Substrate regulation and control method for improving content of intracellular 3-hydroxyvalerate (3HV) of halophilic bacteria

Abstract

The method provided by the invention can be used for effectively synthesizing a high 3-hydroxyvalerate monomer (3-HV) in a proportion. The method comprises the following steps: adding a 3-HV precursor substance into a fermentation system to prepare PHBV; the precursor substance is one or more of short-chain fatty acids (VFAs). By the adoption of the method, it is found that Halomonas venusta has high tolerance to VFAs, so that the yield of PHBV can be increased; and the proportion of the 3-HV monomer in the product PHBV can be adjusted by changing the mixing proportion of the substrate. Finally, the content of the obtained PHBV is as high as 90.81 wt%, the proportion of 3-HV monomers is as high as 49.81%, and the content of the obtained PHBV is far higher than that of other non-engineering pure bacterium fermentation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of synthetic biology, and relates to a purchased Halomonas venusta, and particularly relates to a method for accumulating high 3-hydroxyvaleric acid monomer ratio by using the bacteria. TECHNICAL BACKGROUND

[0002] Petroleum-based plastics are commonly used as substrates for traditional plastic packaging, which can cause resource crisis, environmental pollution, biological safety and other problems during production and use. Therefore, in order to reduce the harm of petroleum-based plastics to the environment and promote sustainable development, biodegradable packaging materials have emerged as the research focus. Biobased polyhydroxyalkanoate (PHA) is a natural polymer-based bioplastic produced by a variety of microorganisms under conditions of nutrient deficiency (such as carbon excess and nitrogen / phosphorus shortage), which has similar properties to petroleum-based derivatives and has strong biocompatibility and biodegradability, and is therefore considered as one of the most promising green and environmentally friendly materials.

[0003] At present, a large number of strains capable of synthesizing PHA have been found, such as Ralstonia eutropha, Pseudomonas putida, Alcaligenes latus, and recombinant Escherichia coli. However, the microbial synthesis of PHA has the disadvantages of high sterilization cost, discontinuous fermentation, high energy consumption, and low yield, which limits the industrial production and application. Halophilic microorganisms are microorganisms that can survive and reproduce in high-salinity (high-salt content NaCl > 0.2 mol / L) environments such as salt lakes, seawater, and saline soils. Due to the special survival mechanism and biological activity of halophilic microorganisms, the PHA synthesis process can be protected from contamination by most mesophilic microbial communities, achieving a fermentation process without the need for sterilization. Therefore, using halophilic bacteria as a chassis microorganism to synthesize PHA not only reduces the energy consumption in the upstream processing process, shortens the time for PHA extraction, but also greatly reduces the synthesis cost of PHA.

[0004] Currently, there are nearly 200 different PHA monomers have been discovered. Among them, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a biodegradable polyester synthesized by microorganisms, which is an important member of the polyhydroxyalkanoate (PHA) family. The introduction of 3-hydroxyvalerate (HV) monomer in its structure can significantly improve the high crystallinity and brittleness of poly-3-hydroxybutyrate (PHB), and endow the material with better mechanical properties and processing performance. PHBV has broad application prospects in packaging materials, medical implants, drug release carriers and other fields. In the synthesis of PHBV, 3HV precursor substances such as volatile fatty acids (VFAs) need to be added. However, high concentration of volatile fatty acids (VFAs) will have an inhibitory effect on the strain, resulting in that the current pure non-engineered bacteria for synthesizing 3-hydroxyvalerate monomer (3-HV) has a low molar ratio (mostly concentrated in 10-20 mol%).

[0005] Therefore, the present application hopes to provide a method for effectively synthesizing a high 3-HV ratio. SUMMARY

[0006] In order to solve these technical problems, the present application provides a method for effectively synthesizing a high 3-hydroxyvalerate monomer (3-HV) ratio in polyhydroxyalkanoate, which is prepared by adding 3-HV precursor substances in the fermentation system to obtain PHBV.

[0007] The present application points out that at least one of the short-chain volatile fatty acids (VFAs) is added in the fermentation system commonly used for producing PHB, which can adjust the 3-HV monomer ratio in the product PHBV and broaden the application range of PHBV material.

[0008] Further, the microorganism used in the fermentation system is Halomonas venusta purchased from China Industrial Microbial Culture Collection Center.

[0009] Further, the Halomonas venusta can be biologically fermented by using carbon sources, including one or more of acetic acid, propionic acid, butyric acid, and valeric acid.

