A method of reducing succinic acid, a by-product of lactic acid fermentation

Abstract

The present invention relates to a method of adding an isocitrate lyase specific inhibitor to a lactic acid fermentation process to block the production of succinic acid as a byproduct of fermentation. The specific inhibitor includes a combination of one or more of glycolic acid methyl ester, malonic acid dimethyl ester, hydroxymalonic acid diethyl ester, 3-nitropropionic acid ethyl ester, mercuric chloride, or 5,5'-dithiobis(2-nitrobenzoic acid) to effectively reduce the amount of succinic acid in the lactic acid.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and mainly relates to a method for producing lactic acid through fermentation. Background Technology

[0002] Currently, petroleum-based chemicals are widely used as raw materials in polymer materials, textiles, paints, and organic solvents. However, against the backdrop of global warming and increasingly scarce petroleum resources, polylactic acid (PLA), as a typical representative of biodegradable materials, is considered one of the main alternatives to petroleum-based plastics and other materials. Utilizing readily available and inexpensive raw materials to prepare D-lactic acid and L-lactic acid with extremely high optical and chemical purity required for PLA processing is essential for the development of the PLA industry and a new driving force for the development and transformation of the organic acid industry.

[0003] To achieve high substrate conversion, high yield, and high optical and chemical purity of the target product through lactic acid fermentation, the metabolic pathway was modified, and genetically engineered strains were used as lactic acid producing bacteria.

[0004] There are three main pathways for the synthesis of succinic acid: first, fumarate is reduced to succinic acid by fumarate reductase; second, succinyl-CoA is hydrolyzed to succinic acid by succinyl-CoA synthase; and third, isocitrate participates in the hemialdehyde cycle to form succinic acid (aceA). Under aerobic metabolism, the formation and decomposition of succinic acid occur simultaneously, and *E. coli* accumulates almost no succinic acid. Under anaerobic metabolism, *E. coli* accumulates succinic acid under the action of fumarate reductase. Lactic acid synthesis often occurs under anaerobic or low-oxygen conditions; therefore, it is necessary to block the synthesis of succinic acid by modifying the pathway. CN 104278003 B, using *E. coli* as the starting strain, knocked out the fumarate reductase gene frdABCD to block the anaerobic succinic acid production pathway. CN 105705630 A, by knocking out the fumarate reductase gene frdA, blocked the anaerobic succinic acid production pathway.

[0005] However, in reality, on the one hand, genetic modification is costly and complex; on the other hand, although the anaerobic pathway for succinic acid production is blocked, some air still exists in the fermenter, or oxygen may be present in the fermentation broth during the early aerobic proliferation stage. Even with aeration stopped for anaerobic culture, the oxygen present under these conditions will still enter the cells and generate succinic acid through the aerobic pathway, leading to its accumulation. If the aerobic pathway for succinic acid production is blocked through genetic engineering, bacteria will be unable to grow. Furthermore, the generation of succinic acid not only reduces the lactic acid conversion rate but also decreases the yield during lactic acid separation and purification, ultimately increasing the cost of high-purity lactic acid. Summary of the Invention

[0006] This invention addresses the problem of the inability to completely inhibit succinic acid production during lactic acid fermentation by using a specific inhibitor of isocitrate lyase (aceA) during fermentation to block succinic acid production.

[0007] To achieve the above effects, the present invention provides a method for reducing succinic acid, a byproduct of lactic acid fermentation, comprising the following steps:

[0008] (1) Cultivate a seed culture of lactic acid producing bacteria, and inoculate the seed culture of producing bacteria into a fermenter containing culture medium for bacterial growth; (2) When the bacterial concentration reaches OD600 of 10-50, add a specific inhibitor of isocitrate lyase, and then enter the acid production stage for lactic acid fermentation; the specific inhibitor is one or a combination of methyl glycolate, dimethyl malonate, diethyl hydroxymalonate, ethyl 3-nitropropionate, mercuric chloride or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB).

[0009] Preferably, the combination of the inhibitors is a mixture of methyl glycolate, dimethyl malonate and mercuric chloride; or a mixture of methyl glycolate, dimethyl malonate, diethyl hydroxymalonate and ethyl 3-nitropropionate; more preferably, a mixture of ethyl 3-nitropropionate, mercuric chloride and 5,5'-dithiobis(2-nitrobenzoic acid).

