WO2012045235A1 - Méthode pour promouvoir la synthèse d'acide docosahexaénoïque par l'ajout de source de carbone - Google Patents

Méthode pour promouvoir la synthèse d'acide docosahexaénoïque par l'ajout de source de carbone Download PDF

Info

Publication number
WO2012045235A1
WO2012045235A1 PCT/CN2011/070241 CN2011070241W WO2012045235A1 WO 2012045235 A1 WO2012045235 A1 WO 2012045235A1 CN 2011070241 W CN2011070241 W CN 2011070241W WO 2012045235 A1 WO2012045235 A1 WO 2012045235A1
Authority
WO
WIPO (PCT)
Prior art keywords
fermentation
dha
acid
added
acetic acid
Prior art date
Application number
PCT/CN2011/070241
Other languages
English (en)
Chinese (zh)
Inventor
黄和
魏萍
纪晓俊
任路静
肖爱华
仝倩倩
Original Assignee
南京工业大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京工业大学 filed Critical 南京工业大学
Publication of WO2012045235A1 publication Critical patent/WO2012045235A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6434—Docosahexenoic acids [DHA]

Definitions

  • the invention belongs to the field of biotechnology and relates to a method for exogenously added factors to promote microbial synthesis of docosahexaenoic acid. Background technique
  • Docosahexaenoic acid is an important omega-3 long-chain polyunsaturated fatty acid, commonly known as "brain gold", which promotes brain cell growth and development, lowers blood fat, lowers blood sugar, protects vision, and fights cancer. Many important physiological functions, such as improving immunity, have been hailed as a new generation of functional health care factors, which have received great attention from the world.
  • the DHA extracted from traditional deep-sea fish oil is unstable due to the variety, season and geographical location of fish, and the high content of cholesterol and other unsaturated fatty acids leads to limited production of DHA, difficulty in separation and purification, and high cost.
  • DHA a high value-added product in the food and pharmaceutical industries.
  • Microbial fermentation production of DHA can overcome the shortcomings of traditional fish oil extraction, can be used for mass production of DHA, and continuously meet people's needs, has broad application prospects, and has attracted the attention of scholars at home and abroad.
  • the microorganisms capable of synthesizing DHA are mainly low-grade marine fungi, such as Thraustochytrium, Schizochytriu, and Cryptophyta.
  • the key precursor of synthetic fatty acids is acetyl-CoA. How much of its content directly affects polyunsaturated fatty acids
  • the mevalonate pathway and the fatty acid synthesis pathway share the same precursor acetyl-CoA. Therefore, by adding a prerequisite substance and inhibiting the mevalonate pathway, more precursor substance acetyl-CoA flows to the fatty acid pathway, thereby facilitating the biosynthesis of polyunsaturated fatty acids.
  • composition of the medium such as “A Vibrio cholerae and a method for producing DHA oil using the same” (200910033869.5), etc.;
  • the technical problem to be solved by the present invention is to provide a method for exogenously adding a factor to promote microbial synthesis of docosahexaenoic acid, which method for the microbial biosynthesis of a fatty acid process shares the same precursor substance with its competing pathway, thereby ensuring more The precursor material flows to the fatty acid synthesis to increase the utilization of the precursor and the fermentation level of DHA, and reduce the production cost.
  • the idea of the present invention is: In vivo (such as Schizochytrium, Thraustochytrium, and Cryptophyceae), the synthesis of long-chain polyunsaturated fatty acids such as DHA is a carbon chain elongation and consumption.
  • the process of energy requires the key precursor acetyl-CoA, and acetyl-CoA is the starting material for fatty acid synthesis.
  • the continuous supply of acetyl CoA is a necessary precursor for the microbial synthesis of polyunsaturated fatty acids.
  • Exogenously added factors promote microbial synthesis of docosahexaenoic acid, and the microorganisms are introduced into the fermentation medium for fermentation to synthesize docosahexaenoic acid, and exogenous addition is added to the fermentation medium before fermentation synthesis a factor, and/or a precursor substance for synthesizing a fatty acid in a fermentation synthesis; wherein the microorganism is any one of Thraustochytrium, Schizochytrium, and Cryptophyta; the exogenous additive factor It is a combination of any one or several of acetic acid, citric acid, and simvastatin; the precursor of the synthetic fatty acid is acetic acid.
  • the addition amount of the exogenous added factor acetic acid in the pre-fermentation period is 3 to 6 mM.
  • the addition amount of the exogenous additive factor citric acid is 2 to 8 mM.
  • the addition amount of the exogenous additive factor simvastatin is 0.5 to 4 ⁇ .
  • the amount of acetic acid added to the precursor of the synthetic fatty acid is 3 to 9 mM.
  • the exogenous addition factor is in the form of a combination of acetic acid and citric acid, the amount of acetic acid added is 6 mM, and the amount of citric acid added is preferably 2 to 4 mM.
  • the exogenous additive factor is in the form of a combination of acetic acid and simvastatin, the amount of acetic acid added is 6 mM, and the amount of simvastatin added is 1 ⁇ .
  • the present invention improves the metabolic process of a DHA-producing strain by adding a fatty acid synthesis precursor substance and an inhibitor of a key enzyme of a competition pathway.
  • the directional regulation of metabolism to the DHA biosynthesis branch is achieved, thereby increasing the biosynthesis level of DHA and increasing the percentage of DHA in total fatty acids.
  • the benefit of this method is to increase the conversion of the substrate, significantly increase the DHA concentration and strength; and the invention is simple to operate, does not require additional labor and equipment, and can increase the conversion rate of the substrate only by a lower additional input. And the concentration of the target product DHA, thereby reducing production costs.