[0010] Further, the fermentation medium components are as follows: acetic acid 10-30 g / L, peptone 1-2 g / L, yeast powder 1-2 g / L, sodium chloride 50-70 g / L, magnesium chloride hexahydrate 5.4-7 g / L, magnesium sulfate heptahydrate 6.8 g / L, anhydrous calcium chloride 1.4-2.8 g / L, potassium chloride 0.7-1.4 g / L, sodium bicarbonate 0.2 g / L, pH 6-8.

[0011] Further, the fermentation medium components are: propionic acid 5-20 g / L, peptone 1-2 g / L, yeast powder 1-2 g / L, sodium chloride 50-70 g / L, magnesium chloride hexahydrate 5.4-7 g / L, magnesium sulfate heptahydrate 6.8 g / L, calcium chloride anhydrous 1.4-2.8 g / L, potassium chloride 0.7-1.4 g / L, sodium bicarbonate 0.2 g / L, pH 6-8.

[0012] Further, the fermentation medium components are: butyric acid 10-40 g / L, peptone 1-2 g / L, yeast powder 1-2 g / L, sodium chloride 50-70 g / L, magnesium chloride hexahydrate 5.4-7 g / L, magnesium sulfate heptahydrate 6.8 g / L, calcium chloride anhydrous 1.4-2.8 g / L, potassium chloride 0.7-1.4 g / L, sodium bicarbonate 0.2 g / L, pH 6-8.

[0013] Further, the fermentation medium components are: valeric acid 10-30 g / L, peptone 1-2 g / L, yeast powder 1-2 g / L, sodium chloride 50-70 g / L, magnesium chloride hexahydrate 5.4-7 g / L, magnesium sulfate heptahydrate 6.8 g / L, calcium chloride anhydrous 1.4-2.8 g / L, potassium chloride 0.7-1.4 g / L, sodium bicarbonate 0.2 g / L, pH 6-8.

[0014] Further, the fermentation medium components are: butyric acid + valeric acid 30 g / L, valeric acid added amount 5-30 g / L, peptone 1-2 g / L, yeast powder 1-2 g / L, sodium chloride 50-70 g / L, magnesium chloride hexahydrate 5.4-7 g / L, magnesium sulfate heptahydrate 6.8 g / L, calcium chloride anhydrous 1.4-2.8 g / L, potassium chloride 0.7-1.4 g / L, sodium bicarbonate 0.2 g / L, pH 6-8.

[0015] Further, the specific steps of the method are:

[0016] (1) Strain activation: inoculate the strain on 60 LB medium, and culture at 27-30℃ for 48h.

[0017] (2) Seed liquid culture: inoculate a single colony in 50ml activated medium, and activate at 27-30℃ in an environment with a rotation speed of 140-190rpm / min for 4-8h to obtain a seed liquid.

[0018] (3) The activated medium components are: peptone 5 g / L, yeast powder 10 g / L, sodium chloride 50-70 g / L, magnesium chloride hexahydrate 5.4-7 g / L, magnesium sulfate heptahydrate 6.8 g / L, calcium chloride anhydrous 1.4-2.8 g / L, potassium chloride 0.7-1.4 g / L, sodium bicarbonate 0.2 g / L.

[0019] (4) PHBV production: the seed liquid obtained in step (2) is inoculated in the above fermentation medium at an inoculation amount of 2-5% (v / v) for fermentation.

[0020] (5) Other fermentation parameters: the time can be 60-144h; the fermentation temperature can be 27-30℃; the shaking flask fermentation is adopted, and the shaking speed can be 150-200rpm / min.

[0021] Compared with the prior art, the present application has the following technical achievements:

[0022] (1) The VFAs as the substrate, it is found that Halomonas venusta has strong tolerance to VFAs (the maximum tolerance of butyric acid can reach 30g / L, and the maximum tolerance of pentanoic acid can reach 15g / L), so that the PHBV yield can be greatly improved.

[0023] (2) The present application realizes a high PHBV content of 90.81wt% and a high 3-HV monomer ratio of 49.81%, which is much higher than the 3-HV monomer ratio in PHBV synthesized by fermentation of other non-engineered pure bacteria, and the adjustable 3-HV monomer ratio in PHBV can realize flexible regulation of the physical and chemical properties, mechanical properties and biodegradation characteristics, so as to meet the needs of different application scenarios. DETAILED DESCRIPTION

[0024] In order to make the skilled in the art more clearly understand the technical solutions described in the present application, the following examples are given for illustration. It should be pointed out that the following examples are only preferred embodiments of the present application, and do not constitute a limitation on the scope of protection required by the present application, and any modification, substitution, combination made without deviating from the spirit and principles of the present application is included in the scope of protection of the present application.

