JP5383959B2 - Erythritol-producing Moniliella strain - Google Patents

Description

background

  Erythritol is a sugar alcohol found in lichens, hemp leaves, and mushrooms. It is also a flavor component in fermented foods, such as wine, soy sauce, or sake. (Sasaki, T. (1989) Production technology of erythritol. Nippon Nogeikagaku Kaishi 63: 1130-1132). Erythritol is a 4-carbon polyol that is sweet (about 70-80% of sucrose), has some properties such as being tooth-friendly, very low in calories and not carcinogenic, and other polyols Is unlikely to cause gastrointestinal upset (Harald and Brexelles (1993) Starch / Starke 45: 400-405).

  Traditional industrial production of erythritol is made by adding catalysts such as hydrogen and nickel to raw raw sucrose under high temperature and pressure. Other methods include meso-tartaric acid (Kent, PW, and Wood, KR (1964) J. Chem. Soc. 2493-2497) or erythrose (Otey, FH and Sloan, JW (1961) Ind. Eng. Chem. 53: 267), and erythritol is obtained by chemical reduction of raw materials. In addition, erythritol is produced by microorganisms. Such microorganisms include hyperosmotic yeasts such as Pichia, Candita, Toluropsis, Trigonopsis, Monilinera, Ourevacidium, and Trichosporon sp. (Onishi, H. (1967) Hakko Kyokaish 25: 495-506; Hajny et al. (1964) Appl. Microbiol. 12: 240-2461 Hattor, K., and Suzuki, T. (1974) Agric. Biol. Chem 38: 1203-1208; Ishizuka, H., et al. (1989) J. Ferment. Bioeng. 68: 310-314.)

Overview

  The invention features an isolate of Monilirela species with an enhanced ability to convert glucose to erythritol. Such strains produce erythritol from glucose with at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, or even higher conversion rates under optimal conditions. can do.

The strains of the present invention include Moniliella isolates from nature, and mutants of Moniliella strains such as American Type Culture Collection (ATCC) with accession numbers PTP-1227, PTA-1228, PTA-1229, PTA- 1230, and Moniliella isolates given PTA-1232. One special mutant strain is N61188-12, an isolate deposited with the American Type Culture Collection accession number PTA-2862 .

  As used herein, the term “mutant” refers to a strain that differs in genetic makeup by at least one nucleotide (eg, substitution, insertion, or deletion) compared to a reference strain or a parent strain. The mutants of the present invention can be made by a number of methods. One method is to select a strain that has a higher conversion rate to erythritol than the parent strain. The strain can be obtained by random mutagenesis of the parent strain. For example, by chemical mutagens, transposons, or radiation. In addition, the mutants of the invention may comprise a recombinant nucleic acid sequence. For example, the mutant may be a strain harboring additional nucleic acid sequences (eg, sequences transformed, transduced or inserted into the parent strain). This additional nucleic acid sequence can encode a polypeptide that is normally or conditionally expressed. Alternatively, the additional nucleic acid sequence may encode a nucleic acid sequence capable of altering cell physiology, such as, for example, an antisense, ribosome, or other nucleic acid sequence. As another example, an inserted nucleic acid is inserted into an endogenous gene and alters its function (eg, enhances or destroys). For example, the inserted nucleic acid may be a knockout construct that inactivates the endogenous gene, or an artificial enhancer or promoter that increases transcription of the endogenous gene. Mutations can disrupt the parent strain's ability to transport, digest or consume erythritol or mannitol.

  The invention also features a method for producing erythritol. This method involves growing a Moniliella strain of the invention, eg, an enhanced mutant in the culture; and purifying erythritol from the supernatant or from the culture, such as a cell mass.

  The details of one or more aspects 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

  Moniliella can ferment simple sugars to produce erythritol, which is used for flavoring in many cooking methods. Screening and mutagenesis are used to identify improved Moniliella strains that yield erythritol product yields with high efficiency. Such strains are ideal for large-scale erythritol production and can be achieved by the methods described herein as examples.

