JPS63216486A - Production of mevalonic acid - Google Patents

Abstract

PURPOSE:To produce mevalonic acid in high efficiency, by culturing a specific microbial strain in a medium added with an organic nitrogen as a sole nitrogen source keeping the carbon source concentration in the culture liquid at a definite level. CONSTITUTION:A microbial strain capable of producing mevalonic acid (e.g. Saccharomycopsis fibuligera IFO 0107, etc.) is cultured in a medium composed of 5-15% carbon source, 0.5-3% organic nitrogen source consisting of peptone, yeast extract, meat extract, casein or soybean flour, 0.01-5% each of inorganic salts such as phosphoric acid salt, potassium salt, magnesium salt and calcium salt and 0.02-0.1% nonionic surfactant and cultured at 20-40 deg.C, preferably 25-35 deg.C under shaking or aeration and agitation. When the pH of the medium reaches >=7 or the dissolved oxygen content of the cultured product passes through the inflection point of the content vs. culture time curve, the substrate is added to the cultured product and the culture is continued while maintaining the carbon source concentration in the cultured liquid to 2-15%, preferably 5-10% to produce the objective mevalonic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing mevalonic acid, and particularly to a method for obtaining mevalonic acid in high yield.

Note that mevalonic acid has two structures, an acid type and a lactone type, but it is known that they can be converted into each other, and hereinafter in this specification, unless otherwise specified, both types will be collectively referred to as mevalonic acid.

[Problems to be solved by the prior art and the invention Mevalonic acid is a substance that was first isolated by Wright et al. Known as an intermediate.

In addition, mevalonic acid plays an important role in the metabolism of living organisms, having a growth-promoting effect on various microorganisms and plants, and is therefore used as a growth promoter for microorganisms, plants, etc., and is also used as a pyrethroid pesticide. , ubiquinone (respiratory coenzyme), dolichol (multiple biosynthetic essential factors), and precursors of fat-soluble vitamins.

Conventionally, these studies have used chemically synthesized racemates, and natural mevalonic acid has been difficult to obtain.

The production of natural mevalonic acid by fermentation is carried out by Applied Microbiology (Applied Microbiology).

Although examples using Saccharomycopsis fibrigera NRRL Y-9069 have been described in 965 (1968) and U.S. Patent No. 3,617,447, the yield is low and
At present, the amount is only 1000 μg/d, and industrial production has not yet been achieved.

Therefore, an object of the present invention is to provide a method for producing mevalonic acid with a high yield that is industrially practicable.

[Means for solving problems]

The present invention aims to achieve the above-mentioned purpose by producing mevalonic acid by culturing microorganisms in a medium. This has been achieved by providing a method for producing mevalonic acid characterized by obtaining the following.

Preferred microorganisms used in the present invention include, for example, Candida genus, Guillemondella (
Gutlliers+ondella), Ranseniaspora, Hansenula, Pichta, Saccharomycopsis
psi) Eli-, Saccharomyces
omyces), genus Cheromyces
es), Endomycops
Examples include microorganisms selected from the group consisting of the genus Is), the genus Endomyces, and the genus Sordaria, particularly Candida.
ida) R, Hansenula spp.
Pichia genus, Saccharomycopsis (S
Examples include microorganisms of the genera Saccharomycopsis, Saccharomyces, and Endosycops-1!ndowyces, and specifically, Candida citrea.
da cHrea), Candida fragicola (C
andida flagicola), candida flagicola
Candida lambica, Candida 5ake, Candida 5cout,
Anzenula Jimenne (+Iansenuladl*5
nnae), Hanssenula californica (Hans
enula carifornica),, A Hansenula holsti (Hansenula holsti)
i) , Hansenula henrisii
henriciL), Hansenula fabianii (H
ansenula fabianii), Hansenula fabianii)
Ransenula m1nuta, Hansenula 5ilvi
cola), Hansenula capsulata (Hansa)
nula capsulata), Anseniaspora p.
eijerinckii), Pichia pinus (Pich
ia pinus), and Plchia ++embranaefacien
s) PichLa 5atto
i), Saccharomycopsis fibrigera (Sac
charomycopsis fibuligera)
, Saccharomycopsis cabuslaris (Saccharomycopsis cabuslaris)
allomycopsis capsularis)
Saccharomyces fermentati
myces fermentati), Saccharomyces de
lbrueckii), Saccharomyces 5aitoanus
) - Saccharomyces cerevisiae (Sacchar
omyces cerevtsiae), Saccharomyces vafe
r), Endomycopsis fibrigera (Endon
ycopsis fibuligera), Endomyces fibuligera (Endoa+yces fib
uligera), Guilliemondella selenospora (G.
uillier mondella 5elenospo
ra), Citelomyces matritellasis (Citer
omyces matriteusis), Sordaria fuimicola (Sordaria ftm1cola)
), and the preferable meat amount is Saccharomycopsis fibrigera (Saccharomycopsis fibrigera).
Copsis fibultgera) ~ Strains belonging to these include, for example, IFO0107, IF
O0103, IFO0104, TFO0105, IFO
0106, IFO0109, IFO0111, IFO1
665, IFO1711, IFO1744, IFO17
45, AHU 4113, IAM 4247, OU
T6071, HUT7234, ATCC208
0, ATCC2082, ATCC208B, ATCC9
947, ATCC20145, ATCC24945, A
TCC44872, ATCC46252, ATCC46
253, ATCC46949, ATCC52921, N
RRL Y-1060,NRRL Y-106
4, NRRL Y-2385, NRRLY-'70
61, NRRL Y-7221, NRRL Y
-7324, NRRL Y-7464, NRRL
Y-9069, DSM 70554, IAM
4025, IFO1342, IFOO800,
IFo 1880. IFo 0980, IFO
1477, IFO1254, TFOO975, IFO0
807, IFO0984, IAM 4945, IF
o 185(1, [AM4307, rFo
0672, IAM 4771, IAM 12
236, IAM 12241, IFo 1
002, IFO1003, IFO1626, IFO1
572, IFO1574, IFo1146, I
FO1213, IFO1287, IFO0954, IF
O0941 and rFo8812 can be mentioned.