[0025] The raw materials, reagents or devices used in the following examples, if not specifically stated, can be obtained from conventional commercial channels, or can be obtained by existing known methods.

[0026] Example 1

[0027] The purpose of this embodiment is to investigate the effect of different concentrations of acetic acid on the accumulation of PHB by Halomonas venusta, and to select the most suitable mixed substrate for Halomonas venusta to accumulate PHBV in combination with Examples 2, 3 and 4.

[0028] The activated medium components are: peptone 5g / L, yeast powder 10g / L, sodium chloride 50-70g / L, magnesium chloride 5.4-7g / L, magnesium sulfate 6.8g / L, anhydrous calcium chloride 1.4-2.8g / L, potassium chloride 0.7-1.4g / L, sodium bicarbonate 0.2g / L.

[0029] The fermentation medium components are: acetic acid 10-30 g / L, peptone 1-2 g / L, yeast powder 1-2 g / L, sodium chloride 50-70 g / L, magnesium chloride 5.4-7 g / L, magnesium sulfate 6.8 g / L, anhydrous calcium chloride 1.4-2.8 g / L, potassium chloride 0.7-1.4 g / L, sodium bicarbonate 0.2 g / L, pH 6-8.

[0030] The specific method is as follows:

[0031] 1. Strain activation

[0032] The glycerol tube stored at -80°C is taken out, inoculated or streaked on 60 LB medium, and cultured at 27-30°C for 48 h.

[0033] 2. Seed liquid culture

[0034] Inoculate a single colony into 50 ml of activated medium, and activate at 27-30°C in an environment with a rotation speed of 140-190 rpm / min for 4-8 h to obtain a seed liquid.

[0035] 3. Fermentation liquid preparation

[0036] Prepare the fermentation medium in a 250 ml conical flask, and then add 50 ml of deionized water to the conical flask. Adjust the pH of the medium to 7 using 1 mol / L HCl and 4.5 mol / L NaOH.

[0037] 4. PHBV accumulation

[0038] Inoculate the seed culture liquid into a 250 ml conical flask at an inoculation amount of 5% (v / v), and culture in a 30°C shaking bed at 180 rpm for 96 h to complete the fermentation experiment. After the fermentation is completed, centrifuge 2 ml of the fermentation liquid at 10,000 rpm for 10 min, wash twice with deionized water, place the centrifuge tube containing the precipitated bacterial cells in a -20°C freezer for 12 h, and then freeze-dry at -60°C for 12 h. After drying, weigh and determine the dry weight (DCW) of the cells. The DCW is calculated by equation (1): Where m (g) is the mass of the centrifuge tube after freeze-drying; m0 (g) is the mass of the empty centrifuge tube after removing the bacterial sample; V (ml) is the volume of the collected fermentation liquid.

[0039] 5. PHA yield determination

[0040] The collected bacterial samples were subjected to esterification treatment, and qualitative and quantitative analysis of PHA was performed using a gas chromatograph mass spectrometer (GC-MS) (7890A, Agilent, USA), wherein the capillary chromatographic column was an HP-INNOWAX type quartz capillary column, the detector was a hydrogen flame ion detector (FID), the sample inlet temperature was 200°C, and the sample injection amount was 1 μL. The monomer structure of PHA was determined according to the peak time of the standard product PHBV (the molar ratio of 3HB to HV was 92:8, purchased from Sigma, USA), and the standard curves of PHB and PHV were obtained. The PHA yield, content, and 3-HV monomer ratio were calculated by formulas (2)-(3). Wherein m pha (g) is the mass of PHA, m sample (g) is the mass of PHA, m PHA yield (g / L) = PHA content * DCW (3)

[0041] 6. Acetic acid concentration determination

[0042] The acetic acid concentration quantitative analysis was determined by colorimetry. The absorbance was determined at a wavelength of 500 nm using a UV spectrophotometer, with a blank reagent as a reference, and the data was brought into the standard curve to obtain the residual concentration of acetic acid. The VFAs utilization rate calculation formula is as follows:

[0043] Wherein S0(g / L) is the original substrate concentration, and S1(g / L) is the residual substrate concentration after fermentation.

[0044] The PHB production conditions obtained by different acetic acid concentrations are shown in Table 1.

[0045] Table 1. PHB accumulation conditions of Halomonas venusta under different acetic acid concentrations.