<Isolation of strains with enhanced erythritol production>
Moniliella isolates can be obtained from natural sources as described in US patent application Ser. No. 09 / 585,926, filed Jun. 2, 2000. For example, Moniliella isolates can be obtained from high sucrose content natural resources including honey, fruit jam, and pollen. Each strain is identified based on its ability to convert glucose to erythritol and its various environmental and physiological tests. As used herein, “conversion rate of glucose to erythritol” is determined by the amount of erythritol produced and the amount of glucose consumed. The resulting percentage is expressed as a percentage. The conversion rate from glucose to erythritol by the strain can be calculated by the following method. The strain was first cultured in a 10 ml broth containing 30% glucose and 1% yeast extract contained in a 50 ml flask for 6 days at 30 ° C. on a rotary shaker at 150 rpm (initial cell concentration 1.105 cells / ml). ). Then, both the erythritol concentration in the medium and the glucose concentration in the medium were measured. The conversion from 1 g glucose to 0.3 g erythritol is referred to as a 30% conversion. Morphological characteristics are measured after growth for 10 days at 20 ° C. in 4% malt extract, 0.5% yeast extract agar. Yast, A Taxonomic Study, Editorial by Kurtzman et al. 4th Ed., Page 765, Elsevier, Amsterdam (1998).

  The mutant of Moniliella strain is Ishizuka, et al. (1989) J. Ferment. Bioeng. 68: 310-314, or a variant thereof (see U.S. Patent No. 5,036,011). One variation of mutagenesis in Moniliella cells using N-methyl-N nitrosoguanidine (NTG) is described below. Moniliella cells are inoculated into a broth containing 30% glucose and 1% yeast extract and cultured overnight at 150 rpm in a 30 ° C. rotary shaker. The culture is diluted 1: 100 in a broth containing 10 ml of 30% glucose and cultured at 150 rpm in a 30 ° C. rotary shaker for one day. The culture broth is centrifuged at 3000 rpm for 15 minutes to form a cell mass, and the supernatant is discarded. The cell mass is washed with 0.1M pH 7.0 sterile phosphate buffer solution (PBS). Centrifuge the supernatant and discard the supernatant again. Resuspend cells in PBS with 150 μg / ml NTG for 10 minutes.

  After NTG treatment, Moniliella cells are grown in glucose lysate for 3 hours. The culture medium is diluted appropriately, inoculated into a medium containing 65% glucose, and cultured at 30 ° C. for 6 hours. Colonies are randomly selected, inoculated into a broth containing 30% glucose, and cultured overnight at 30 ° C. in a rotary shaker at 150 rpm. 30% glucose (10 ml) is inoculated with a 1: 100 dilution of this overnight culture and cultured for 4 days at 30 ° C. in a rotary shaker at 150 rpm. Then, the culture medium is centrifuged at 12000 rpm for 10 minutes. The supernatant is diluted appropriately and the residual glucose is measured using the DNS method (see below). The culture solution with high glucose consumption (in other words, low glucose remaining amount) is further analyzed to determine the amount of erythritol produced. The HPLC method described below can be used to quantify erythritol production. Culture media showing an increase in erythritol production are subjected to further confirmation. For example, individual colonies are obtained for culture, grown again as described above, and analyzed again. Selected colonies may be improved according to these steps by the next step of mutagenesis.

<Measurement of residual glucose>
Collect broth after 4 days of culture and centrifuge at 12000 rpm for 10 minutes. Dilute the supernatant appropriately. Add 1 ml of each diluted solution to 0.5 ml DNS (dinitrosalicylic acid) reagent. DNS reagents (eg, a, 1% 3,5-dinitrosalicylic acid (DNS). B. 0.2% phenol; c. 0.05% NaHSO ₃ or 0.025% Na ₂ S ₂ O ₃ ; d.1% NaOH; e. 0.5 % Potassium tetrahydrogen tartrate) was prepared and used according to the method described in MIller, GL (1958) Anol. Chem. 31: 425-428. This mixture is mixed well and incubated at 100 ° C. for 5 minutes. After cooling at room temperature, 9 ml of water is added and the absorbance at 540 nm (OD540 nm) is measured. The concentration of glucose was measured by using an absorbance at 540 nm and comparing to a reference curve obtained by measuring pure glucose at various concentrations.