[0010] The lactic acid producing strains mentioned in step (1) are selected from one or more of the following: recombinant Escherichia coli, Bacillus coagulans, Bacillus subtilis, Lactobacillus delbrueckii, Lactobacillus bulgaricus, Aspergillus niger, and yeast.

[0011] The seed liquid mentioned in step (1) is a shake flask culture seed liquid and / or a seed tank culture seed liquid. The culture medium for the shake flask culture seed liquid is LB medium. The shake flask culture conditions are as follows: LB medium is placed in an Erlenmeyer flask, lactic acid producing bacteria glycerol tubes are inoculated, and cultured on a shaker at 30-37℃ and 100-500r / min for 10-14h.

[0012] The culture conditions for the seed culture in the seed tank are as follows: the seed culture from the shake flask is inoculated into the seed tank containing liquid culture medium at an initial inoculation rate of 3-5%. After inoculation, the initial volume of the seed tank is 25%-60% of the working volume. The temperature is controlled at 30-37℃. The pH is maintained at 6.8-7.5 by adding an alkaline neutralizing agent. The dissolved oxygen is controlled at 20-60%. The aeration rate is 0.1 vvm-2.0 vvm, and the rotation speed is 200-1000 r / min. When the cell concentration reaches OD600 of 10-30, it is used as the seed culture for inoculation into the fermenter.

[0013] The liquid culture medium is M9 culture medium, and the working volume of the seed tank is 40-60%, preferably 50-60%.

[0014] The dissolved oxygen in the seed tank is controlled at 20-60%, preferably 20-40%, and more preferably 20-30%.

[0015] The alkaline neutralizing agent is selected from one or more of calcium hydroxide, sodium hydroxide, magnesium hydroxide, and ammonia water, with ammonia water being preferred.

[0016] The ventilation gas is sterile air.

[0017] In step (1), the temperature during the bacterial growth stage is controlled at 37-45℃, pH at 6.8-7.5, aeration rate at 0.1-2.0 vvm, rotation speed at 200-1000 r / min, and dissolved oxygen at 10-30%.

[0018] The lactic acid fermentation process described in step (2) also includes a process of adding an alkaline neutralizing agent to maintain the pH at 6.0-7.5.

[0019] The alkaline neutralizing agent is one or more of calcium hydroxide, sodium hydroxide, magnesium hydroxide, and ammonia water, with calcium hydroxide being preferred.

[0020] The fermentation acid production stage described in step (2) also includes a process of continuously feeding or batch feeding of glucose.

[0021] The culture conditions for the acid production stage in step (2) are: temperature control of 37-50℃, preferably 37-45℃, more preferably 40-45℃, and rotation speed control of 100-500rpm, more preferably 100-300rpm.

[0022] When the OD600 in step (2) is 15-45, more preferably 20-30, a succinic acid-specific inhibitor is added.

[0023] In step (2), the concentration of the specific inhibitor is 0.01-20 mM, preferably 0.03-15 mM.

[0024] Preferably, the combination of the multiple inhibitors is a mixture of ethyl 3-nitropropionate, mercuric chloride and 5,5'-dithiobis(2-nitrobenzoic acid), with an effective concentration of 0.01-0.05 mM, preferably 0.03-0.05 mM, and the mixture composition ratio is preferably (5-7):(2-3):(1-2).

[0025] The beneficial effects of this invention are as follows:

[0026] Compared to current lactic acid production processes, this invention adds a specific inhibitor of isocitrate lyase during lactic acid fermentation, which not only does not affect lactic acid production but also effectively reduces succinic acid synthesis, with an inhibition rate of up to 60-100%. Detailed Implementation

[0027] To further illustrate the beneficial effects of the technical solution of the present invention, the present invention will be described in detail below with reference to the embodiments, but this does not constitute a limitation on the present invention.

