 2% or greater, sodium chloride; by culture in nutrient medium
including
greater than about 4%, preferably 6% or greater, ammonium sulfate; by culture
in
nutrient medium including greater than about 5%, preferably about 8% or
greater,
ammonium glutamate; or the like.
The strains of methanol utilizing Bacillus are preferably isolated from
environmental sources such as soil, dry soil, fresh water marsh soil, or bog
muck.
As stated above, methanol utilizing Bacillus used in the present invention are
also
characterized by utilization of an oxidative pathway that provides for
conversion of
methanol to COZ as shown in Figure 3. This pathway also provides precursor
compounds that can serve as building blocks for cellular components such as
amino
acids.
The invention can further employ methanol utilizing Bacillus strains
characterized metabolically by amino acid synthetic pathways utilizing a
methanol
metabolite such as formaldehyde and as shown in Figure 3. Briefly, methanol is
converted to formaldehyde by an NAD linked methanol dehydrogenase that is
uniquely present in this bacterium. Pyruvate, a product of the ribulose
monophosphate pathway, can serve as a precursor to the production of alanine,
aspartic acid, lysine, lysine, and arginine in three separate pathways.
The methylotrophic bacteria employed in the present invention include a
strain of methanol utilizing Bacillus, preferably, having the characteristics
as set
forth in Table I, below.
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TABLEI
Characteristics of Some Strains of Methanol Utilizing Bacillus
Spore localization subterminal
5 Survival after 10 min. at 80C +
Sporulation at 37C +
Optimum temperature for growth 45-55C
Carbon and energy sources:
Methanol +
Mannitol +
Glucose +
Nitrogen Source:
Ammonium +
The invention can be practiced using any number of salt tolerant strains of
methanol utilizing Bacillus. One ~f skill in the art can practice the
invention using
any number of salt tolerant methanol utilizing Bacillus strains that are gram
positive;
form spores at a subterminal to central position; grow at 35 °C to 65
°C, with
optimum growth at about 55 °C; and grow on methanol.
Salt tolerant strains of methanol utilizing Bacillus include strains isolated
from natural or environmental sources, such as soil, dry soil, fresh water
marsh soil,
bog muck, or pasteurized bog muck and that have the characteristics described
above. The salt tolerant strains of methanol utilizinc Bacillus isolated from
natural
or environmental sources can include auxotrophic Bacillus. As used herein,
auxotroph refers to an organism requiring specific growth factors in addition
to the
carbon source present in minimal nutrient media. The salt tolerant strains of
methanol utilizing Bacillus can include laboratory generated auxotrophic
mutants of
Bacillus strains or amino acid analog resistant Bacillus strains. Auxotrophic
mutants
and amino acid analog resistant strains can be generated as described
hereinbelow.
Isolation and Characteristics of Bacillus methanolicus Strains
The salt tolerant methanol utilizing Bacillus of the invention include salt
tolerant strains of the species Bacillus methanolicus. Characteristics of
strains of
bacteria classified as B. methanolicus can be found in Arfman et al., Int. J.
Syst.
Bact., 42:439 (1992), which is hereby incorporated by reference. Although
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fermentation of substrates can vary among the strains as shown by Adman et
al.,
there are several characteristics that identify a bacterium as a strain of
B. methanolicus. These characteristics include: ( 1 ) the bacteria are gram
positive;
{2) the bacteria foam spores at a subterminal to central position; (3) growth
is
obligately aerobic and occurs at temperatures 35-60°C, with optimum
growth at
55°C; (4) growth on methanol is exhibited; (5) utilizes a ribulose
monophosphate
pathway to convert methanol to carbon dioxide; and (6) has a G/C content of
about
44% to about 52%. Many strains of B. methanolicus are rod shaped. Typically,
strains of B. methanolicus are motile during part of their life cycle.
The methylotrophic bacteria employed in the present invention include a
strain of Bacillus methanolicus, preferably, having the characteristics as set
for th in
Table II, below.