<Measurement of erythritol concentration>
The amount of erythritol in the supernatant can be quantified by HPLC and TLC, and for example, the erythritol production ability of the strain can be measured. HPLC analysis was performed with a Hewlett Packard H4033A analyzer using an Ion-300 chromatography column with 0.1N sulfuric acid as the mobile phase, at a flow rate of 0.4 ml / min and a temperature set at 75 ° C. For the TLC analysis, the method of Neissner et al. (Neissner, et al. 1980. Herstellung, aanalyse und DC-ternnung 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 ° C. for 20 minutes before use. The developing solution is ethyl methyl ketone: acetone: water (amount of 100: 10: 10), and the color generating reagent is KMnO ₄ concentrated sulfuric acid solution.

  The erythritol purified from the supernatant by HPLC or TLC is further purified by extraction and then dried under reduced pressure. The further purified product and reference erythritol are acetylated according to the method of Shindou et al. Erythritol is commercially available from, for example, Merk, Germany. The final product can be analyzed by GC-MS to determine if the repurified product is consistent with the reference standard.

<Large-scale production of erythritol>
According to the specific examples provided below, one of ordinary skill in the art can optimize erythritol production by Monellaa mutants by identifying the preferred pH, temperature, and carbon source for growth and fermentation. Similar analysis can be used to optimize aeration, agitation speed, culture volume, and culture time.

  To produce erythritol on a large scale, 0.2 ml of Moniliella cells maintained in glycerol is added to 50 ml broth in a 500 ml flask and incubated for about 24 hours at 150 rpm in a 30 ° C. rotary shaker. From this culture, 2 ml is inoculated into a 50 ml broth in a second 500 ml flask. The second culture is incubated for 48 hours at 30 ° C. in a rotary shaker at 150 rpm. This second broth is inoculated into a 2L broth in a 5L fermenter (NBS. Edison, New Jersey, USA). The culture conditions are as follows. Aeration: 1 VVM; Agitation speed: 500 rpm; Temperature: 30 ° C .; Culture period: 5-7 days.

For these purposes, the broth may contain 1% yeast extract along with 30%, 35%, 40%, 45%, 50% glucose. In addition, KM72 and KM72F (ShinEtsu, Shin-Etsu Chemocal Co., Ltd. 6-1, Ohtemachi 2-Tyoume, Chiyodaku, Tokyo, Japan) can be used as antifoaming agents.

<Purification of erythritol>
The culture medium from the fermenter is stretched to separate the culture supernatant from the cell mass. The supernatant is decolorized by plating on activated carbon (eg, powdered charcoal, as selected from local suppliers). Desalted and deproteinized by continuously passing the decolored supernatant over cation exchange resin, DAISON, WA30 (Mitubishi) and anion exchange resin, AMBERLITE IR120NA (Rohm and Company). The selected solution is concentrated by the following instrument: EYELA Rotary Vacuum Evapolator NN Series; EYELA Waterbath SB-450; and EYELA, Aspirator A-3 (Tokyo Rikakiki Co. LTD). The concentrated solution is crystallized at room temperature. Optionally, the crystals are washed or recrystallized in aqueous alcohol and water (eg, at 4 ° C.) to remove trace impurities.

<Confirmation of erythritol purity>
To confirm the chemical identity of the purified product, the NMR spectrum of the purified product is obtained from a standard product such as Mercury Co. (NJ, USA) or other commercial supplier of erythritol. Compare with NMR spectrum. Samples are dissolved in 100% D ₂ O and placed on an NMR spectrometer (Bruker AM-500, Germany). The following conditions are used for the ¹ HNMR spectrum: 400.135 MHz; pulse length: 4.0 μs; acquisition time: 1.245 seconds; pulse delay: 1 second; chemical shift: D ₂ O was 0 ppm. The following conditions were used for the 13C NMR spectrum: 100.536 MHz; pulse length: 5.0 μs; acquisition time: 0.623 seconds; pulse delay: 2 seconds; chemical shift: 10 mM DSS at 0 ppm. Those skilled in the art can obtain the mutants of the present invention to produce erythritol and use it extensively based on the guidance of the following specific examples, but the following examples are for illustrative purposes only: The scope of the present invention is not limited. All publications cited herein are hereby incorporated by reference in their entirety.