[0028] Main raw material information of the embodiments of the present invention:

[0029] Methyl glycolate was purchased from alfa, product number A17870;

[0030] Dimethyl malonate was purchased from CATO, catalog number CCFD200172;

[0031] Diethyl hydroxymalonate was purchased from Sigma-Aldrich, catalog number 86320;

[0032] 3-Nitropropionic acid was purchased from Sigma-Aldrich, catalog number N5636;

[0033] 5,5'-Dithiobis(2-nitrobenzoic acid) was purchased from Sigma-Aldrich, catalog number D8310;

[0034] Mercuric chloride was purchased from Shanghai Testing Equipment Co., Ltd., item number 10013616;

[0035] The recombinant Escherichia coli strain was purchased from the China General Microbiological Preservation Center, strain number CGMCC 11059.

[0036] LB medium: 10g peptone, 5g yeast extract, 10g NaCl, adjust pH to 7.4 with 1M sodium hydroxide solution, bring volume to 1L with deionized water, and sterilize at 121℃ for 20min;

[0037] M9 culture medium: First, prepare 1M MgSO4, dissolve 2.46g of MgSO4·7H2O in 10ml of deionized water, and autoclave for later use; prepare 1M CaCl2, dissolve 2.191g of CaCl2·6H2O in 10ml of deionized water, and autoclave for later use; then prepare 5×M9 salt solution, dissolve 12.8g of Na2PO4·7H2O, 3.0g of KH2PO4, 0.5g of NaCl, and 1.0g of NH4Cl in 200ml of deionized water, and sterilize at 121℃ for 15min; prepare 20% glucose solution, dissolve 4g of glucose in 16ml of deionized water, and filter sterilize through a 0.22μm filter; in a sterile room, add 200ml of 5×M9 salt solution, 2ml of 1M MgSO4, 20ml of 20% glucose solution, and 0.1ml of 1M CaCl2 to 1L of sterile deionized water.

[0038] Yeast powder and peptone were purchased from oxoid, and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0039] 1. Shake-flask seed culture

[0040] 50 mL of LB medium was placed in a 250 mL Erlenmeyer flask and inoculated with a glycerol tube containing recombinant Escherichia coli (CGMCC 11059). The flask was then incubated at 37 °C and 250 rpm for 11 h on a shaker.

[0041] 2. Seed culture experiment

[0042] The shake flask seed culture was inoculated into 1.5L of M9 liquid medium at an initial inoculation rate of 5%. First, a 1M MgSO4 fermenter was prepared. After inoculation, the initial volume of the fermenter was 25%-60% of the working volume. The temperature was controlled at 37℃, the pH was maintained at 6.8-7.5, the dissolved oxygen was controlled at 20%, the aeration rate was 0.1vvm-2.0vvm, and the rotation speed was 200-1000r / min. When the cell concentration reached OD600 of 20, it was used as the seed culture for inoculation into the fermenter.

[0043] 3. Fermentation test in a fermenter

[0044] The seed culture was inoculated into a fermenter containing M9 liquid medium at a 5% inoculation rate. The initial volume of the fermenter after inoculation was 25%-60% of the working volume. Fermentation was carried out using a two-stage fermentation method (Tian Kangming et al., Chinese Journal of Biotechnology, 29:111-114, 2013). During the cell growth stage of fermentation, the temperature was controlled at 37℃, the pH was maintained at 6.8-7.5, the aeration rate was 0.1-2.0 vvm, the stirring speed was 200-1000 r / min, and the dissolved oxygen was controlled at 20%. When the cell concentration reached OD600 of 30, the lactic acid fermentation stage began, with the temperature controlled at 42℃, the stirring speed adjusted to 200 r / min, and a 25 wt% calcium hydroxide suspension added to maintain the pH at 6.0-7.5. During the acid production stage of fermentation, glucose is fed continuously or in batches. In the batch feeding method, glucose solution with a final concentration of 6wt% is added in four batches, with a total addition amount of 25% of the initial fermentation volume. In the fed-flow method, the sugar concentration is maintained by controlling the flow rate of the sugar solution, with the total amount of sugar solution being 25% of the initial fermentation volume.

