Vibrio sodium-requiring strain utilizing ethanol, and construction method and application thereof

Abstract

The application discloses an ethanol-utilizing Vibrio natriegens strain, which is obtained by domesticating and culturing a starting strain of Vibrio natriegens (ATCC 14048) in an M9 medium added with ethanol for multiple generations to obtain ethanol-utilizing capacity. The application also discloses a construction method of the strain. The application further discloses a 3-hydroxypropionic acid production strain and application thereof to fermenting and producing 3-hydroxypropionic acid. The application selects non-food carbon source ethanol as a substitute for food-based materials such as glucose as a substrate, which is cheap, easy to obtain and widely available, and can reduce the use of fossil fuels. Meanwhile, the process of biosynthesizing 3-HP by using the substrate ethanol belongs to a carbon fixation step, and the carbon fixation can reduce the emission of free carbon to a certain extent, thereby providing a feasible solution for treating excessive carbon emission.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically, it relates to an ethanol-utilizing sodium-dependent Vibrio strain that produces 3-hydroxypropionic acid from ethanol, its construction method, and its application. Background Technology

[0002] In recent years, renewable and non-food carbon sources such as formic acid, acetic acid, crude glycerol, methane, methanol, ethanol, and syngas have been increasingly developed as alternative carbon sources for industrial biotechnology. Ethanol is a common and inexpensive non-food carbon source that can be produced through biological or chemical methods. Ethanol can be converted into acetyl-CoA by microorganisms, serving as an alternative substrate for the production of acetyl-CoA with high atom economy, and further producing acetyl-CoA-derived chemicals such as sterols, fatty acids, 3-hydroxypropionic acid, poly(3-hydroxybutyric acid), and amino acids.

[0003] *Vibrio natriegens* is a Gram-negative marine bacterium capable of growing in high-salt environments. It is a novel chassis cell that has emerged in recent years for application in biotechnology and synthetic biology. *V. natriegens* exhibits an extremely rapid growth rate, and its broad substrate spectrum and rapid substrate uptake are crucial. Studies have shown that *V. natriegens* can utilize more than dozens of common carbon sources, including D-glucose, D-mannose, glycerol, rhamnose, sucrose, D-mannitol, N-acetylglucosamine glucose, gluconic acid, heptaate, malic acid, and phenylacetic acid, on minimum-salt media, but cannot grow using ethanol. Based on the excellent biological characteristics of *Vibrio natriegens*, various natural products and proteins have been efficiently biosynthesized within it.

[0004] 3-Hydroxypropionic acid (3-HP) is a three-carbon achiral organic acid, a structural isomer of lactic acid, but with more reactive chemical properties. As one of the most promising chemical products of the 21st century, 3-hydroxypropionic acid can be used to produce a wide variety of chemical derivatives, such as acrylic acid, 1,3-propanediol, methacrylates, acrylamide, malonic acid, and so on.

[0005] Currently, the main methods for producing 3-hydroxypropionic acid include chemical synthesis and biosynthesis. Chemical synthesis methods, such as propionic acid hydrolysis and the 2-cyanoethanol acid-base method, cause significant environmental pollution. Biosynthesis, on the other hand, utilizes inexpensive biomass resources and offers advantages such as mild reaction conditions, simple operation, fewer byproducts, environmental friendliness, and lower production costs. Traditional biosynthesis methods for 3-hydroxypropionic acid primarily use glucose and glycerol as substrates for fermentation. However, utilizing non-grain carbon source ethanol to produce 3-hydroxypropionic acid can effectively overcome many obstacles related to raw material innovation, cost-effectiveness, and large-scale production by developing and utilizing non-grain biomass. Summary of the Invention

[0006] The inventors of this case discovered through long-term research that by adaptively acclimating sodium-dependent Vibrio strains, that is, by continuously subculturing sodium-dependent Vibrio strains in a medium supplemented with ethanol, the beneficial mutations and traits of sodium-dependent Vibrio in terms of ethanol uptake and utilization pathways can be accumulated. This not only improves the growth of sodium-dependent Vibrio in ethanol medium, but also improves the ability to produce products using ethanol as a substrate. Furthermore, this acclimation method is reproducible.