In addition, IFO, ATCC, NRRL, DSM, AHU, I
AM, OUT, and HUT are the strains preserved by the Fermentation Research Institute, the American Type Culture Collection, the ARS Northern Regional Research Center, the Teutje Sammlung Fono Microorganismen, and the Faculty of Agriculture, Hokkaido University. preserved strains,
Shown are strains preserved at the Institute of Applied Microbiology, the University of Tokyo, strains preserved at the Department of Fermentation Engineering, Faculty of Engineering, Osaka University, and strains preserved at the Department of Fermentation Engineering, Faculty of Engineering, Hiroshima University.

Regarding the medium composition used in the present invention, only organic nitrogen sources such as peptone, yeast extract, meat extract, casein, soybean flour, etc. are used as the nitrogen source, and unless an inorganic nitrogen source is added, ordinary nitrogen sources are used. A specific medium composition includes, for example, a carbon source of 5 to 15% by weight and an organic nitrogen source of 0.5 to 15% by weight.
Examples include a medium containing 3% by weight and water (the balance), and also a medium containing 0.01 to 5% by weight each of inorganic salts such as phosphates, potassium salts, magnesium salts, and calcium salts.

Note that it is preferable to include 0.02 to 0.1% by weight of a nonionic surfactant in the medium, since this increases the amount of mevalonic acid obtained.

In addition, in the method for producing mevalonic acid of the present invention, if the carbon source concentration of the culture solution (culture filtrate) in the culture being cultivated is maintained at 2 to 15% by weight, preferably 5 to 10% by weight, mevalonate This is preferable because the amount of acid obtained can be increased.

A method for maintaining the carbon source concentration within the above range includes a method of adding a substrate containing at least a carbon source to the culture during cultivation.

When adding the above-mentioned substrate, it is preferable to add it to the culture so that the carbon source concentration in the culture solution (culture filtrate) is maintained within the above-mentioned range. However, -
Since it takes a long time to measure the carbon source concentration in (a), it is best to use the pH of the culture or the amount of dissolved oxygen as an indicator for determining the timing of substrate addition.

When pH is used as an indicator, the point at which the pH becomes 7 or higher is used as an indicator, and when the amount of dissolved oxygen is used as an indicator, the point when the amount of dissolved oxygen decreases once and starts to rise again, that is, the curve of dissolved oxygen amount. The point immediately after passing the minimum value (with saturation as the initial value) becomes the index.

The amount of substrate to be added at this time may be determined by measuring the carbon source concentration at the time of the index in advance, and adding an amount corresponding to that.

The substrate to be added may be one containing at least a carbon source, and may be the same or different from the medium composition before culturing, but preferred carbon sources include, for example, glucose, fructose, maltose, malto extract, glycerin,
Examples include acetate. Further, the form of the substrate when added is not limited as long as it is sterile, but it is preferable to use it as an aqueous solution from the viewpoint of ease of sterilization, etc. In that case, the concentration is also not limited, but it should be at a high concentration (for example, 40% or more). ) is preferable.

Preferred specific embodiments of the manufacturing method of the present invention are as follows.