TABLE II
Characteristics of Some Strains of Bacillus methanolicus
Spore localization subterminal
Survival after 10 min. at 80C +
Sporulation at 37C +
Optimum temperature for growth 45-55C
Carbon and energy sources:
Methanol ++
Mannitol ++
Glucose +
Nitrogen Source:
Ammonium +
Nitrate -
Nitrate reduction -
Nitrate respiration -
Hexulose phosphate synthase +
DNA base ratios (moles% G+C) 44-52
Examples of a bacteria used in the invention include methanol utilizing
Bacillus methanolicus strains HEN9 and DFS2. Methanol utilizing Bacillus
strains
HEN9 and DFS2 isolated in the manner described herein from fresh water marsh
soil
and well-drained deciduous forest soil, respectively. Methanol utilizing
Bacillus
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~'~~~nolicus H~19 has been deposited with the American Type Culture Collection
in Rockville, MD, aad given Accession No. 202) 80. Methanol utilizing c' ,~
rnethanolicus DFS2 bas boon deposited with the American Type Culture
CoIlxtiors
in Rockville,1~, and given Accession No. 202181. Additional examples include
the amino acid analog resistant mutants of DFS2 and HEN9, DMYB-10 aad MS-38,
respectively.
The invention can be practiced using aay number of strains of salt tolerant
ACillLC m-,a~.t~h~nnliM~e_ pne of skill in the art can practice the invention
using arty
number of salt tolerant $~illus mechanolicus strains that arc ribulosc
monophosphate pathway utilizing and gram positive; form spores at a
subterminal to
central position; grow ai 35 °C to 60 °C, with optimum growth at
55 °C; grow on
methanol; and have a G!C content of about 44% to about 52%,
Strains ofHacillus methanolicL have a highly construed 16s RNA. Fewer
than 10 bases of the l6s RNA typically vary between strains ofillus
~~c. In general, a greater than 1% difference between sequences of 16s
RNA indicates that the samples compared are not of the same species. A
differexice
of less then 1 % generally indicates that the samples compared are from the
same
species. Strains of ~, methanoIicus that produce lysine at of at least about 3
g/L
typically show a difference in 16s RNA sequence of less than 0.9%, preferably
lrss
than about 0.3%, more preferably less than about 0.2%. The role and
interpretation
of 16s RNA sequences is described in Stackcbrandt et al. Intl. J. of
Systematic
Bacteriology 44(4):846-849 (1994), the disclosure of which is incorporated
herein
by reference.
Salt tolerant strains of Bmethanalicus include strains isolated from natural
or environmental sources, such as soil, dry soil, fresh water marsh soil, bog
muck, or
pasteurized bog muck and that have the characteristics described above. The
salt
tolerant strains of B;methanolicus isolated from natural or environmental
sources
can include auxotrophic H. meth annli~, As used herein, auxotroph refers to an
organism requiring specific growth factors in addition to the carbon source
present in
minimal nutrient media, The salt tolerant strains of methanol utilizing ~,
methanolicus can include or be used to produce laboratory generated
auxotruphic
mutants of ~, me mo icuc s~~~ or amino acid analog resistant B, methanoli:cus
r~~;~~I~i~~~J ~'~EEf
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strains. Auxotrophic mutants and amino acid analog resistant strains can be
generated as described hereinbelow.
Media for Growth of Methanol Utilizing Bacillus
As described herein "aqueous nutrient media" refers to a water based
composition including minerals and their salts necessary for growth of the
bacterium
used in the present invention. Preferred nutrient media contains an effective
amount
of a phosphate source, a nitrogen source, a sulfate source, calcium, and trace
elements. As described herein "trace elements" refers to elements essential
for
growth in trace concentrations i.e., minute fractions of 1 percent (1000 ppm
or less).
As indicated in Tables I, II, and III, the bacterium used in the present
invention can
utilize a number of carbon and energy sources for growth other than methanol;
including glucose or mannitol; however the preferred carbon and energy source
is
methanol.
A satisfactory media for culturing the bacterium employed in the present
invention is a minimal salts media, such as that described in Example 1 or the
like.
In a preferred embodiment, such as Example l, minimal salts media to grow the
bacterium used in the present invention includes from about 20 to about 500 mM
ammonium sulfate; from about 10 to 125 mM potassium phosphate, from about 0.1-
1.5 mM calcium chloride; and salts of magnesium, and the trace metals: iron,
copper, manganese, zinc, molybdenum, borate and cobalt in concentrations as
stated
in Example 3. The amount of methanol needed for growth can vary. The amount of
methanol in the media can range from about 0.05% wt/vol. to about 5% wt/vol.,
with
amounts of from about 0.2% wt/vol. to about 0.5% wt/vol. preferred. The media
should contain at least 0.05% wt/vol. methanol. Optimal growth of the
bacterium
takes place at 45-55°C within a pH range of about 6.0-8Ø No growth
occurs when
the pH is 5Ø Optimal growth of the bacteria also requires methionine,
preferably at
about 0.01 mM to about i 0 mM. Optionally, the bacteria can require one or
more
vitamins or biotin for growth. Typical vitamins are included in the MV medium
described in Example 1. When grown in minimal salts media with methanol and
methionine the bacterium used in the present invention can grow at a rate from
about
0.2 hr-' to about 2.5 hr'' at a temperature of about 50°C to
60°C.
