DD267999A1 - PROCESS FOR THE PREPARATION OF 2-OXOGLUTARIC ACID BY YEAST - Google Patents

Abstract

The invention relates to a fermentation process for the preparation of 2-oxoglutaric acid with a stable, efficient yeast mutant of the strain Yarrowia lipialytica H 222-27-11 ZIMET 43856 under aerobic, submerged conditions. The mutant is capable of utilizing different sources of carbon, such as n-paraffins, glucose, and the like. a., 2-Oxoglutersaeure in high concentration and at high speed in the culture medium to accumulate. Under the conditions of an optimal strain-specific thiamine concentration of 0.05-0.15 mg / l of thiamine hydrochloride are at a p H value of preferably 3.0-4.0 and a Geloestsauerstoffkonzentration of 50-90%, especially based on n Paraffins, 125 g / l 2-oxoglutaric acid produced in 90 hours. The application of the invention is in the field of microbial product synthesis.

Description

Field of application of the invention

The invention relates to a process for the preparation of 2-OxoglutarsSure by yeasts with suitable carbon sources untei aerobic, submortional conditions and is thus classified in the field of technical microbiology.

Characteristic the bekannton Standen dot Technik

2-oxofluoric acid is finding increasing interest as a starting material for advanced chemical syntheses. It can be prepared chemically only in complex multi-step process.

It is known that 2-oxoglutaric acid can be accumulated in the culture medium both by bacteria and by yeasts. As a source of carbon dioxide, both glucose and paraffins are used. With the bacterium Arthrobacter paraffineus No. 2411 (ATCC 15691) e.g. a maximum of 60g / l of 2-oxoglutaric acid with a Produktbildjngsgeschwindigkeit of 0.8g / l h and a yield voii 74%, based on the paraffin used

(GB 1,192,905, 1965, Prior. JP 38921, 1964, Kyowa Hakko Kogyo Co., Ltd.).

Lower performance was achieved with the same strain as a result of L-glutamic acid byproduct formation (US 3,450,599, 19fl5, US Pat. Prior. JP 3947169, 1964, Kyowa Hakko Kogyo Co., Ltd.). It is known that thiaminauxotrope yeasts, especially of the genus Candida lipolytica under thiamine deficiency conditions on

Preferably, the base of η-paraffins precipitates 2-osoglutaric acid and, based on glucose, preferentially pyruvic acid (Ermakova, J. T., et al., Frikladnaja Biochimija i Mikrobiologie 22 (1986) 3,341-347).

The with yeasts of the genus Candida, ζ. R. Candida lipolytica, acid concentrations De'ragnn reached a maximum of 48 g / l at one Product turnover rate of O.eg / l.hund with a yield of 60% (GB 1,187,006,1968, Prior. JP 4104,1968, Ajlnlmoto Co., Inc.). With the introduction of the diploid strain of Candida lipolytica D 1805 (FR 2.207.989.1973, Inst. Francais du Petrole des Carburonts et Lubricants, Paris) these performance parameters could be far exceeded, so that a

2-Oxoglutarsiurekonzentration of 185g / l in 240h (productivity 0.9g / l · h) with a yield of 80% ^ based on the Paratfinmenge used, could be achieved.

The disadvantages of the listed methods are the low product formation rates and low final concentrations as well as the Formation of by-products, e.g. L-glutamic acid (GB 1,187,006 and US 3,450,593). Of diploid efficiency> st

It is known that reversion can occur in lower power haploid forms.

Object of the invention

The aim of the invention is to produce with a yeast from different carbon sources by means of a fermentation process 2-Oxoglutarslure in high final concentrations and with high product formation rate.

Essence of the invention

The object of the invention is to select a specially prepared yeast mutant and to cultivate it under specific process conditions. According to the invention, the object is achieved by optimally treating the yeast strain Yarrowia iipolytica H 222-27-11 (ZIMET 43856) in known nutrient media at a pH-Wo: i <6.5, preferably from 3.0-4.0 , strain-specific thiamine concentration of 0.05-0.1 5μg / l thlamol hydrochloride, a temperature of <35 ⁰ C, preferably 28-32'C and a dissolved oxygen concentration of 50-90% cultured.

The strain is characterized by the fact that he on the most different assimilable Kohlenstoffq'jellen such. B. η-paraffins, glucose, glucos >> naltigen hydrolyzates, invert sugar, triglycerides, ethanol, glycerol, acetic acid or acetate in the presence of a mineral Nihrsalzlösung and precisely dosed growth factors and vitamin B1 wichst and 2-Oxoglutartä. · Β accumulates. Especially on the basis of η-paraffins are produced large amounts of 2-Oxoglutarsfiure high speed production and high yield, which exceed the previously known. Thus, the rate of production of 2-oxoglutaric acid from n-paraffins averages 1.4 g / l h, i. Production productivity is about 50% higher than the described best performance with a diploid strain, which achieved the yield achieved by about 10%.