That is, a microorganism is inoculated into a medium containing a carbon source, a meta-nitrogen source, a nonionic surfactant with a cell membrane solubilizing effect, and an inorganic salt in a predetermined amount, and the microorganism is heated at 20-40°C, preferably at 25-40°C.
Incubate with shaking at 35°C or 100-500 rpm,
Preferably 200-400 rpm, 0.2-1.5VV
M, preferably 0.5 to 1, aerated agitation culture with OVVM, and after culturing for a predetermined period of time, the above-mentioned The substrate is fed (added) to the culture, and the substrate is fed as many times as necessary, and the culture is terminated when it is judged that the production amount of mevalonic acid does not increase any further.After the culture is finished, the culture is Remove bacterial cells using known methods such as centrifugation and filtration, and concentrate using reverse osmosis, vacuum distillation, etc., countercurrent distribution method using butanol or ethyl acetate, or silica gel, diamond ion, ion exchange resin, etc. Mevalonic acid can be obtained by a combination of known purification techniques such as column chromatography, molecular distillation, and crystallization.

It should be noted that an antifoaming agent or the like may be added to the culture medium in the method of the present invention, if desired, within the scope of the purpose of the present invention.

In addition, the carbon source concentration in the culture solution is measured by an enzymatic method using glucose oxidase, etc. In this case, the "culture solution" refers to the solution portion of the culture from which Kokutai, etc. are removed by centrifugation, filtration, etc. say.

〔Example〕

Examples of the present invention are shown below, but the present invention is not limited thereto.

In addition, the quantitative determination of mevalonic acid in Examples and Comparative Examples was performed as follows.

[Mevalonic acid determination method]

Take 0.8- of the sample solution in a subic tube and
Adjust the pH to 2 using the solution. NazsOa for this
After adding 2.0 m of ethyl acetate and stirring, remove the upper layer. Add 2.0 m of ethyl acetate to the lower layer and stir, then remove the upper layer and combine with the previous upper layer. Repeat this operation to obtain a total of 6.0 m of ethyl acetate layer, which is evaporated to dryness. This dried product was quantified by gas chromatography using 3,4-dimethoxybenzaldehyde as an internal standard.

The conditions for gas chromatography are as follows.

Column size: diameter 31n length 10100O made of stainless steel) Column liquid phase: 10% Thermon-3000 Column support: chromosorb WAW-0MC38
0-100 mesh column temperature: 180°C Injection temperature: 230°C Carrier gas jNt (40m/min) [Example 1,
Comparative Example 1] Glucose 10% by weight, malt extract 1% by weight
, peptone 0.5% by weight, yeast extract 0.
1% by weight, KHz PO40, 3% by weight, Mg 304
・Prepare media 201 and 200- consisting of 7 Hz 00.05% by weight, (:, aCOs 1% by weight, and the balance water,
One platinum loop of Saccharomycopsis fibrigera IFO 1744 was inoculated into the 200-liter medium and incubated at 28°C for 3 days with shaking.
Culture was carried out with aeration at 8° C., rotation speed 30 Qrpm, and aeration rate 2 (H!/min).

The amount of mevalonic acid obtained after culturing for 6 days was 11
It was 60 μg/−.

In addition, 0.3% by weight of ammonium chloride was added to the above medium.
When cultured with the addition of mevalonic acid (Comparative Example 1), the amount of mevalonic acid was 910 gg/d after 6 days of culture.

[Example 2] The same culture medium as in Example 1 was grown at 28°C and at a rotation speed of 309 rpm.
After 12017 minutes of aeration, the culture was carried out with aeration while measuring the glucose concentration, pH, and amount of dissolved oxygen.

After measuring the amount of glucose in the culture solution on the third day of culture and confirming that the glucose concentration was 5% or less, at this point, 2.0 kg of a 50% by weight aqueous solution of glucose was added and the culture was continued. 1! continued. Furthermore, on the 6th day of culture and the 9th day of culture, a 50% aqueous solution of glucose was added at 2.0%.
Kir was fed to the cells, and the culture was continued for a total of 12 days, and then the culture was completed. After the cultivation was completed, mevalonic acid obtained from the culture was measured using the quantitative method described above, and the amount of mevalonic acid was 201Q.
It was 11g/-.

Figure 1 shows the culture time and dissolved oxygen m in this example.
(* Sum - 100), pHb glucose amount (wt%
), and the amount of mevalonic acid (wt%). From this graph, it can be seen that immediately after the glucose concentration in the culture solution became 5% or less on the third day of culture, the pH exceeded 7, and dissolved It can be seen that the amount of oxygen has passed through the minimum value.

[Example 3] Culture was carried out in the same manner as in Example 2 except that yeast extract was replaced with meat extract.