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B. Formation of Auxotro_phs
As described hereinabove, an auxotrophic, salt tolerant strain of methanol
utilizing Bacillus can be isolated from a natural or environmental source.
Auxotrophic mutants of salt tolerant, methanol utilizing Bacillus can be
formed in
the laboratory. As used herein, amino acid auxotrophic mutant refers to salt
tolerant
strains of methanol utilizing Bacillus mutagenized to require one or more
amino
acids for growth and to produce lysine. Mutant refers to a sudden heritable
change
in the phenotype of a strain, which can be spontaneous or induced by known
10 mutagenic agents, including radiation and various chemicals. Typically, the
mutant
is also salt tolerant.
Auxotrophic mutants of the present invention can be produced using a
variety of mutagenic agents including radiation such as ultra-violet light, x-
rays,
chemical mutagens, site-specific mutagenesis and transposon mediated
mutagenesis.
15 Examples of chemical mutagens are ethyl methane sulfonate (EMS),
diepoxyoctane,
N-methyl-N-nitro-N'-nitrosoguanine (NTG) and nitrous acid.
The present invention is also directed to production of amino acid analog
resistant isolates or mutants of salt tolerant strains of methanol utilizing
Bacillus that
overproduce and excrete various amino acids. As defined herein "amino acid
20 analog" refers to a compound structurally similar to an amino acid but
which does
not react with the biosynthetic enzymes and genetic control elements in the
same
way as the natural amino acid. Examples of such structurally similar analogs
and
their related amino acid are S-methyl-DL-tryptophan (MT), p-
fluorophenylalanine,
5-fluoro-DL-tryptophan (FT), S-2-aminoethyl-L-cysteine (AEC), methyllysine,
25 hydroxylysine, hydroxynorvaline (threonine antagonist), and ethionine.
Typically,
the amino acid resistant isolate or mutant of the salt tolerant strain is also
salt
tolerant.
Amino acid producing mutants of methanol utilizing Bacillus of the present
invention are produced by treating the bacteria with an amount of mutagenic
agent
30 effective to produce mutants that overproduce lysine and, optionally,
additional
amino acids. While the type and amount of mutagenic agent to be used can vary,
use of EMS and NTG in amounts from about 10 and 50 ltgxml-', respectively is
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preferred. After mutagenic treatment, isolates of the treated bacterium are
tested for
growth on nutrient media containing one or more amino acids. One suitable
medium
to select amino acid excreting mutants is minimal salt or minimal vitamin
media of
the type described in Example 1 or the Like. Auxotrophic isolates are
identified by
5 their ability to grow only on minimal media containing one or more specific
amino
acids and, optionally, one or more vitamins.
Auxotrophic mutants of salt tolerant strains of methanol utilizing Bacillus
can be generated readily using UV irradiation. Briefly, a salt tolerant strain
is grown
to mid exponential phase (A600 = 0.5-0.6) in a media containing methionine.
The
10 culture is then exposed to UV irradiation at 254 nm, preferably for a
period of time
less than one minute. Mutagenized cells are left to grow in the dark for 3
hours.
Cells were then selected by growth in the presence of increasing amounts of
s-2-aminoethyl-L-cysteine.
Other methods of mutagenesis are known to those of skill in the art, and
could be readily employed to produce auxotrophic mutants of the invention. For
example, generation of mutants of aspartokinase or diaminopimelate
decarboxylase
could lead to overproduction of lysine. Techniques such as transposon mediated
mutagenesis and site specific mutagenesis can be conducted on salt tolerant
strains
of methanol utilizing Bacillus, as described by Bohanon et al., "Isolation of
auxotrophic mutants of methylophilus methylotrophus by modified marker
exchange", Appl. Environ. Microbiol., 54:271-273 (1988) and Simon et al., "A
broad host range mobilization system for in vitro genetic engineering:
Transposon
mutagenesis in gram negative bacteria", Bio/Technology, 1:784-791 (1983),
which
are hereby incorporated by reference.
Auxotrophic or other mutants of salt tolerant strains of methanol utilizing
Bacillus can also be treated alternatively or additionally with an amino acid
analog
to select for mutants which overproduce specific amino acids. In one preferred
embodiment, amino acid producing mutants are first treated with the chemical
mutagenic agent EMS (10 pgxml-' or NTG (SOpgXml-') or UV irradiation to
produce
amino acid auxotrophic or other mutants. Amino acid auxotrophic or other
mutants
are then treated with increasing amounts of the amino acid analog AEC to
further
select mutants for lysine or amino acid production.
