WO1999020783A1

WO1999020783A1 - Production of lysine using salt tolerant, methanol utilizing bacillus

Info

Publication number: WO1999020783A1
Application number: PCT/US1998/021838
Authority: WO
Inventors: Frederick J. Schendel; Richard S. Hanson; Richard Dillingham
Original assignee: The Regents Of The University Of Minnesota
Priority date: 1997-10-17
Filing date: 1998-10-15
Publication date: 1999-04-29
Also published as: CA2307019A1; KR20010031152A; AU1091999A

Abstract

A method of producing lysine by culturing a biologically pure salt tolerant Bacillus methanolicus which exhibits sustained growth at 50 °C using methanol as a carbon and energy source is provided.

Description

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. 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; Hanson, 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 115:205-210 (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. Arfman 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 by Snedecor and Cooney; however, Snedecor and Cooney, were unable to isolate a pure culture capable of growth on methanol. It is extremely 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 methanol at temperatures above 50°C has occurred. Accordingly, there is a need for a method of producing lysine using a salt tolerant Bacillus which exhibits sustained growth on methanol at a temperature of at 50°C. There is also a need for an inexpensive method of producing lysine on an industrial scale.

Summary of the Invention

The invention provides using microorganisms in a method for producing lysine. The method involves culturing salt tolerant, methanol utilizing Bacillus methanolicus in media with methanol as a carbon source and recovering lysine from the nutrient media. In one embodiment, salt tolerant, methanol utilizing Bacillus methanolicus culture is in medium including methionine until lysine is produced at a concentration of at least about 3 g/L, preferably more than about 30 g/L. In another embodiment, the lysine producing methanol utilizing Bacillus methanolicus is an auxotrophic mutant. The Bacillus methanolicus can also be an amino acid analog resistant isolate or mutant of a salt tolerant Bacillus methanolicus culture. The method is especially useful to produce lysine on an industrial scale from an inexpensive and readily available substrate such as methanol.

Strains of salt tolerant, methanol utilizing Bacillus methanolicus used in the invention have the following characteristics: (1) gram positive; (2) spore forming with spores present at a subterminal to central position; and (3) growth is obligately aerobic and occurs at temperatures 35-65°C, with optimum growth at about 55°C. According to the invention, the salt tolerant strain Bacillus methanolicus or amino acid resistant isolate or mutant thereof exhibits sustained growth at 50°C in 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 μm.

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. The bar represents 10 μm.

FIGURE 3 shows the amino acid biosynthetic pathways employed by strains of methanol utilizing Bacillus methanolicus.

Detailed Description 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 subterminal 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 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 CO₂ 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.

TABLE I

Characteristics of Some Strains of Methanol Utilizing Bacillus

Spore localization subterminal

Survival after 10 min. at 80°C +

Sporulation at 37°C +

Optimum temperature for growth 45-55°C

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 of 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 utilizing 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 fermentation of substrates can vary among the strains as shown by Arfman 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 form 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 forth in Table II, below.

TABLE II Characteristics of Some Strains of Bacillus methanolicus

Spore localization subterminal

Survival after 10 min. at 80°C +

Sporulation at 37°C +

Optimum temperature for growth 45-55°C

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 methanolicus HEN9 has been deposited with the American Type Culture Collection in Rockville, MD, and given Accession No. . Methanol utilizing

Bacillus methanolicus DFS2 has been deposited with the American Type Culture

Collection in Rockville, MD, and given Accession No. . Additional examples include the amino acid analog resistant mutants of DFS2 and HEN9, DMY8-10 and M5-38, respectively.