[0007] Therefore, in a first aspect, the present invention provides an ethanol-utilizing sodium-dependent Vibrio strain, which is obtained by acclimatizing and cultivating sodium-dependent Vibrio (V. natriegens) ATCC 14048 as the starting strain in M9 medium supplemented with ethanol for multiple generations to develop ethanol utilization ability.

[0008] According to a preferred embodiment of the present invention, the strain is Vibrio natriegens VYII-28D1, with accession number CCTCC M 20232715.

[0009] A second aspect of the present invention provides a method for constructing the above-mentioned ethanol-utilizing sodium-dependent Vibrio strain, comprising the following steps:

[0010] S1: Inoculate Vibrio natriegens ATCC 14048 into M9 medium supplemented with ethanol and culture until the strain grows, then perform continuous subculture.

[0011] S2: When the average growth rate of the strains from 0 to 24 h no longer changes, increase the concentration of ethanol in the culture medium to increase the screening pressure, and the resulting strains will continue to be passaged.

[0012] S3: Repeat step S2 until the desired ethanol-tolerant strain is obtained.

[0013] A third aspect of the present invention provides a 3-hydroxypropionic acid producing strain, wherein the producing strain is obtained by transforming the above-mentioned ethanol-utilizing sodium-dependent Vibrio strain with key enzymes from Chloroflexus aurantiacus and Corynebacterium glutamicum in the 3-hydroxypropionic acid production pathway, namely acetyl-CoA carboxylase and malonyl-CoA reductase for the production of 3-hydroxypropionic acid from acetyl-CoA, thereby constructing an exogenous 3-HP synthetic pathway.

[0014] According to the present invention, the acetyl-CoA carboxylase is encoded by the dtsR1 and accBC genes derived from Corynebacterium glutamicum, the sequences of which are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively.

[0015] According to the present invention, the malonyl-CoA reductase is encoded by a mutated mcr* gene derived from Chloroflexus aurantiacus, the sequence of which is shown in SEQ ID No. 3.

[0016] In a fourth aspect, the present invention provides the application of the above-described 3-hydroxypropionic acid producing strain for the fermentation production of 3-hydroxypropionic acid.

[0017] A fifth aspect of the present invention provides a method for producing 3-hydroxypropionic acid, wherein the above-described 3-hydroxypropionic acid producing strain is used for fermentation.

[0018] According to a preferred embodiment of the present invention, the fermentation medium is M9 medium supplemented with biotin, and ethanol is also added to the medium. The culture conditions are 30°C and 220 rpm. During the culture process, IPTG is added as an inducer to induce the fermentation, and NaOH or H2SO4 is used to maintain the pH value at around 7.0.

[0019] According to another preferred embodiment of the invention, yeast extract is also added to the fermented M9 culture medium.

[0020] The present invention has the following beneficial effects:

[0021] 1. This invention obtained sodium-dependent Vibrio vYⅡ-28D1, which can use ethanol as the sole carbon source to grow in a basal salt medium, through laboratory adaptive domestication.

[0022] 2. The constructed exogenous expression pathway for 3-hydroxypropionic acid was transformed into VYⅡ-28D1, and the resulting strain VYⅡ-28D1(M*DA) was able to produce 3-hydroxypropionic acid in a medium with ethanol as the carbon source.