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Optionally, these mutants can be exposed to other lysine analogs such as
hydroxylysine and methyllysine, and mixtures thereof or other amino acid
analogs
such as HNV (a threonine analog). This selection process can involve a single
exposure to an amino acid analog or mixtures of amino acid analogs or multiple
5 selection steps. Preferably, between selection steps, rapidly growing
isolates are
assayed for lysine production. Isolates producing the greatest amount of
lysine can
be further selected with the same or different amino acid analogs. In
addition, the
isolates can optionally be grown in the presence of increasing amounts of
lysine and
then grown in media without lysine and assayed for production of lysine.
Isolates
10 that can rapidly grow in the presence of lysine while still retaining the
capacity to
excrete lysine are preferred.
While not in any way meant to limit the invention, it is believed that
isolates
that can rapidly grow in the presence of the desired amino acid and still
overproduce
the desired amino acid may no longer exhibit feedback inhibition of amino acid
15 biosynthetic enzymes with the end product of the pathway. It is envisioned
that the
present invention can be employed to produce amino acid auxotrophs and/or
amino
acid analog resistant mutants of methanol utilizing Bacillus that are capable
of
producing most, if not all, of the known amino acids.
20 C. Method of Lysine Production
To produce lysine from salt tolerant methanol utilizing Bacillus, the
organism is cultured in an aqueous nutrient medium including methanol as a
carbon
source. The medium also contains a phosphate source, a sulfate source, a
nitrogen
source, calcium and trace elements in amounts such as indicated in Example 3.
As
25 previously described a satisfactory media is a minimal salts media, such as
described
in Example 1 or the like. When cultivated on minimal salts media of the type
described in Example 1, salt tolerant methanol utilizing Bacillus strains can
grow at
cell densities up to about 60 g/1 dry wt.
The amount of methanol needed for production of lysine can vary. Methanol
30 can range from about 0.05% wt/vol. to 5% wt/vol. with an amount of from
about
0.3% to about 2% wtlvol. methanol preferred. Methanol concentrations can also
be
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expressed in units of molarity. In molar units, methanol concentration is
preferably
about 20 mM to about 800 mM, preferably about I00 mM.
Controlling the concentration of oxygen in the media during culturing of
methanol utilizing Bacillus is also advantageous. Preferably, oxygen levels
are
maintained at about 10% to about 45% saturation. Sparging with air or with
pure
oxygen regulates the concentration of oxygen in the media.
Many nitrogen sources can be used in the aqueous nutrient media, such as
ammonium chloride, ammonium sulfate and ammonium nitrate. The preferred
nitrogen sources are ammonia, ammonium chloride, or (NH4)ZS04 required in
amounts of at least 20 mmoles/L.
Maintaining a level of methionine in the media during culturing can increase
the rate and level of cell growth and provide greater production of lysine.
The
concentration of methionine can range of about 0.01 mM to about I O mM,
preferably from about 0.05 mM to about 2 mM. Methionine is advantageously
1 S added coupled with the methanol feed. An auxotrophic strain or auxotrophic
mutant
can require different or additional amino acids.
Employing salt tolerant, methanol utilizing Bacillus, lysine can be produced
in substantial quantities. That is, quantities of lysine from at least 20 g/L
to about
100 g/L, preferably from about 25 g/1 to about 90 g/L, preferably from about
80 g/L
to about 90 g/L. The present invention is believed useful to produce lysine
either
singly or in combination with many of the 19 amino acids, including glutamic
acid,
aspartic acid, and/or alanine. In one embodiment, salt tolerant strains can
produce
from about 30 to about 70 g/1 of lysine. The yield of lysine can also be
expressed as
a fraction of the carbon source that is converted to lysine. For example,
yield of
lysine can be expressed as carbon conversion of methanol to lysine in percent.
The
carbon conversion of methanol is typically at least about 10%, preferably
about at
least 30% to about 50%, or more.
Salt tolerant methanol utilizing Bacillus can produce lysine when grown in
batch culture. However, fed-batch or semi-continuous feed of methanol, trace
elements, and, optionally, rnethionine enhances lysine production. Lysine
production by salt tolerant methanol utilizing Bacillus can be further
enhanced by
using continuous culture methods in which trace elements are fed
automatically.