The amount of mevalonic acid obtained as a result was 2070 μg/d.

[Example 4] Culture was carried out in the same manner as in Example 2 except that peptone was replaced with casein. The amount of mevalonic acid obtained as a result was 197
It was 0 μg/d.

[Example 5] Culture was carried out in the same manner as in Example 2 except that Saccharomycopsis fibrigera IFO1744 was replaced with Saccharomycopsis fibrigera IFO0107. The amount of mevalonic acid obtained as a result was 8590 μg/d.

[Example 6] The culture 22I2 obtained in Example 2 was rotated at a rotation speed of 500 rpm.
The mixture was centrifuged to obtain filtrate 141. This was concentrated to 41 using reverse immersion i31*, and then adjusted to pH 2 using 50% by weight phosphoric acid water.
41 and extracted three times with ethyl acetate, and the ethyl acetate layer 1
I got 21. Add this to 51 liters of 0.02N sodium hydroxide solution.
After adjusting the pH to 2 with 50% by weight phosphoric acid water, it was passed through a Diaion 1 (P-20 column (11)).The effluent was extracted three times with ethyl acetate in 41, Layer 121 was obtained. This was dehydrated with anhydrous sodium sulfate and then evaporated to dryness. This dried product was dissolved in a small amount of acetone/benzene (1/7) and separated by silica gel column chromatography (150 g). The fraction containing mevalonic acid was dried to obtain 14.4 g of a colorless oily substance.

Specific optical rotation (α) I’--21,6° (C-2,31, ethanol).

〔Effect of the invention〕

According to the method for producing mevalonic acid of the present invention, mevalonic acid can be obtained in an industrially high yield.

[Brief explanation of the drawing]

Figure 1 shows the relationship between the culture time and the amount of dissolved oxygen (taiO-100 and 4), pH, amount of glucose (wt%), and amount of mevalonic acid (wt%) in Example 1 of the present invention. In the figure, 1 indicates the amount of mevalonic acid, 2 indicates the amount of glucose, 3 indicates the pH, and 4 indicates the amount of dissolved oxygen. Cultivation date fll (Sunday) Procedural amendment April 6, 1988 Manufacturing method of mevalonic acid 7, ? ! Correct content (1) r on page 3, line 11 (JAC3, 78, 5273
, 1956) J. (Journal of the American Chemical Society)
the AmericanChe+wical 5oc
iety) Vol. 78, pp. 5273-5275, 1956
2013)”. (2) "Diaion" on page 12, line 15 was corrected to "porous polymer resin." (3) “Diaion HP-” on page 17, lines 12-13
204 was corrected to "Diaion HP-20 (registered trademark)". (4) Correct “Silica gel column chromatography (150g) J” on page 17, lines 17 to 18 to “Silica gel column chromatography (Wakogel C-200 (registered trademark), 1500g) J. +51 No. 181 (line 10 to "Amount of mevalonic acid (wt%)" in line 11 is changed to "Amount of mevalonic acid (μg/sl) J and Masaaki."

Claims (7)

[Claims] (1) When producing mevalonic acid by culturing microorganisms in a medium, the culture is performed using a medium to which only an organic nitrogen source is added as a nitrogen source, and then mevalonic acid is obtained from the culture. A method for producing mevalonic acid. (2) As an organic nitrogen source, one or more substances selected from the group consisting of peptone, yeast extract, meat extract, and casein soy flour are used. The method for producing mevalonic acid described in (1). (3) The culture is carried out by adding a substrate containing at least a carbon source to the culture.
1) The method for producing mevalonic acid according to any one of items 1). (4) Carbon source concentration in the culture solution (culture filtrate) is 2 to 1
The method for producing mevalonic acid according to claim (3), characterized in that the substrate is added so that the concentration is maintained at 5% by weight. (5) The method for producing mevalonic acid according to claim (3), characterized in that the substrate is added when the pH of the culture reaches 7 or higher. (6) Mevalonic acid according to claim (3), characterized in that the addition of the substrate is carried out at the time when the curve indicating the amount of dissolved oxygen in the culture (versus the number of days of culture) has passed an inflection point. manufacturing method. (7) Candida as a microorganism
Genus, Guilliermondella
a) Genus Hanseniaspora
a) Genus Hansenula, Genus Pichia, Genus Saccharomycopsis (Sacc)
genus haromycopsis, Saccharomyces (Sa
genus ccharomyces, genus Citellomyces
eromyces), Endomycopsis (Endo mycopsis)
genus mycopsis, Endomyces
The method for producing mevalonic acid according to claim 1, characterized in that a microorganism selected from the group consisting of the genus Ces and Sordaria is used.