  • C. cohnni ATCC 30556 ⁇ Schizochytrium and Thraustochytrium seed culture medium: D-glucose 40g/L, yeast extract 2 g/L, sodium glutamate 10 g/L, MgCl 2 3 g/L, CaCl 2 -2H 2 0 1 g/L, KH 2 P0 4 4 g/L, KC1 2 g/L, NaCl 15 g/L, MgS0 4 7H 2 0 5 g/L, FeCl 3 0.1 g/L.
  • D-glucose 40g/L D-glucose 40g/L, yeast extract 2 g/L, sodium glutamate 10 g/L, MgCl 2 3 g/L, CaCl 2 -2H 2 0 1 g/L, KH 2 P0 4 4 g/L, KC1 2 g/L, NaCl 15 g/L, MgS0 4 7H 2 0 5
  • Schizochytrium and Thraustochytrium fermentation medium D-glucose 40g/L, yeast extract 2 g/L, sodium glutamate 10 g/L, MgCl 2 3 g/L, (NH4) 2 S0 4 6 g/L, KH 2 P0 4 4 g/L, KC1 2 g/L, NaCl 15 g/L, MgS0 4 -7H 2 0 5 g/L, FeCl 3 0.1 g/L.
  • D-glucose 40g/L yeast extract 2 g/L, sodium glutamate 10 g/L, MgCl 2 3 g/L, (NH4) 2 S0 4 6 g/L, KH 2 P0 4 4 g/L, KC1 2 g/L, NaCl 15 g/L, MgS0 4 -7H 2 0 5 g/L, FeCl 3 0.1 g/L.
  • Cryptophyta seed and fermentation medium D-glucose 25g/L, yeast extract 4g/L, NaCl 16g/L, MgS0 4 -7H 2 0 10 g/L, KH 2 P0 4 11 g/L, KNO3 5 g /L, (NH 4 ) 2SO4 12 g/L, VH 6 mg/L, VB12 lg/L.
  • the fermentation culture methods used in the following examples are as follows:
  • the seeds were cultured for three generations.
  • the first two generations of seeds were carried out in a 250 mL flask, the volume of the liquid was 50 mL, and the third generation seeds were carried out at 500 mL.
  • the liquid volume was 100 mL, and the inoculation amount was 5% (v/v) per generation.
  • the third-generation seeds were inserted into a triangular flask (500 mL) with a liquid volume of 100 mL of fermentation medium at a rate of 9% (v/v); at 25 ° C, 150 r
  • Example 1 Example 1:
  • DHA accounts for the dry weight of cells containing ⁇ t (%) 14.67 15.52 16.61 12.43
  • DHA accounts for ⁇ t(%) of total fatty acids 36.51 38.61 39.34 35.8
  • DHA accounts for ⁇ t (%) of total fatty acids 32.22 35.12 37.11 28.56
  • DHA production (g/L) 5 5.58 6.89 3.9
  • Acetic acid is a direct source of acetyl-CoA, and the amount of acetyl-CoA is directly related to fatty acid synthesis. It can be seen from Tables 1 and 2 that each parameter increases first and then decreases with increasing acetic acid concentration. Biosynthesis of DHA is facilitated at an acetic acid concentration of 3 mM to 6 mM. Especially at the acetic acid concentration of 6 mM, DHA production, DHA accounted for the highest cell dry weight content and DHA accounted for the highest percentage of total fatty acids.
  • Example 2 Example 2:
  • DHA accounts for the dry weight of cells 14.67 16.65 18.33 15.56 13.47 DHA accounts for total fatty acid content 36.51 39.02 40.34 37.85 34.89
  • DHA accounts for the dry weight of cells. 14.67 16.61 18.33 18.69
  • DHA production (g/L) 9.01 10.27 11.02 11.55
  • a indicates that it is added before fermentation
  • b indicates that the fermentation is added later in the pre-fermentation without adding precursor substances, and acetic acid is added at the later stage, as shown in Table 3, and it is found that when added When the concentration of acetic acid is 3 to 9 mM, it is advantageous for the synthesis of DHA.
  • the concentration of acetic acid before fermentation is 6 mM, or only when the concentration of acetic acid is 6 mM at the late stage of fermentation, the yield of DHA and the percentage of total fatty acids and total dry weight are the highest.
  • DHA accounts for the dry weight of cells 14.67 17.09 19.90 17.89 16.12 14.82 DHA accounts for total fatty acid content 36.51 39.26 40.75 38.35 37.41 33.82
  • DHA accounts for the dry weight of cells 9.77 15.33 17.38 12.70 10.63 7.87 DHA accounts for total fatty acid content 38.33 40.22 42.98 39.87 38.41 35.82
  • DHA accounts for the dry weight of cells ( ⁇ ) 14.67 16.61 19.90 21.43
  • DHA accounts for ⁇ t (%) of total fatty acids 36.51 39.34 40.75 39.87
  • Acetyl-CoA carboxylase is the key rate-limiting enzyme in fatty acid synthesis, and citric acid is an acetyl-CoA carboxylase activator. Inhibitor of mevalonate 5-pyrophosphate de-plugase. This enzyme is an important enzyme in the mevalonate pathway. The addition of an appropriate amount of citric acid not only inhibits the mevalonate pathway, but also allows more acetyl-CoA to flow to fatty acid synthesis, and also increases the activity of acetyl-CoA carboxylase and increases the fermentation cycle.
  • the concentration of citric acid of 2 to 8 mM is favorable for the biosynthesis of DHA.
  • DHA yield and DHA accounted for the highest dry weight of cells.
  • DHA accounted for the highest percentage of total fatty acids.
  • Example 6 Considering the more precursor acetyl-CoA flow to the synthesis of fatty acids, the optimal acetic acid and superior citric acid concentration were added to the medium before fermentation. The glucose concentration in the medium was measured using a biosensor, and the fermentation was stopped when the residual glucose concentration in the medium was Og/L. The results are shown in Table 9. Table 9 Effect of exogenous acetic acid and citric acid on the fermentation of Schizochytrium
  • DHA accounts for the dry weight of cells. 14.67 16.61 17.59 16.33 19.65 16.29
  • DHA yield (g/L) 9.01 10.27 10.89 9.99 12.2 9.56
  • c indicates the addition of acetic acid
  • e indicates the addition of citric acid.
  • Table 9 when 6 mM acetic acid and 2 mM citric acid are added, the DHA content is not added relative to the two. And adding only one of them has been improved. However, DHA accounted for only a percentage of total fatty acids, which was higher than when neither was added. When 6 mM acetic acid and 4 mM citric acid were added, DHA accounted for a decrease in total fatty acid content relative to the addition of only 4 mM citric acid, but It is improved by others.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)