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The pH is preferably maintained at a pH of about 5.5 to 7.2, more preferably
about
6.0 to 6.8. Production of lysine by salt tolerant strains is maximized when
the
bacterium employed in the present invention is grown to the required cell
densities
by using continuous addition of methanol, methionine, and trace elements to
culture
S media together with controlling pH by addition of ammonia.
In a preferred version, a salt tolerant strain, such as DFS2, is grown in a 20
liter fed batch fermentor in MV media. Methanol is fed continually to maintain
a
dissolved methanol concentration of about 100 mM. The pH of the culture is
maintained at about 6.5-7, and dissolved oxygen at about 10% to about 45% air
saturation. The strain of bacteria is typically grown for 16-48 hours. Lysine
is
produced and excreted into the media.
While not in any way meant to limit the invention, it is believed that
deregulation of certain key enzymes in the biosynthetic pathways shown in
Figure 3,
provides for overproduction of amino acids, such as lysine.
If desired, the lysine produced in the culture can be separated using known
extraction procedures such as ion exchange chromatography. In a preferred
method
the fermentation broth including the methanol utilizing Bacillus strain,
culture media
components and amino acids produced is dried directly to produce a material
containing cells, media components and ore or more over produced essential
amino
acids which are useful as an animal feed or animal feed supplement. The
fermentation broth can be dried by, for example, the method reported in G.L.
Solomons, Materials and Methods in Fermentation. (Academic Press, N.Y. 1964).
The present invention may be better understood with reference to the
following examples. These examples are intended to be representative of
specific
embodiments of the invention, and are not intended as limiting the scope of
the
invention.
Examples
Example 1 -- Isolation and Characterization of
Salt Tolerant Methanol Utilizing Bacillus Strains
Growth and Sporulation Media: Minimal salts medium (MS) contained in
one liter of distilled water: KZHP04, 3.8 g; NaHZP04~Hz0, 2.8 g; (NH4)ZS04,
3.6 g;
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MgS04~7H20, 0.5 g; FeS04~7Hz0, 2 mg; CuSO,.5H20, 40 pg; H3B03; 30 lsg;
MnS04~4Hz0, 200 pg; ZnS04~7H20, 200 pg; Na2Mo04, 40 pg; CaC12.2H20, 5.3 pg;
CoC12.6H20, 40 fig. The pH of this medium was adjusted to 7.0 prior to
autoclaving. The phosphates were reduced by 50% when MS medium was used for
continuous cultures.
The minimal vitamin medium (MV) was MS medium supplemented with
thiamine.HCl, D-calcium pantothenate, riboflavin, d-biotin, nicotinic acid,
and
pyridoxine HC1, each at 100 pg/1; p-aminobenzoic acid at 20 pg/l; lipoic acid,
folic
acid, and B,2 at 10 pg/1. Yeast extract medium (MY) was MS medium
supplemented with yeast extract O.S g/l. All media (MV and MY) contained 0.4%
(vol/vol) methanol unless otherwise stated.
Isolation of Strains. Two wild-type, salt tolerant strains of thermotolerant
methylotrophic spore forming bacilli, designated DFS2 and HEN9, have been
isolated. DFS2 was isolated from well-drained deciduous forest soil and HEN9
was
cultivated from wet marsh soil using the following methods.
One gram of each soil sample was suspended in 5 mL of water; this
suspension was heated at 80°C for 10 minutes prior to decanting into
250 milliliter
Erlenmeyer flasks. The flasks held 20 rnL of Minimal Vitamin (MV) medium
containing 60 grams per liter (6%) of ammonium sulfate, 1% methanol and
vitamin
mixture. The vitamin mixture included d-biotin (100,ug/1), thiamine
(100,ug/1),
riboflavin ( 100,ug/1), pyridoxine ( 1 OO~g/1), pantothenate ( 100~/l),
nicotinic acid
( l OO,ug/1), p-aminobenzoic acid (20,ug/1), folic acid ( l O,ug/1), vitamin B
12 ( l O,ug/1)
and lipoic acid ( 1 O,ug/1). The flasks were closed using rubber stoppers
vented by 20
gauge hypodermic needles and incubated at 50°C and rotated 350 rpm.
When growth was evident, as determined by increased turbidity, 10%
transfers were made into the same medium. Beginning with the 10th of such
sequential transfers, aliquots were subcultured onto Minimal Vitamin Agar (MV
Agar) and MV Agar supplemented with 0.05% Yeast Extract (MY Agar).
After 2 days incubation at 50°C, plates were examined using a
stereoscope to
find nonsporulating colonies. These colonies were subcultured for isolation
onto
separate agar media of the same composition until pure cultures were achieved.