[0046] The results show that: under the conditions of acetic acid concentration of 10-30, Halomonas venusta can produce PHB. With the increase of acetic acid concentration, the DCW and PHB yield show a trend of first increasing and then decreasing, wherein when the acetic acid concentration is 15 g / L, the DCW and PHB yield are the highest, which are 2.91, 1.10 g / L respectively, and the PHB content is 37.29 wt%. In addition, with the increase of acetic acid concentration, the utilization rate of the strain to acetic acid shows a trend of first increasing and then decreasing, and the utilization rate is the highest when the acetic acid concentration is 15 g / L, which is 76.53%.

[0047] Example 2

[0048] The purpose of this example is to investigate the effect of different concentrations of propionic acid on PHBV accumulation by Halomonas venusta and to select the optimal mixed substrate for PHBV accumulation by Halomonas venusta in combination with Examples 1, 3, and 4. The propionic acid concentrations were set according to Table 2, and the other methods were the same as in Example 1.

[0049] The PHBV production obtained at different propionic acid concentrations is shown in Table 2.

[0050] Table 2 PHBV accumulation by Halomonas venusta at different propionic acid concentrations.

[0051] The results show that at propionic acid concentrations of 5-20 g / L, Halomonas venusta can grow at propionic acid concentrations of 5-20 g / L, but cannot synthesize PHBV at a propionic acid concentration of 20 g / L, because propionic acid inhibits the growth of the strain and the accumulation of PHBV. In addition, as the propionic acid concentration increases, the DCW, PHBV yield, and PHBV content first increase and then decrease, with the DCW and PHBV yield being highest at a propionic acid concentration of 10 g / L, at 0.87 and 0.33 g / L, respectively, and the PHBV content being 37.29 wt%, and the 3-HV monomer molar ratio being 25.47 mol%. In addition, as the propionic acid concentration increases, the utilization rate of the strain for propionic acid decreases, with the highest utilization rate being 91.00% at a propionic acid concentration of 5 g / L.

[0052] Example 3

[0053] The purpose of this example is to investigate the effect of different concentrations of butyric acid on PHB accumulation by Halomonas venusta and to select the optimal mixed substrate for PHB accumulation by Halomonas venusta in combination with Examples 1, 2, and 4. The butyric acid concentrations were set according to Table 3, the fermentation broth extraction time was 84 h, and the other methods were the same as in Example 1.

[0054] The PHB production obtained at different butyric acid concentrations is shown in Table 3.

[0055] Table 3 PHB accumulation by Halomonas venusta at different butyric acid concentrations.

[0056] The results show that under the condition of butyric acid concentration of 10-40 g / L, Halomonas venusta can grow and accumulate PHB under the condition of butyric acid concentration of 10-40 g / L. With the increase of butyric acid concentration, the DCW shows an increasing trend. However, the PHB yield and the PHB content show a trend of first increasing and then decreasing, wherein when the butyric acid concentration is 30 g / L, the PHB yield is the highest, which is 1.81 g / L, the PHB content is 73.98 wt%, and the DCW is 2.45 g / L. In addition, with the increase of butyric acid concentration, the utilization rate of butyric acid by the strain shows a decreasing trend, but when the butyric acid concentration is less than 30 g / L, the utilization rate of butyric acid by the strain does not change significantly.

[0057] Example 4

[0058] The purpose of this example is to investigate the effect of different concentrations of valeric acid on the accumulation of PHBV by Halomonas venusta and to select the most suitable mixed substrate for Halomonas venusta to accumulate PHBV in combination with Examples 1, 2 and 3. The valeric acid concentration is set according to Table 4, the extraction time of the fermentation broth is 96 h, and the other methods are the same as in Example 1.

[0059] The PHBV production obtained under different valeric acid concentrations is shown in Table 4.

[0060] Table 4 Accumulation of PHBV by Halomonas venusta under different valeric acid concentrations.

[0061] The results show that under the condition of valeric acid concentration of 10-30 g / L, Halomonas venusta can grow and accumulate PHBV under the condition of valeric acid concentration of 10-30 g / L. In addition, with the increase of valeric acid concentration, the DCW, the PHBV yield and the PHBV content show a trend of first increasing and then decreasing, wherein when the valeric acid concentration is 15 g / L, the DCW and the PHBV yield are the highest, which are 1.70 g / L and 1.01 g / L, respectively, and the PHBV content is 59.45 wt%. When the valeric acid concentration is 10-20 g / L, the molar proportion of 3-HV monomer does not change significantly, which can reach a maximum of 55.01 mol%, but the problem is that the PHBV yield is not high, which also leads to a low 3-HV yield. In addition, with the increase of valeric acid concentration, the utilization rate of valeric acid by the strain does not change significantly, which can all reach more than 70%.

