WO2012045235A1

WO2012045235A1 - Method of promoting synthesis of docosahexaenoic acid by adding carbon source

Info

Publication number: WO2012045235A1
Application number: PCT/CN2011/070241
Authority: WO
Inventors: 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: 2010-10-09
Filing date: 2011-01-13
Publication date: 2012-04-12
Also published as: CN101979623A; CN101979623B

Abstract

Provided is a method of promoting synthesis of docosahexaenoic acid by a marine microorganism, which includes the addition of any one or any combinations of acetic acid, citric acid and simvastatin in an initial fermentation medium, or the supplement of acetic acid in the fermentation process. The said marine microorganism is selected from Thraustochytrid sp., Schizochytrium sp. and Crypthecodinium sp.. The method is simple, environmentally friendly, and cost effective.

Description

Method for promoting the synthesis of docosahexaenoic acid by adding a carbon source

Technical field

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 (DHA) 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. As the source of fish oil raw materials becomes increasingly scarce, it is difficult to achieve the widespread use of 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.

(Crythecodinium cohnii) and so on.

The key precursor of synthetic fatty acids is acetyl-CoA. How much of its content directly affects polyunsaturated fatty acids

(eg DHA, DPA) content. Due to another competitive pathway, 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.

At present, regarding the research on DHA for microbial fermentation, in summary, the relevant patents that have been published in China are mainly concentrated in the following three aspects:

1. Mutagenesis screening methods for DHA-producing strains, such as the Industrial Application of Marine Fungi Schizochytrium OUC88 (200410075426.X), China Ocean University, and a docosahexaenoic acid production at Nanjing University of Technology. Strain and its mutagen screening method and its application (200910033493.8), etc.;

2. Regarding the composition of the medium, such as “A Vibrio cholerae and a method for producing DHA oil using the same” (200910033869.5), etc.;

3, on the extraction and refining of oils and fats, such as Nanjing University of Technology, "a process for extracting and refining DHA-rich fatty acids from Cryptophyta" (200710025079.3), Inner Mongolia Jindawei Pharmaceutical Co., Ltd., etc. Algae fermentation broth Method for extracting DHA unsaturated fatty acids"(200910159368.1);

However, although the above methods have improved the synthesis ability of DHA to some extent, they have not increased the potential of microbial synthesis of DHA from the source, especially by improving the metabolic process to promote microbial synthesis of DHA. There have been reports of promoting the synthesis of DHA by microorganisms by simply adding exogenous substances. Summary of the invention

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.

In order to solve the above technical problems, 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. Adding exogenous added factors such as prodrugs of synthetic fatty acids and inhibitors of key enzymes of competition pathways to the fermentation medium before fermentation culture; or, in order to ensure sufficient precursor material flow to fatty acid synthesis in the late stage of fermentation, to culture A prerequisite for the addition of synthetic fatty acids to the base.

The specific technical solutions adopted are as follows:

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.

Among them, the addition amount of the exogenous additive factor simvastatin is 0.5 to 4 μΜ.

The amount of acetic acid added to the precursor of the synthetic fatty acid is 3 to 9 mM.

Among them, it is preferred that 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.

Among them, it is preferred that 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 μΜ.

Among them, it is preferred to add acetic acid to the medium in the pre-fermentation period in an amount of 6 mM, and in the latter stage of fermentation, that is, when the glucose concentration in the fermentation medium is lowered to 20 to 25 g/L, acetic acid is added in an amount of 6 mM. Advantageous Effects: 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. detailed description

The invention can be better understood in light of the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions, and results described in the examples are merely illustrative of the invention and are not intended to limit the invention as described in the claims. . The strains used in the following examples are:

Schizochytrium sp. HX-308, CCTCC No: M 209059;

Thraustochytrium sp. ATCC No: 26185;

C. cohnni: ATCC 30556 ο 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. (Refer to "A Schizochytrium and Method for Producing DHA Grease Using It", Application No. 200910033869.5).