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After purification, clones were subcultured into MV containing 0.05% Yeast
Extract
(MY). After overnight incubation at 50°C and 350 rpm, methanol
utilization was
confirmed using gas chromatography. Salt tolerance of new isolates was
determined
by observing for growth in MY medium containing 20 g/1 sodium chloride, 60 g/1
5 ammonium sulfate, or $0 g/1 ammonium glutamate.
Characterization of Strains. Cells of DFS2 were large, short, curved rods
that occurred single or in short chains. The DFS2 cells demonstrated a quick
vibrating form of motility and formed spores readily when the temperature is
dropped from 50°C to 37°C. In contrast, cells of HEN9 formed
long filaments
10 during lag phase which broke up during exponential growth into longer
curved rods
that occurred in single or short chains. No motility has been observed with
HEN9
and this strain does not efficiently form spores after temperature downshift.
Table III illustrates some distinguishing characteristics of these salt-
tolerant,
wild-type, methanol utilizing strains of Bacillus.
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Table III
Distinguishing characteristics of salt-tolerant, wild-type, methanol utilizing
strains
of Bacillus, DFS2 and HEN9.
DFS2 HEN9 MGA3 NOA2
Substrates:
Methanol + + + +
Ethanol - - -
Dextrose + + + +
Fructose - - - -
Maltose - + + +
Mannitol + + + +
Raffinose + - - -
Ribose - - - -
Sorbitol + + - -
Trehalose + - - -
Salt Tolerance
2% sodium chloride + + - -
6% ammonium sulfate + + - -
8% ammonium glutamate + + - -
Catalase weak weak weak weak
Motility + - + +
Stimulation of Growth
Arginine - + - +
Methionine + + + +
5 Strains MGA3 and NOA2 are strains methanol utilizing Bacillus of the
species Bacillus methanolicus. These strains are not salt tolerant, as
demonstrated
by the data in the table.
Example 2--Lysine Determination
10 Lysine was determined by HPLC using pre-column derivatization with o-
phtalaidehyde (OPA) and fluorescence detection of the OPA-amino acid
derivative.
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Culture supernatants were diluted 50-1000 fold with methanol, and then
centrifuged
for 2-5 minutes at high speed to remove any precipitated protein. The sample
(25 pL) was then mixed with o-phtalaldehyde (Pierce #26015) (50 pL), then
injected
onto a 5 p particle size C-18 reverse phase column (Alltech #28066).
Separation of
the OPA amino acids was carried out using a flow rate of 1 mL/min and a non-
linear
gradient from 10-50% methanol in 50 mM potassium phosphate (pH 6.8).
Example 3 - Lysine Overproduction by Salt Tolerant
Methanol Utilizing Bacillus in a Stirred Reactor
Generally in a stirred reactor cells can be cultured with growth rates from
0.5-
1 Amax using the following concentration ranges of nutrients: ammonium sulfate
from 20-500 mM, sulfate from 0.1-500 mM, methanol from 20-800 mM, phosphate
from 10-I25 mM, magnesium from 0.5-20 mM, manganese from 2-100 mM, iron
from 10-800 mM, calcium from 0.1-1.5 mM, chloride from 0-80 mM, zinc from I-
20 mM, cobalt from 0.1-20 mM, copper from 0.1-20 mM, molybdate from 0.2-40
mM, borate from 0.4-8 mM, and methionine from about 0.01 mM to about 10 mM.
Lysine was over produced in the aerated stirred reactor by culturing the
appropriate salt tolerant strain, such as DFS2, under defined conditions.
Growth of
DFS2, or another salt tolerant strain, in the bioreactor requires control of
methanol
levels, dissolved oxygen levels, pH, and temperature. and addition of
methionine.
All experiments were carried out at 50°C with methanol levels
controlled at 100
mM, dissolved oxygen levels maintained by supplementation of the air sparge
with
pure oxygen, pH controlled by the addition of either anhydrous ammonia or 30%
ammonium hydroxide.
The reactor was hatched with phosphate salts and ammonium sulfate in a
medium including in each liter: KzHP04, 4.1 g; NaH,P04.H,0, 1.5 g; (NH4)zS04,
2.1 g; methionine, 0.5 mM; and antifoam SAG-471, 0.5 mL. After sterilization,
trace metals and methanol were added so the media included the following
ingredients in each liter of media: MgS04~7H20, 0.25 g; FeCl,.4H20, 7.9 mg;
CuClz.