[0045] 4. Fermentation process analysis

[0046] Sample preparation: Take 1 mL of the fermentation broth to be tested and mix it with 50 μL of 50% concentrated sulfuric acid. Centrifuge at 8000 r / min for 10 min. Take an appropriate amount of supernatant, add acetonitrile to make up to 5 mL, mix well, and centrifuge at 10000 r / min for 5 min. Dilute the supernatant with 5 mM sulfuric acid and filter it through a 0.22 μm organic microporous membrane for analysis and determination of relevant components.

[0047] (1) Glucose concentration determination: After diluting the sample with deionized water, the glucose concentration was determined using an SBA-40C biosensor, and the average value of three parallel data was taken.

[0048] (2) OD detection: During the cell growth stage, the fermentation broth was diluted with deionized water and the OD600 was measured. During the lactic acid fermentation stage, the fermentation broth was diluted with 1M hydrochloric acid and the OD600 was measured.

[0049] (3) Determination of D-lactic acid, L-lactic acid and succinic acid content: HPLC was used. The chromatographic conditions were as follows: HPX-87H organic acid analytical column, column temperature 65℃, detection wavelength 210nm, mobile phase 5mM sulfuric acid solution, flow rate 0.8mL / min, and injection volume 10μL. All data are the average of three parallel experiments.

[0050] Inhibition rate = (N1-N2) / N1*100%

[0051] Where N1: succinic acid content in the control group, and N2: succinic acid content detected.

[0052] Example 1

[0053] Effect of methyl glycolate on lactic acid fermentation

[0054] The shake-flask seed culture of lactic acid producing bacteria was inoculated into 1.5 L of M9 liquid medium and cultured at 37℃ and 200-1000 rpm for 7 h. Then, the culture was inoculated into M9 liquid medium with a glucose concentration of 30 g / L at an initial OD value of 0.3. After inoculation, the initial volume of a 50 L fermenter was 25 L. The temperature was controlled at 37℃, the aeration rate at 0.5-2.0 vvm, the stirring speed at 200-1000 rpm, the dissolved oxygen at 20%, and the pH was maintained using ammonia. 6.5-7.0; When the cell concentration reaches OD600 of 30, add 0-10mM methyl glycolate to a final concentration, close the ventilation, set the stirring speed to 200r / min, and control the fermentation temperature to 42℃. Add the total glucose (based on the initial fermentation volume, the total amount of glucose added is approximately 240g / L) to the fermenter in 4 portions, and add 25wt.% calcium hydroxide suspension to maintain the pH at 7.0. Stop fermentation when the residual sugar concentration is below 0.2g / L before the end of fermentation.

[0055] Under these fermentation conditions, the total amount of glucose added remained constant, and the fermentation broth was used to test the content of lactic acid and succinic acid.

[0056] Table 1 Effect of methyl glycolate on lactic acid fermentation

[0057] 0 145 2000 0 1 145.33 1565 21.75 3 145.43 1435 28.25 5 145.76 1000 50 7 145.93 780 61 10 146.24 375 81.25

[0058] Example 2

[0059] The effect of dimethyl malonate on lactic acid fermentation

[0060] The lactic acid fermentation process is the same as in Example 1. When the cell concentration reaches OD600 of 25, dimethyl malonate with a final concentration of 0-20 mM is added.

[0061] Table 2 Effect of methyl malonate on lactic acid fermentation

[0062] 0 142 1820 0 10 142.43 1250 31.32 12 142.55 1105 39.29 15 142.83 732 59.78 18 142.90 645 64.56 20 142.96 560 69.23

[0063] Example 3

[0064] Effect of diethyl hydroxymalonate on lactic acid fermentation

[0065] The lactic acid fermentation process is the same as in Example 1. When the cell concentration reaches OD600 of 20, dimethyl malonate with a final concentration of 0-1 mM is added.

[0066] Table 3 Effects of diethyl hydroxymalonate on lactic acid fermentation

[0067] 0 137.5 1750 0 0.2 137.90 1230 29.71 0.3 138.05 1025 41.43 0.5 138.30 700 60.00 0.7 138.56 355 79.71 1 138.57 345 80.29

[0068] Example 4

[0069] The effect of 3-nitropropionic acid on lactic acid fermentation

[0070] The lactic acid fermentation process is the same as in Example 1. When the cell concentration reaches OD600 of 30, 0-0.1 mM 3-nitropropionic acid is added.