【Example】
<Isolation of Moniliera mutant>
The erythritol-producing bacterium Moniliella PTA-1230 was mutagenized using NGT by the method described above. The process was repeated with the modified erythritol producing bacterium isolated in one step used as the parent strain for the next step. The N61188-12 mutant (ATCC deposit no. PTA-2862 ) was isolated after six stages of mutagenesis.

  N61188-12 mutant strain and the parent strain PTA-1230 in a broth containing 35% glucose and 1% yeast extract at 37 ° C. in a rotary shaker at 25 ° C., 30 ° C., 34 ° C., and 150 rpm. Cultured for days.

At each of these temperatures, the conversion of glucose to erythritol was 43.9%, 61.4%, 17.8%, and 2.2% for the N61188-12 mutant, respectively, and 18,9% for the parent PTA-1230 30.5%, 17.9%, and 7.7%. At 25 ° C. and 30 ° C., the erythritol production of the N61188-12 mutant was at least twice that of PTA-1230. The 61.4% yield observed in the N61188-12 mutant was unexpected, as it stood fairly close to the theoretical upper limit of 68% for the complete conversion of glucose to erythritol. is there.

  For confirmation purposes, pure erythritol was obtained from the fermentation broth of N61188-12 mutant strain using the above method. Pure erythritol obtained from N61188-12 was analyzed by nuclear magnetic resonance as described above. Its spectrum was identical to that of the reference erythritol, indicating that the recovered, purified and crystallized product was genuine erythritol.

<Optimization of erythritol production conditions>
Erythritol production was measured simultaneously in parental strains PTA-1230 and N61188-12 under conditions with varying pH, temperature (see above) and carbon source.

PH:
Parental strains PTA-1230 and N61188-12 were cultured at 30 ° C. for 6 days in a broth containing 35% glucose and 1% yeast extract adjusted to various pHs using a rotary shaker at 150 rpm. The erythritol production of PTA-1230 was 31.2%, 39.3%, 38.4%, 34.4%, and 34.2% at pH of 3.0, 4.0, 5.0, 6.0, and 7.0, respectively. The erythritol production was 56.6%, 59.4%, 58.5%, 60.3%, and 57.3%, respectively.

Glucose concentration:
Culture broths containing 1% yeast extract with 20%, 30%, 35%, 40%, and 50% glucose were prepared. Both PTA-1230 and N61188-12 strains were cultured in the broth at 30 ° C. for 6 days using a rotary shaker at 150 rpm. At each of these glucose concentrations, the erythritol production of PTA-1230 was 40.6%, 37.1%, 34.5%, 29.4%, and 19.2%, respectively, whereas the erythritol production of the N61188-12 strain was 56.6%, respectively. %, 57.5%, 62.8%, 55.6% and 35.8% (Table 10). The optimal production amount in the PTA-1230 strain was when the glucose solution was 20%, and the production amount decreased as the glucose concentration increased.

The optimum production in the N61188-12 strain was at 35% glucose broth. The yields obtained with other glucose concentrations, such as 20%, 30%, and 40% glucose solutions, were similar to each other, but those obtained from 50% glucose solutions were reduced to 35.8%. At high glucose concentrations, eg, 40% and 50%, erythritol production in the N61188-12 strain was almost double that of the PTA-1230 strain. These results indicate that the erythritol production ability of the N61188-12 strain was unexpectedly improved compared to the wild type PTA-1230 strain.

Carbon source:
A culture broth was prepared containing 35% glucose, maltodextrin, maltose, either sucrose, fructose, or lactose, and 1% yeast extract as the nitrogen source. Both PTA-1230 and N61188-12 were cultured in the broth at 30 ° C. for 6 days using a 150 rpm rotary shaker. In each of glucose, maltodextrin, maltose, sucrose, fructose, or lactose, the PTA-1230 strain produced 120.8, 44.5, 0, 154.0, 111.0, and 0 g / L erythritol, while the N61188-12 strain 220.0, 15.1, 22.8, 239.4, 211.4, and 0 g / L. These results indicated that sucrose had the highest efficiency in both strains, followed by glucose and fructose. It should be noted that the PTA-1230 strain cannot use maltose and lactose to produce erythritol, whereas the N61188-12 strain can use maltose to produce erythritol but not lactose. In the PTA-1230 strain, erythritol production was 27.5% higher when sucrose was used as the carbon source, but it was only 9% higher in the PTA-1230 strain under the same conditions. It was.