2H20, 15 fig; CaClz.2H20, 15 mg; CoC12.6H20, 81 pg; MnC12~4Hz0, 20 mg; ZnCl2,
273 p.g; NaZMo04, 97 fig; H~BO3; 61 ~g and 100 mM methanol.
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The methanol feed typically contained trace metals in each liter of methanol
at
levels of about: MgClz.6H~0, 3.5 g; FeClz.4H~0, 0.78 g; MnCl2-4H20, 0.5 g;
CuCl2~
2H20, 13 mg; CoC12.6Hz0, 19 mg; Na,Mo04~2Hz0 22 mg; ZnCl2, 22 mg. Trace
metals are fed with the methanol by adding a concentrated solution of the
metals to
the methanol. Methionine was fed from a 150 mM solution at a rate 25% the rate
of
methanol addition.
All reactor runs were carried out in a 20L Biolafitte reactor equipped with
the
controls described above.
Production of Lysine by Strain DFS2
The growth of DFS2 in the 201 Biolafitte reactor was carried out under the
conditions for growth and lysine production described above. Methionine was
fed
as needed to keep the cells growing. After the run was completed, 40 hours,
the
reactor was analyzed with the following results: The cell dry weight (CDW) was
24
g/L, glutamate was produced at 13 g/L, and lysine was produced at 4.3 g/L. In
another run CDW was 26 g/L, glutamate was produced at 21 g/L, and lysine was
produced at 3.3 g/L.
EXAMPLE 4
Lysine Production by Amino Acid Analog Resistant
Isolates of Salt Tolerant Bacillus methanolicus
Batches of the salt tolerant strains of B. methanolicus DFS2 and HEN9 were
mutagenized and grown in the presence of AEC (S-2-aminoethyl-L-cysteine) to
select isolates resistant to this amino acid analog. Selection was performed
as
follows. Wild type salt tolerant strains DFS2 and HEN9 were grown to midlog
phase, MNNG was added to 40 pg/L, and incubation continued. Cells were
collected by centrifugation, resuspended, and grown in the presence of AEC.
Agar
dilution plates contained 0, 50, 100, 150, or 200 mg/L of AEC. AEC resistant
strains selected for further studies grew in the presence of at least 50 mg/L
AEC.
Two such isolates DMYB-10 (AEC resistant DFS2) and MS-38 (AEC resistant
HEN9) were selected for further study of amino acid production.
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For production of amino acids, the isolates were cultured as described in
Example 4 with the following exceptions. The Complex Medium was medium as
described in Example 3, except CuCI,~2H20 is at 50 p.g/L. The Complex Medium
was, in certain experiments. also supplemented with methionine at 0.5 mM. In
other
experiments the Complex Medium was supplemented with a vitamin mixture. The
vitamin mixture included d-biotin. 100 Itg/1; thiamine~HCI, 100 p.g/1;
riboflavin,
100 pg/1; pyridoxine~HCI. 100 ug/1; pantothenate, pg/1; nicotinic acid, 100
p.g/1; p-
aminobenzoate, 20~tg/1: folic acid, 10 p,g/1; vitamin B12, IO ltg/1; and
lipoic acid,
p,g/ 1.
10 These isolates produced amino acids at levels reported in Tables IV and V.
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Table IV - - Production of Lysine and Other Amino Acids by Strain DMYB-10
(AEC Resistant DFS2) in Complex Medium with O.SmM Methionine at pH 6.5 and
Fed With 150mM Methionine
Time Aspartate Glutamate GlutamineAlanine Lysine
hr. g/1 g/1 g/1 g/1 g/1
0
13 0.04 0.15 1.26
I7 0.05 0.71 0.08 5.30
20 3.76 7.45
24 11.71 0.90 8.86
34 26.31 5.53 0.53 10.46
Time OD 500 CDW Acetate Methanol Methionine
hr. g/1 g/1 g/1 g/1
0 0.10 0.03 0.00
13 29.20 9.11 0.49 23.17 0.21
17 71.00 22.15 0.89 71.14 0.61
20 128.00 39.94 1.31 121.02 0.99
24 116.00 36.19 I.50 175.92 1.38
34 125.00 39.00 7.88 253.88 1.83
Carbon Carbon
Time Conversion ProductivityConversionProductivity
hr. lys/meOl-I lys/l/hr glu/meOH glu/1/hr
0 0.00 0.00
13 0.07 0.10 0.01 0.01
17 0.10 0.31 0.01 0.04
20 0.08 0.37 0.03 0.19
24 0.07 0.37 0.07 0.49
34 0.05 0.31 0.11 0.77
5
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Table V - - Production of Lysine and Other Amino Acids by Strain MS-38 (AEC