The invention can be practiced using any number of strains of salt tolerant Bacillus methanolicus. One of skill in the art can practice the invention using any number of salt tolerant Bacillus methanolicus strains that are ribulose monophosphate pathway utilizing and gram positive; form spores at a subterminal to central position; grow at 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 of Bacillus methanolicus have a highly conserved 16s RNA. Fewer than 10 bases of the 16s RNA typically vary between strains of Bacillus methanolicus. In general, a greater than 1% difference between sequences of 16s RNA indicates that the samples compared are not of the same species. A difference of less than 1% generally indicates that the samples compared are from the same species. Strains of B. methanolicus 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 less than about 0.3%, more preferably less than about 0.2%. The role and interpretation of 16s RNA sequences is described in Stackebrandt 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 B. methanolicus 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 B. methanolicus. 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 auxotrophic mutants of B. methanolicus strains or amino acid analog resistant B. methanolicus 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 1 , 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 6.0-8.0. No growth occurs when the pH is 5.0. Optimal growth of the bacteria also requires methionine, preferably at about 0.01 mM to about 10 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^"1 to about 2.5 hr^"1 at a temperature of about 50°C to 60°C. B. Formation of Auxotrophs

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 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. 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 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 5-methyl-DL-tryptophan (MT), p-fluorophenylalanine, 5-fluoro-DL-tryptophan (FT), S-2-aminoethyl-L-cysteine (AEC), methyllysine, 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 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 μgxml^"1, respectively is 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 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 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, 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 μgxml^"1 or NTG (50μg^χml^"') 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. 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 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 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 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.

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 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 can range from about 0.05% wt/vol. to 5% wt/vol. with an amount of from about 0.3% to about 2% wt/vol. methanol preferred. Methanol concentrations can also be expressed in units of molarity. In molar units, methanol concentration is preferably about 20 mM to about 800 mM, preferably about 100 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)₂SO₄ 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 10 mM, preferably from about 0.05 mM to about 2 mM. Methionine is advantageously 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, methionine 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. 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 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 one 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: K₂HPO₄, 3.8 g; NaH₂PO₄ H₂O, 2.8 g; (NH₄)₂SO₄, 3.6 g; MgSO₄-7H₂O, 0.5 g; FeSO₄-7H₂O, 2 mg; CuSO₄-5H₂O, 40 μg; H₃BO₃; 30 μg; MnSO₄-4H₂O, 200 μg; ZnSO₄-7H₂O, 200 μg; Na₂MoO₄, 40 μg; CaCl₂.2H₂O, 5.3 μg; CoCl₂.6H₂O, 40 μg. 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 μg/1; p-aminobenzoic acid at 20 μg/1; lipoic acid, folic acid, and B,₂ at 10 μg/1. Yeast extract medium (MY) was MS medium supplemented with yeast extract O.5 g/1. All media (MV and MY) contained 0.4% (vol/vol) methanol unless otherwise stated.

Isolation of Strains. Two wild-type, salt tolerant strains of thermo tolerant 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 mL 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/yg/l), thiamine (100 g/l), riboflavin (100 g/l), pyridoxine (100 g/l), pantothenate (100μg/l), nicotinic acid (100 g/l),/?-aminobenzoic acid (20 g/l), folic acid (10 g/l), vitamin B12 (lOμg/1) and lipoic acid (10 g/l). 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. 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 ammonium sulfate, or 80 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 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.

Table III Distinguishing characteristics of salt-tolerant, wild-type, methanol utilizing strains of Bacillus, DFS2 and HEN9.