Abstract

L'invention concerne une méthode pour promouvoir la synthèse de l'acide docosahexaénoïque par un micro-organisme marin, qui comprend l'ajout de l'un quelconque ou de toute combinaison d'acide acétique, d'acide citrique et de simvastatine dans un milieu de fermentation initiale, ou la supplémentation en acide acétique dans le procédé de fermentation. Ledit micro-organisme marin est choisi parmi Thraustochytrid sp., Schizochytrium sp. et Crypthecodinium sp. La méthode est simple, sans danger pour l'environnement et peu coûteuse.
PCT/CN2011/070241 2010-10-09 2011-01-13 Méthode pour promouvoir la synthèse d'acide docosahexaénoïque par l'ajout de source de carbone WO2012045235A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201010504635.7 2010-10-09
CN2010105046357A CN101979623B (zh) 2010-10-09 2010-10-09 外源添加因子促进微生物合成二十二碳六烯酸的方法

Publications (1)

Publication Number Publication Date
WO2012045235A1 true WO2012045235A1 (fr) 2012-04-12

Family

ID=43600161

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2011/070241 WO2012045235A1 (fr) 2010-10-09 2011-01-13 Méthode pour promouvoir la synthèse d'acide docosahexaénoïque par l'ajout de source de carbone

Country Status (2)