Another essential feature of the strain is that it uses different C sources, such as. B. nPa affine and glucose in the mixture can recycle simultaneously or that you use different C sources at different times, such. For the growth phase paraffin and for the product formation phase glucose or vice versa. The strain retains its ability to produce 2-O-oglutarsfiure in high volumes at high speed and high yield, unchanged over a long test period of several years.

-2- 267

The following is the characterization of the new strain: The strain H 222-27-11, ZIMET 43656 represents a methlonfnunabhingfge revertant of methionine,

citric acid homologs mutant H 222-27 .

1. Morphological characteristics Colonies after * J8h on YEP-G (1% yeast extract), 2% peptone, 2% glucose, pH 5.5-6.0): about 2 mm rum, round, white gray, smooth (later wrinkled) with smooth Edge. Cells: distinct dimorphism, yeast cells oval, of different size hyphae forming pseudomyzel

2. Physiological properties Subtrate utilization: glucose +

Fructic f

Acetate +

Ethanol +

n-alkanes +

Citric Acid + L-Sotbose -

Glycerine +

galactose

lactose

Maltota

Seccharoio -

Strength KNOi

Maximum growth temperature: 25-32'C Vitamin requirement: thiamin-needy 3. Genetic properties Kernstalus: haploid

Pairing type: A

Genotype: A, melA, SUPA, SP01 In the following examples, the invention is explained in more detail;

example 1 To medium follow the composition NH ₄ Cl 2%, ΚΗ, ΡΟ, 0, i%. MgSO ₄ 7H ₁ O 0.1%. FeSO ₄ .7H ₂ O 10 mg / l, ZnSO ₄ .7H ₁ O 44 mg / l, dissolved in distilled water.

700mi η-paraffins of the chain C ₁₁ -C ₁ I are added.

The culture medium is sterilized at 120 ° C. for 30 minutes. After cooling, 0.3 g of thiamine hydrochloride (in 10 ml of dost. Water) and separately sterilized by filtration, and 300 ml of a 24-h grown Yarrowia culture

lipolytica H 222-27-11, ZIMET 43856 added as inoculum.

The medium for the inoculum has the following composition: NH ₄ Cl 0.3%, KH] PO ₄ 0.07%, MgSO ₄ .7H, 00.035%, yeast extract 0.1%, η-paraffins C ₁₁ -C ₁₁ 2%, trace elements: CuSO ₄ 5HjC

1 mg / 1 _MnSO4 · 4 H ₁₀₁₅ my / I, ZnCl ₂ 10mg / l, CoSO ₄ · 7H ₂ O 4 mg / l, H] BOJ 40mg / l, dissolved in distilled water.

The fermentation is carried out in a 12 l stirred fermentor. Fermentation conditions: sterile mode of formation, Air volume 50-250l / h, ZO'C. Feeding 1500-2000U / min, pH value of the growth phase 4.0-5.0, pH value of the production phase

3.5, pH regulation with 40% NaOH.

About 10 hours after growth of the culture, the accumulation of 2-OxogluiarsSuro begins, at first slowly, later

with a high product formation rate of on the average 1.4 g / l · h.

After another 90 hours, the fermentation is stopped. The concentration of 2-oxoglutaric acid formed in the Culture medium is 125 g / l (taking into account the dilution of the supplied liquor volume). The yield obtained, booted to par used paraffin, including the paraffin consumption for the Biomass formation, cheats 90%. Example 2 It will proceed, as in the example! 1 has been described. 50 hours after the start of product formation, another 350ml

η-paraffins added in a sterile procedure. The fermentation is continued for 100 hours. After a total of 150 hours of production, the 2-oxoglutaric acid concentration is 195 g / l.

BaIpIeI 3

The procedure is as described in Example 1, except that instead of paraffin for product formation 450 g / l Glucoto be used. The thiamine concentration is increased to 0.3 μg of thiamine hydrochloride.

After a total of 120 hours, the fermentation is stopped. In the culture medium 36g / l 2-Cxoglutarsäure and

16g / l pyruvic acid determined.

Example 4

Method according to Example 1, wherein instead of 700 ml paraffin at the beginning of the fermentation, a mixture of paraffin (300 ml) and glucose (75 g / l) are given as carbon sources. After 90 hours, the fermentation is stopped. The concentration of 2-oxoglutaric acid formed is 60 g / l.

Claims (2)

-1- 267 99'Si claim: Process for the production of 2-Oxoglutaric acid by yeasts, characterized in that a stable Leistungsrr.utante the yeast Yarrowia lipolytica H 222-27-11 ZIMET 43856 at a pH <6.0, preferably boi 3.0-4.0 and a temperature of <37 ° C, preferably 2 & -32 ° C under aerobic submerged conditions in a known manner is cultivated.