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. (Refer to "A Schizochytrium and Method for Producing DHA Grease Using It", Application No. 200910033869.5). 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. (See "Streaming strategy for the production of DHA by high-density fermentation with Crypthecodinium cohnii", Food and Fermentation Industries, 2007 Vol. 33 No. 1. PP: 25-27). 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:

Before the fermentation, a certain amount of acetic acid was added to the fermentation medium of Schizochytrium and Thraustochytrium, respectively. The glucose concentration in the medium was measured by a biosensor, and the fermentation was stopped when the residual glucose concentration in the medium was Og/L. The experimental results are shown in Tables 1 and 2: Table 1 Effect of exogenous acetic acid on the fermentation of Schizochytrium

0 3 6 12

Dry cell weight (g/L) 61.4 61.73 61.84 58.88

Total oil production (g/L) 30.61 30.67 30.78 28.61

DHA accounts for the dry weight of cells containing 逦 t (%) 14.67 15.52 16.61 12.43

DHA accounts for 脂肪酸 t(%) of total fatty acids 36.51 38.61 39.34 35.8

DHA production (g/L) 9.01 9.58 10.27 7.32 Table 2 Effect of exogenous acetic acid on the fermentation of Thraustochytrium

Exogenous acetic acid concentration (mM)

0 3 6 12

Dry cell weight (g/L) 40 40.12 40.21 39.11

Total oil production (g/L) 18.34 18.44 18.56 17.11

DHA accounts for the dry weight of cells. t(%) 12.5 13.91 17.14 9.97

DHA accounts for 脂肪酸 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:

Considering that with the consumption of glucose, the fatty acid synthesis precursors are also reduced. In order to further increase the yield of DHA, under the premise of acetic acid without adding acetic acid or adding the optimal concentration in the fermentation, in the late stage of fermentation, When the glucose concentration is 20 to 25 g/L, acetic acid is added to the fermentation liquid, and the glucose concentration in the medium is measured by a biosensor, and the fermentation is stopped when the residual glucose concentration in the medium is Og/L. The results are shown in Tables 3 and 4 below: Table 3 Effect of Precursor Substance Acetic Acid on Fermentation of Schizochytrium

Exogenous acetic acid concentration (mM)

0 3 6 9 12

Dry cell weight (g/L) 61.4 61.33 60.12 59.88 58.58

Total oil production (g/L) 30.61 31.67 32.78 30.78 28.09

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 production (g/L) 9.01 10.21 11.02 9.32 7.89 Table 4 Effect of precursor acetic acid on the fermentation of Schizochytrium

, . Exogenous acetic acid concentration (mM)

0 6a+0b 0a+6b 6a+6b

Dry cell weight (g/L) 61.4 61.84 60.12 61.81 Total oil production (g/L) 30.61 30.78 32.78 29.12

DHA accounts for the dry weight of cells. 14.67 16.61 18.33 18.69

DHA accounts for 36.51 39.34 40.34 45.00

DHA production (g/L) 9.01 10.27 11.02 11.55 Note: 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. Considering that in the first embodiment, when 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. On this basis, this time, this time, EXAMPLES The addition of 6 mM acetic acid in the pre- and post-fermentation period resulted in an increase in DHA production and percentage of total fatty acids, especially as DHA accounted for a significant increase in total fatty acid content (as shown in Table 4). Example 3:

Before the fermentation, a certain amount of simvastatin was added to the fermentation medium of Schizochytrium and Crypthecodin. 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 Tables 5 and 6. Table 5 Effect of exogenous simvastatin on the fermentation of Schizochytrium

Exogenous simvastatin concentration

0 0.5 1 2 4 8 Cell dry weight (g/L) 61.4 61.6 61.4 61.22 60.68 58.04 Total oil production (g/L) 30.61 30.74 30.7 30.31 30.3 29.96

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 production (g/L) 9.01 10.53 12.22 10.95 9.78 8.60 Table 6 Effect of exogenous simvastatin on the fermentation of Cryptophyta

Exogenous simvastatin concentration

0 0.5 1 2 4 8 Cell dry weight (g/L) 22.01 21.99 21.98 21.03 20.88 19.56 Total oil production (g/L) 10.82 10.78 10.22 10.11 10.02 9.56

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 production (g/L) 2.15 3.37 3.82 2.67 2.22 1.54 Acetyl-CoA is a common precursor of the mevalonate pathway and fatty acid synthesis pathway, and simvastatin is an inhibitor of the mevalonate pathway key enzyme HMG-COA reductase. When the appropriate amount of simvastatin is added, the mevalonate pathway is inhibited, so that more acetyl-CoA flows to the synthesis of fatty acids, which is more favorable for the synthesis of DHA. As shown in Tables 5 and 6, simvastatin at 0.5 to 4 μM is beneficial to the biosynthesis of DHA, and DHA production is highest when the concentration of simvastatin is ΙμΜ. Example 4:

Considering the conversion of more precursor acetyl-CoA to fatty acids, the optimal concentration of acetic acid and simvastatin was added to the medium before fermentation. The glucose concentration in the medium was measured by a biosensor, and the fermentation was stopped when the residual glucose concentration in the medium was Og/L. The results are shown in Table 7.