Resistant HENS} in Complex Medium with Complete Vitamins at pH 6.5, 50
°C and
Fed With 150mM Methionine
TimeAspartateGlutamateAlanine Lysine o-methylHse
hr. g/1 g/1 g/1 g/1 g/l
0.0
12.50.06 1.44 0.00 2.87 0.00
19.00.12 3.43 0.00 5.47 0.00
23.00.10 3.65 0.00 8.59 0.00
28.0 4.5I 0.00 11.09 0.00
36.0 i .57 0.00 12.79 0.00
TimeCDW Acetate 3C VFA Isobutyrate4C VFA Isovalerate
hr. g/I g/I g/1 g/1 g/I g/1
0.0 0.06
12.58.46 0.53 0.09 0.13 0.12
19.020.59 0.57 0.07 0.22 0.20
23.040.56 0.97 0.11 0.33 0.28
28.042.43 I.00 0.13 0.35 0.11 0.32
36.013.42 1.90 0.18 0.45 0.12 0.38
Carbon Carbon
TimeConversionProductivityConversionProductivityMethanol Methionine
hr. lys/MeOH lys/1/hr gIu/MeOHglu/I/hr g/t g/1
0.0 0.00 0.00
12.50.12 0.23 0.05 0.12 30.75 0.28
19.00.09 0.29 0.05 0.18 76.41 0.62
23.00.08 0.37 0.03 0.16 139.75 1.02
28.00.08 0.40 0.03 0.16 193.83 1.35
36.00.07 0.36 0.01 0.04 237.52 1.53
EXAMPLE 5
16s rRNA Sequence Comparisons for Various
Strains of Wild Type Bacillus methanolicus
Samples of several strains of B. methanolicus were isolated and subjected to
analysis of their 16s RNA sequences to determine the degree to which these
strains
were similar to each other, distinct from certain known strains of B.
methanolicus,
and distinct from other species of Bacillus. Strains MGA2, NOA2, HEN9, DFS2,
TSL32, and PB 1 were analyzed. PB 1 is the ATCC type strain, ATCC number
51375.
The analysis was conducted by MIDI Labs (Newark, DE) employing standard
techniques known in the art for comparison of 16s RNA sequences. Briefly, the
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entire 16S rRNA gene was PCR amplified from genomic DNA isolated from a
bacterial colony. The amplification products were purified by ultrafiltration.
Agarose gel electrophoresis provided an indication of quality and quantity of
the
amplification products. The 16S rRNA amplification products were sequenced
using the AmpliTaq FS DNA polymerase and dRhodamine dye terminators. Gel
filtration removed unwanted reagents and the desired products were prepared
for
sequencing by electrophoresis. Data were analyzed using PE Applied Biosystem's
DNA editing and assembly software. Sample sequences were identified using PE
Applied Biosystem's MicroSeqTM microbial identification software and database.
Each of the B. methanolicus strains tested revealed 16S rRNA sequences at
least 3.9% different from the nearest known strain of Bacillus, Bacillus
niacini. The
ATCC strain of B. methanolicus showed no less than 0.81% difference compared
to
each of the other strains of B. methanolicus. Not surprisingly, each of the B.
methanolicus subjected to selection for glutamate production (MGA2, NOA2,
1 S HEN9, DFS2, and TSL32) showed only small differences in the range of 0.03%
to
0.1% when compared pairwise to one another. Each of the percent differences is
shown in Table VI.
Table VI - - Percent Differences in 16S rRNA Sequence From Pairwise
Comparisons of Strains
BacillusPB MGA3 NOA2 HEN9 DFS2 TSL32
Niacini 1
Bacillus 4.10 3.87 3.90 3.93 3.93 3.90
Niacini
PB 1 4.10 0.88 0.84 0.81 0.81 0.84
MGA3 3.87 0.88 0.03 0.06 0.06 0.10
NOA2 3.90 0.84 0.03 0.03 0.03 0.06
HEN9 3.93 0.81 0.06 0.03 0.00 0.03
DFS2 3.93 0.81 0.06 0.03 0.00 0.03
TSL32 3.90 0.84 0.10 0.06 0.03 0.03
All publications and patent applications in this specification are indicative
of
the level of ordinary skill in the art to which this invention pertains. A11
publications
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and patent applications are herein incorporated by reference to the same
extent as if
each individual publication or patent application was specifically and
individually
indicated by reference.
It will be apparent to one of ordinary skill in the art that many changes and
modifications can be made in the invention without departing from the spirit
or
scope of the appended claims.