[0071] Table 4. Effects of 3-nitropropionic acid on lactic acid fermentation

[0072]

[0073]

[0074] Example 5

[0075] The effect of mercuric chloride on lactic acid fermentation

[0076] The lactic acid fermentation process is the same as in Example 1. When the cell concentration reaches OD600 of 25, mercuric chloride with a final concentration of 0-0.1 mM is added.

[0077] Table 5. Effects of mercuric chloride on lactic acid fermentation

[0078] 0 142 1820 0 0.01 142.13 1645 9.62 0.03 142.62 1005 44.78 0.05 142.85 705 61.26 0.07 142.91 630 65.38 0.1 142.99 522 71.32

[0079] Example 6

[0080] Effects of 5,5'-dithiobis(2-nitrobenzoic acid) on lactic acid fermentation

[0081] The lactic acid fermentation process is the same as in Example 1. When the cell concentration reaches OD600 of 30, 5,5'-dithiobis(2-nitrobenzoic acid) with a final concentration of 0-0.1 mM is added.

[0082] Table 6. Effects of 5,5'-dithiobis(2-nitrobenzoic acid) on lactic acid fermentation

[0083] 0 145 2000 0 0.01 145.48 1365 31.75 0.03 145.80 956 52.20 0.05 145.95 752 62.40 0.07 146.06 612 69.40 0.1 146.10 555 72.25

[0084] Example 7

[0085] Effect of combination C on lactic acid fermentation

[0086] The lactic acid fermentation process is the same as in Example 1. When the cell concentration reaches OD600 of 30, a final concentration of 0-0.05 mM of combination C is added. C is a mixture of ethyl 3-nitropropionate, mercuric chloride and 5,5'-disulfidebis(2-nitrobenzoic acid) in a ratio of 5:3:2.

[0087] Table 7 Effects of Combination C on Lactic Acid Fermentation

[0088] 0 145 2000 0 0.01 145.31 1593 20.35 0.02 145.58 1235 38.25 0.03 145.92 795 60.25 0.04 146.23 387 80.65 0.05 146.53 0 100

[0089] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A method for reducing succinic acid, a byproduct of lactic acid fermentation, comprising the following steps: (1) Cultivate the seed culture of lactic acid producing bacteria and inoculate the seed culture of producing bacteria into a fermenter containing culture medium for bacterial growth; the lactic acid producing bacteria are selected from recombinant Escherichia coli; (2) When the bacterial concentration reaches OD600 of 10-50, add a specific inhibitor of isocitrate lyase, and then enter the acid production stage for lactic acid fermentation; the specific inhibitor is a mixture of ethyl 3-nitropropionate, mercuric chloride and 5,5'-dithiobis(2-nitrobenzoic acid) in a ratio of (5-7):(2-3):(1-2).

2. The method as described in claim 1, characterized in that, In step (2), the concentration of the specific inhibitor is 0.01-20 mM.

3. The method as described in claim 1, characterized in that, In step (2), the concentration of the specific inhibitor is 0.03-15 mM.

4. The method according to any one of claims 1-3, characterized in that, In step (1), the temperature during the bacterial growth stage is controlled at 37-45℃, pH at 6.8-7.5, aeration rate at 0.1-2.0 vvm, rotation speed at 200-1000 r / min, and dissolved oxygen at 10-30%.

5. The method according to any one of claims 1-3, characterized in that, The lactic acid fermentation process described in step (2) also includes a process of adding an alkaline neutralizing agent to maintain the pH at 6.0-7.5; the alkaline neutralizing agent is one or more of calcium hydroxide, sodium hydroxide, magnesium hydroxide, and ammonia water.

6. The method according to any one of claims 1-3, characterized in that, The fermentation acid production stage described in step (2) also includes a process of continuously feeding or batch feeding of glucose.

7. The method according to any one of claims 1-3, characterized in that, The acid production stage culture conditions described in step (2) are a temperature control of 37-50℃ and a rotation speed control of 100-500rpm.

8. The method according to any one of claims 1-3, characterized in that, When the OD600 in step (2) is 15-45, a succinic acid-specific inhibitor is added.

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