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 Lysine was determined by HPLC using pre-column derivatization with o- phtalaldehyde (OP A) and fluorescence detection of the OPA-amino acid derivative. 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 μL) was then mixed with o-phtalaldehyde (Pierce #26015) (50 μL), then injected onto a 5 μ 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 μmax 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 10-125 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 1- 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 batched with phosphate salts and ammonium sulfate in a medium including in each liter: K₂HPO₄, 4.1 g; NaH₂P0₄ H₂O, 1.5 g; (NH₄)₂SO₄, 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: MgSO₄-7H₂O, 0.25 g; FeCl₂-4H₂O, 7.9 mg; CuCl₂- 2H₂O, 15 μg; CaCl₂.2H₂O, 15 mg; CoCl 6H₂O, 81 μg; MnCl₂-4H₂O, 20 mg; ZnCl₂, 273 μg; Na₂MoO₄, 97 μg; H₃BO₃; 61 μg and 100 mM methanol. The methanol feed typically contained trace metals in each liter of methanol at levels of about: MgCl₂-6H₂O, 3.5 g; FeCl₂-4H₂O, 0.78 g; MnCl₂-4H₂O, 0.5 g; CuCl₂- 2H₂O, 13 mg; CoCl₂.6H₂O, 19 mg; Na₂MoO₄-2H₂0 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 μg/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 DMY8-10 (AEC resistant DFS2) and M5-38 (AEC resistant HEN9) were selected for further study of amino acid production. 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 CuCl₂»2H₂O is at 50 μg/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 μg/1; thiamine^»HCI, 100 μg/1; riboflavin, 100 μg/1 ; pyridoxine^»HCI. 100 μg/1 ; pantothenate, μg/1; nicotinic acid, 100 μg/1; p- aminobenzoate, 20μg/l ; folic acid, 10 μg/1; vitamin B12, 10 μg/1; and lipoic acid, 10 μg/1. These isolates produced amino acids at levels reported in Tables IV and V.

Table IV - - Production of Lysine and Other Amino Acids by Strain DMY8-10 (AEC Resistant DFS2) in Complex Medium with 0.5mM Methionine at pH 6.5 and

Fed With 150mM Methionine

Time Aspartate Glutamate Glutamine Alanine Lysine hr. g/i g/i g/i g/i g/i 0

13 0.04 0.15 1.26

17 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/i g/i g/i g/i

0 0.10 0.03 0.00

13 29.20 9.1 1 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 1 16.00 36.19 1.50 175.92 1.38

34 125.00 39.00 7.88 253.88 1.83

Carbon Carbon

Time Conversion Productivity Conversion Productivity hr. lys/meOH lys/l/hr glu/meOH glu/l/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

Table V - - Production of Lysine and Other Amino Acids by Strain M5-38 (AEC Resistant HEN9) in Complex Medium with Complete Vitamins at pH 6.5, 50 °C and

Fed With 150mM Methionine

Time Aspartate Glutamate Alanine Lysine o-methylHse hr. g i g/i g/i g/i g/i

0.0

12.5 0.06 1.44 0.00 2.87 0.00

19.0 0.12 3.43 0.00 5.47 0.00

23.0 0.10 3.65 0.00 8.59 0.00

28.0 4.51 0.00 11.09 0.00

36.0 1.57 0.00 12.79 0.00

Time CDW Acetate 3C VFA Isobutyrate 4C VFA Isovalerate hr. g/i g/i g/i g/i g i g/i

0.0 0.06

12.5 8.46 0.53 0.09 0.13 0.12

19.0 20.59 0.57 0.07 0.22 0.20

23.0 40.56 0.97 0.1 1 0.33 0.28

28.0 42.43 1.00 0.13 0.35 0.11 0.32

36.0 13.42 1.90 0.18 0.45 0.12 0.38

Carbon Carbon

Time Conversion Productivity Conversion Productivity Methanol Methionine hr. lys/MeOH lys/l/hr glu/MeOH glu l/hr g/i g/i

0.0 0.00 0.00

12.5 0.12 0.23 0.05 0.12 30.75 0.28

19.0 0.09 0.29 0.05 0.18 76.41 0.62

23.0 0.08 0.37 0.03 0.16 139.75 1.02

28.0 0.08 0.40 0.03 0.16 193.83 1.35

36.0 0.07 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 PB1 were analyzed. PB1 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 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 MicroSeq™ 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, 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

All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications 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.