Byproduct accumulation:
In the carbon source, the concentrations of metabolic byproducts glycerol, pentitol, and alcohol were monitored. For example, when glucose is used as the carbon source, the concentration of glycerol and pentitol in the culture broth of the PTA-1230 strain was 35.4 and 17.2 g / L, respectively, whereas the culture broth of the N61188-12 strain was In it, there was no glycerol and the pentitol content was 3.8 g / L. The results for other carbon sources are listed in Table 1.

No alcohol was produced during fermentation of strain PTA-1230 using glucose as the carbon source. However, in other carbon sources, both PTA-1230 and N61188-12 strains produced alcohol 5 days after planting. However, on the sixth day, alcohol was consumed. The only exception was when fructose was used to culture the N61188-12 strain, and some alcohol remained (0.7 g / L) even on day 6. Collectively, these results indicate that when sucrose is used in the culture of the N61188-12 strain, the ability to convert to erythritol is high without accumulating by-products.

各炭素源は35%で存在し；窒素源は1% 酵母抽出液であり；インキュベーションは150rpmで6日間であった。

Each carbon source was present at 35%; the nitrogen source was 1% yeast extract; incubation was for 6 days at 150 rpm.

Rough characteristics of the mutant N61188-12 strain:
Strain PTA-1230 and mutant N61188-12 were grown and compared under various conditions. The cell morphology was substantially the same. However, the mutant strain on the plate grew to only a quarter of the size of the PTA-1230 strain. Of note, the two strains have different physiological characteristics. These differences reflect the ability of the fungus to ferment and digest different sugars. Mutant N61188-12 can ferment galactose (Table 2), but not PTA-1230. In addition, the N61188-12 strain cannot digest erythritol and mannitol in contrast to the PTA-1230 strain (Table 3).

細胞密度：
温度、pH、炭素源、およびグルコース濃度といったさまざまな条件下において、N61188-12株の培養ブロースの濁度(A660)は、PTA-1230株より少ない。全てにおいて(炭素源としてマルトースおよびラクトースを除く)、N61188-12株の培養ブロースの濁度は、PTA-1230株の31％から77％の間であった。ほとんどの場合、N61188-12株の濁度は、PTA-1230株の50％より少なかった。このように、N61188-12株は、細胞の成長に当てられる炭素源の比率を低下させ、その代わりに、より多くの比率の炭素源をエリスリトールに変換させたと推定される。

Cell density:
Under various conditions such as temperature, pH, carbon source, and glucose concentration, the turbidity (A660) of the culture broth of N61188-12 strain is less than that of PTA-1230 strain. In all (except maltose and lactose as carbon sources), the turbidity of the culture broth of the N61188-12 strain was between 31% and 77% of 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 estimated that the N61188-12 strain reduced the proportion of the carbon source devoted to cell growth, and instead converted a larger proportion of the carbon source to erythritol.

<Other aspects>
A number of aspects of the invention have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other aspects are within the scope of the claims.

Claims (4)

An isolate of the genus Moniliella which has a conversion rate of glucose to erythritol of at least about 55% and is N61188-12 deposited with the American Type Culture Collection under the accession number PTA-2862 .
2. The isolate of claim 1 wherein the conversion rate of glucose to erythritol is at least about 60%.
A method of manufacturing erystole,
Glucose to erythritol conversion is at least about 55%, and the Moniliella spp., N61188-12, deposited with the American Type Culture Collection under the accession number PTA-2862, is grown in culture. by a method comprising converting the glucose to erythritol,
A method comprising purifying erystol from said culture medium.
4. The method of claim 3 , wherein the conversion rate is at least about 60%.