Table 7 Effect of exogenous acetic acid and simvastatin on the fermentation of Schizochytrium Exogenous acetic acid and simvastatin concentration (mM+μΜ)

0c+0d 6c+0d Oc+ld 6c+ld

Dry cell weight (g/L) 61.4 61.84 61.4 61.64

Total oil production (g/L) 30.61 30.78 30.7 31.32

DHA accounts for the dry weight of cells (逦) 14.67 16.61 19.90 21.43

DHA accounts for 脂肪酸 t (%) of total fatty acids 36.51 39.34 40.75 39.87

DHA production (g/L) 9.01 10.27 12.22 13.21

Note: c indicates the addition of acetic acid; d indicates the addition of simvastatin. As can be seen from Table 7, Simultaneous addition of 6 mM acetic acid and ΙμΜ simvastatin, DHA yield and DHA account for the dry weight of the cells are relatively neither added or only added First, there has been an increase, but DHA accounts for a decrease in the total fatty acid content relative to the simvastatin supplemented with lipids. Example 5

A certain amount of citric acid was added to the fermentation medium of Schizochytrium separately before fermentation. The glucose concentration in the medium was measured by a biosensor, and the fermentation was stopped when the residual glucose concentration in the medium was Og/L. The results are shown in Table 7. Table 8 Effect of exogenous citric acid on the fermentation of Schizochytrium

.. Exogenous citric acid concentration (mM)

0 2 4 8 12 Cell dry weight (g/L) 61.4 61.92 61.16 61 61 Total oil production (g/L) 30.61 30.95 29.12 28.79 27.32 DHA accounted for the dry weight of cells 14.67 17.59 16.33 15.10 12.79 DHA accounted for total fatty acid content 36.51 38.61 42.65 41.17 35.57

DHA production (g/L) 9.01 10.89 9.99 9.21 7.8 Fermentation cycle (h) 69 57 58 61 70 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. As can be seen from Table 7, the concentration of citric acid of 2 to 8 mM is favorable for the biosynthesis of DHA. When the concentration was 2 mM, DHA yield and DHA accounted for the highest dry weight of cells. When the concentration was 4 mM, 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

Exogenous acetic acid and citric acid and concentration (mM+mM)

0c+0e 6c+0e 0c+2e 0c+4e 6c+2e 6c+4e Cell dry weight (g/L) 61.4 61.84 61.92 61.16 62.08 58.67 Total oil production (g/L) 30.61 30.78 30.95 29.12 31.07 29.45

DHA accounts for the dry weight of cells. 14.67 16.61 17.59 16.33 19.65 16.29

DHA accounts for 36.51 39.34 38.61 42.65 37.52 39.66

DHA yield (g/L) 9.01 10.27 10.89 9.99 12.2 9.56 Note: c indicates the addition of acetic acid; e indicates the addition of citric acid. As shown in 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.

Claims

Claim

1. Exogenously added factors promote microbial synthesis of docosahexaenoic acid, and the microorganisms are introduced into a fermentation medium for fermentation to synthesize docosahexaenoic acid, which is characterized in that fermentation fermentation synthesis feed medium is used. Adding an exogenous additive factor, and/or adding a precursor substance for synthesizing a fatty acid to the fermentation synthesis; wherein the microorganism is any one of Thraustochytrium, Schizochytrium, and Cryptophyta; The exogenous additive factor is any one or a combination of several of acetic acid, citric acid, and simvastatin; and the precursor of the synthetic fatty acid is acetic acid.

The method for promoting microbial synthesis of docosahexaenoic acid by the exogenous addition factor according to claim 1, wherein the amount of the exogenous added factor acetic acid is 3 to 6 mM.

The method according to claim 1, wherein the exogenous added factor citric acid is added in an amount of 2 to 8 mM.

The method according to claim 1, wherein the exogenous addition factor simvastatin is added in an amount of 0.5 to 4 μM.

5. The method according to claim 1, wherein the method for promoting microbial synthesis of docosahexaenoic acid is characterized in that a precursor substance of a synthetic fatty acid is added at a later stage of fermentation synthesis, that is, the glucose concentration in the fermentation medium is lowered to The precursor substance of the synthetic fatty acid is added at 20 to 25 g/L.

The method for promoting microbial synthesis of docosahexaenoic acid by the exogenous addition factor according to claim 1, wherein the amount of acetic acid added to the precursor of the synthetic fatty acid is from 3 to 9 mM.