Claims

WHAT IS CLAIMED IS:

1. A method for producing lysine, the method comprising the steps of: culturing a biologically pure culture of a salt tolerant Bacillus methanolicus in nutrient medium comprising methanol as a carbon source at a temperature of about 45┬░C to about 60┬░C until lysine is produced at a concentration of at least about 3 g/L; and recovering lysine from the nutrient media.

2. A method according to claim 1, wherein identifying characteristics of Bacillus methanolicus comprise that a strain of said bacterium: is gram positive; forms spores at a subterminal to central position; grows at 35 ┬░C to 60 ┬░C, with optimum growth at 55 ┬░C; and grows on methanol as a sole carbon and energy source.

3. A method according to claim 1, wherein identifying characteristics of Bacillus methanolicus comprise that a strain of said bacterium: is ribulose monophosphate pathway utilizing and gram positive; forms spores at a subterminal to central position; grows at 35 ┬░C to 60 ┬░C, with optimum growth at 55 ┬░C; grows on methanol; and has G/C content of about 44% to about 52%.

4. A method according to claim 3, wherein the identifying characteristics of Bacillus methanolicus further comprise that a strain of said bacterium is rod shaped.

5. A method according to claim 1, wherein the salt tolerant strain of Bacillus methanolicus comprises strain DFS2.

6. A method according to claim 1 , wherein the temperature is about 50┬░C.

7. A method according to claim 1 , wherein lysine is produced at a concentration of at least about 40 g/L.

8. A method according to claim 1, wherein lysine is produced with a carbon conversion of methanol to lysine of at least about 20%.

9. A method according to claim 8, wherein the carbon conversion is about 30%.

10. A method according to claim 1 , wherein in the step of culturing, the oxygen level is maintained at about 10% to about 45% saturation.

11. A method according to claim 1 , wherein the in the step of culturing, the pH is maintained at about 6.0 to about 7.5.

12. A method according to claim 1, wherein the nutrient medium further comprises methionine or a vitamin mixture.

13. A method for producing lysine, the method comprising the steps of: culturing a biologically pure culture of an amino acid resistant mutant of a salt tolerant Bacillus methanolicus in nutrient medium comprising methanol as a carbon source at a temperature of about 45┬░C to about 60┬░C until lysine is produced at a concentration of at least about 3 g/L; and recovering lysine from the nutrient media.

14. A method according to claim 13, wherein identifying characteristics of Bacillus methanolicus comprise that a strain of said bacterium: is gram positive; forms spores at a subterminal to central position; grows at 35 ┬░C to 60 ┬░C, with optimum growth at 55 ┬░C; and grows on methanol as a sole carbon and energy source.

15. A method according to claim 13, wherein identifying characteristics of Bacillus methanolicus comprise that a strain of said bacterium: is ribulose monophosphate pathway utilizing and gram positive; forms spores at a subterminal to central position; grows at 35 ┬░C to 60 ┬░C, with optimum growth at 55 ┬░C; grows on methanol; and has G/C content of about 44% to about 52%.

16. A method according to claim 15, wherein the identifying characteristics of Bacillus methanolicus further comprise that a strain of said bacterium is rod shaped.

17. A method according to claim 13, wherein the salt tolerant strain of Bacillus methanolicus comprises strain DFS2.

18. A method according to claim 13, wherein the amino acid resistant mutant comprises DMY8-10 or M5-38.

19. A method according to claim 13, wherein the temperature is about 50┬░C.

20. A method according to claim 13, wherein lysine is produced at a concentration of at least about 40 g/L.

21. A method according to claim 13, wherein lysine is produced with a carbon conversion of methanol to lysine of at least about 20%.

22. A method according to claim 21 , wherein the carbon conversion is about 30%.

23. A method according to claim 13, wherein in the step of culturing, the oxygen level is maintained at about 10% to about 45% saturation.

24. A method according to claim 13, wherein the in the step of culturing, the pH is maintained at about 6.0 to about 7.5.

25. A method according to claim 13, wherein the nutrient medium further comprises methionine or a vitamin mixture.