[0062] Example 5

[0063] According to the embodiments 1, 2, 3, 4, it is found that the optimal VFAs for the strain is butyric acid, and the optimal concentration of butyric acid is 30 g / L. The embodiments 2 and 4 show that pentanoic acid can more effectively support the incorporation of 3-HV monomers in Halomonas venusta than propionic acid. Therefore, in order to increase the yield of 3-HV while maintaining a high proportion of 3-HV monomers, this embodiment selects butyric acid and pentanoic acid as mixed substrates to investigate the effect of different proportions of butyric acid and pentanoic acid on the accumulation of PHBV by Halomonas venusta. The concentrations of pentanoic acid and butyric acid are set according to the contents of Table 5, and the total fermentation time is 84 h. Other methods are the same as those in embodiment 1.

[0064] The PHBV production obtained by different proportions of butyric acid and pentanoic acid is shown in Table 5.

[0065] Table 5 Effect of different proportions of butyric acid and pentanoic acid on the accumulation of PHBV by Halomonas venusta.

[0066] The results show that as the proportion of pentanoic acid in the total acid increases, although the DCW shows a downward trend, the PHBV yield and PHBV content are increasing, and when the proportion of pentanoic acid to butyric acid is 2:1, the PHBV yield and content are the highest, being 1.68 g / L and 90.81 wt%, respectively. At the same time, when the proportion of pentanoic acid to butyric acid is 2:1, the proportion of 3-HV monomers reaches 49.81 mol%, and the 3-HV yield is 0.83 g / L. The proportion of 3-HV monomers and the yield are significantly improved compared with pure propionic acid and pure pentanoic acid fermentation. In addition, the utilization rate of the strain to the mixed acid changes insignificantly with the change of the proportion of the mixed acid, and can reach more than 75%. Therefore, the addition of a certain amount of butyric acid in the pentanoic acid fermentation system can further promote the accumulation of PHBV by Halomonas venusta and effectively improve the proportion and yield of 3-HV in PHBV.

[0067] The above embodiments only express several embodiments of the present application, which are more specific and detailed, but cannot be understood as limiting the protection scope of the present application. It should be said that, for those skilled in the art, on the basis of the concept of the present application, many modifications and improvements can be made, which all belong to the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. A method for synthesizing a high proportion of 3-hydroxyvalerate monomers from *Halomonas venusta* using volatile fatty acids, characterized in that... The method uses volatile fatty acids as the carbon source, and the carbon-nitrogen ratio in the fermentation medium is (10-100):1; the carbon-phosphorus ratio is (100-1000):1; the NaCl concentration is 20-100 g / L; and the pH value is 4-9.

2. The method according to claim 1, characterized in that, The carbon-nitrogen ratio is (10-50):1; the carbon-phosphorus ratio is (100-1000):

1.

3. The method according to claim 1, characterized in that, The carbon source is one or two of acetic acid, propionic acid, butyric acid or valeric acid, and the concentration in the culture medium is 1-60 g / L.

4. The method according to claim 1, characterized in that, The fermentation medium consists of the following components: acetic acid 10-30 g / L (propionic acid 5-20 g / L, butyric acid 10-40 g / L, valeric acid 10-30 g / L, butyric acid + valeric acid 30 g / L), peptone 1-2 g / L, yeast extract 1-2 g / L, sodium chloride 50-70 g / L, magnesium chloride hexahydrate 5.4-7 g / L, magnesium sulfate heptahydrate 6.8 g / L, anhydrous calcium chloride 1.4-2.8 g / L, potassium chloride 0.7-1.4 g / L, sodium bicarbonate 0.2 g / L, and pH 6-8.

5. The method according to claim 1, characterized in that, Inoculate the strain into 250ml or 500ml Erlenmeyer flasks at an inoculation rate of 2-5% (v / v).

6. The method according to claim 1, characterized in that, The fermentation conditions were: fermentation temperature 27–30℃, rotation speed 140–190 rpm / min, and fermentation time 72–120 h.

7. The application according to claim 3, characterized in that, Polyhydroxy fatty acid esters were isolated from the strain using sulfuric acid, methanol, and chloroform or dichloromethane, and then quantitatively analyzed using external standard or internal standard methods.

8. The application of the method according to claims 1 to 7 in the production of polyhydroxyalkanoates (PHA).

9. The application according to claim 8, characterized in that, The application is specifically in the production of polyhydroxybutyrate (PHB) or polyhydroxybutyrate valerate (PHBV).

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