Country Link
CN (1) CN101979623B (fr)
WO (1) WO2012045235A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105803005A (zh) * 2016-04-29 2016-07-27 中国科学院天津工业生物技术研究所 外源调控因子促进裂殖壶菌合成角鲨烯的方法
CN109504646A (zh) * 2017-09-15 2019-03-22 武汉藻优生物科技有限公司 一种获得高dha含量的裂殖壶藻的方法及沉降罐
CN113817783A (zh) * 2021-09-24 2021-12-21 吉林中粮生化有限公司 提高微生物生产有机酸效率的外源添加物及其应用
EP4198136A3 (fr) * 2021-12-16 2023-08-30 Indian Oil Corporation Limited Procédés et formulations pour améliorer des lipides de grande valeur
CN114774484A (zh) * 2022-05-10 2022-07-22 南京师范大学 提高油脂中多不饱和脂肪酸含量的方法和微生物油脂的制备方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001004338A1 (fr) * 1999-07-14 2001-01-18 The University Of Hull Culture de crypthecodinium cohnii pour la synthese d'acide gras polyinsature
EP1359224A1 (fr) * 2002-05-01 2003-11-05 Ato B.V. Procédé de production d'acides gras polyinsaturés par des microorganismes marins
CN101812484A (zh) * 2009-03-20 2010-08-25 厦门汇盛生物有限公司 高密度培养裂殖壶菌发酵生产dha的方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001004338A1 (fr) * 1999-07-14 2001-01-18 The University Of Hull Culture de crypthecodinium cohnii pour la synthese d'acide gras polyinsature
EP1359224A1 (fr) * 2002-05-01 2003-11-05 Ato B.V. Procédé de production d'acides gras polyinsaturés par des microorganismes marins
CN101812484A (zh) * 2009-03-20 2010-08-25 厦门汇盛生物有限公司 高密度培养裂殖壶菌发酵生产dha的方法

Also Published As

Publication number Publication date
CN101979623A (zh) 2011-02-23
CN101979623B (zh) 2012-07-04

Similar Documents

Publication Publication Date Title
Huang et al. A fermentation strategy for producing docosahexaenoic acid in Aurantiochytrium limacinum SR21 and increasing C22: 6 proportions in total fatty acid
Wu et al. Effect of culture conditions on docosahexaenoic acid production by Schizochytrium sp. S31
US20150104557A1 (en) Method for the cultivation of microorganisms of the genus thraustochytriales by using an optimized low salt medium
CN108004149B (zh) 一种海洋原生生物及利用其发酵生产高附加值脂质产品的方法
Nisha et al. Effect of culture variables on mycelial arachidonic acid production by Mortierella alpina
Peng et al. A temperature-shift strategy for efficient arachidonic acid fermentation by Mortierella alpina in batch culture
WO2012045235A1 (fr) Méthode pour promouvoir la synthèse d'acide docosahexaénoïque par l'ajout de source de carbone
US20130096332A1 (en) Method for increasing the content of docosahexaenoic acid in fat-containing materials or in fats and oils
Dashti et al. Batch culture and repeated-batch culture of Cunninghamella bainieri 2A1 for lipid production as a comparative study
US20170327851A1 (en) Microalgae Aurantiochytrium sp. LA3 (KCTC12685BP) and Method for Preparing Bio-Oil Using the Same
US20210310032A1 (en) Microbial oils with high levels of omega-3 fatty acids
CN102676409A (zh) 一种酿酒酵母及其补料分批发酵生产s-腺苷甲硫氨酸的工艺
CN1358839A (zh) 利用海洋微藻异养培养生产长链多不饱和脂肪酸
US20240052385A1 (en) Method for increasing yield of eicosapentaenoic acid in schizochytrium sp.
CN106636264B (zh) 一种利用盐生红胞藻产藻红蛋白和多不饱和脂肪酸的方法
CN102618592A (zh) 一种利用类波氏真眼点藻生产epa的方法
JP2022002522A (ja) 粗製グリセリンを用いたバイオマス及び油分の高密度生産
CN114621983B (zh) 提高裂殖壶菌的dha产量的方法和微生物油脂的制备方法
CN114703238B (zh) 一种裂殖壶菌产二十二碳六烯酸发酵方法及其应用
CN105331670A (zh) 裂壶藻与寇氏隐甲藻混合发酵的方法
CN117604046A (zh) 一种单细胞油脂生产工艺
Athalye Production of Eicosapentaenoic acid from biodiesel derived crude glycerol using fungal culture
KR20160063888A (ko) 종자박을 이용한 미세조류로부터 도코사헥사엔산 생산 방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11830191

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11830191

Country of ref document: EP

Kind code of ref document: A1