[0023] 3. Using ethanol—a non-grain-based carbon source—as a substrate to replace grain-based materials such as glucose is inexpensive, readily available, and widely sourced, which can reduce the use of fossil fuels. At the same time, the process of biosynthesizing 3-HP using ethanol as a substrate is a carbon fixation step, and carbon fixation will reduce the emission of free carbon to a certain extent, providing a feasible solution for dealing with excessive carbon emissions. Attached Figure Description

[0024] Figure 1 This is a metabolic diagram of sodium-dependent Vibrio spp. producing 3-hydroxypropionic acid (3-HP) from ethanol. In the diagram: adhP is alcohol dehydrogenase, mhpF is aldehyde dehydrogenase, adhE is alcohol dehydrogenase / aldehyde dehydrogenase, acc is acetyl-CoA carboxylase, mcr is malonyl-CoA reductase, CIT is citrate, ICIT is isocitrate, AKG is α-ketoglutarate, SUCCoA is succinylo-CoA, SUC is succinate, MAL is malate, OAA is oxaloacetate, and FUM is fumarate.

[0025] Figure 2 The growth curves of ethanol-utilizing strain VYⅡ-28 and sodium-dependent Vibrio wild-type strain Vna are shown in M9 basal salt medium containing 20 g / L ethanol.

[0026] Figure 3 This is a gel image used to verify the transformation of the 3-HP plasmid.

[0027] Preservation Matters

[0028] The ethanol-utilizing strain VYⅡ-28D1 obtained in this invention was deposited on December 29, 2023, at the China Center for Type Culture Collection (CCTCC) of Wuhan University, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCM20232715. Detailed Implementation

[0029] The present invention will be described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are all available through conventional commercial channels.

[0031] In the following examples, the pMB1 plasmid was constructed using the method disclosed in (Wu Fengli, Liang Yanxia, ​​Zhang Yuanyuan, Huo Yanan, Wang Qinhong. Construction of a novel, fast-growing, sodium-dependent Vibrio genome editing system [J]. Chinese Journal of Biotechnology, 2020, 36(11):11).

[0032] The culture medium formulations used in the following examples are as follows:

[0033] LB3 medium composition (per liter): 10g tryptone, 5g yeast extract (YE), 30g NaCl. The amount of agar powder added to the solid medium is 1.5%.

[0034] M9 culture medium composition (per liter): Na2HPO4·12H2O 15.12g, KH2PO4 3g, NaCl 15g, MgSO4·7H2O 0.49g, CaCl2 0.011g, NH4Cl 1g, and 1% vitamin B1 1mL, and trace elements 0.1mL.

[0035] Cell concentration was determined by spectrophotometry at 600 nm.

[0036] Example 1: Construction of ethanol-utilizing strains

[0037] *V. natriegens* ATCC 14048 (wild-type Vna) was cultured in M9 medium supplemented with 10 g / L ethanol at 30°C and 220 rpm to enable its growth in ethanol-rich media. After 120 hours of wild-type culture, the strain emerged and was then subjected to continuous subculturing to improve its tolerance to and ability to utilize ethanol. The specific procedures for continuous subculturing are as follows:

[0038] Glyceryl bacteria of *V. natriegens* ATCC 14048 (containing 50% glycerol, stored at -80℃) were streaked onto LB3 agar plates and incubated overnight at 30℃. Single colonies from the plates were inoculated into tubes containing 5 mL of LB3 liquid medium and incubated overnight on a shaker at 30℃ and 220 rpm. The wild-type bacterial culture was then transferred to a 250 mL Erlenmeyer flask containing 50 mL of M9 medium and incubated on a shaker with 10 g / L ethanol at an inoculation rate of 1%. Cell concentration (OD) was measured every 12 hours. 600 The pH value was maintained at 7 using 3MH2SO4 during the culture process. After 24-48 hours of culture (entering the stable growth phase), glycerol bacteria were prepared for preservation. The culture conditions were 30℃ and 220 rpm. This process was the first subculture.

[0039] Subsequently, the bacterial culture was transferred to another 250mL Erlenmeyer flask containing 50mL of M9 medium. Ethanol was added at a concentration of 10g / L, with an inoculum size of 1%. After culturing for 24-48 hours (entering the stationary phase), glycerol culture was prepared for preservation at 30℃ and 220rpm. Cell concentration (OD) was measured every 12 hours.600 The pH value was maintained at 7 using 3MH2SO4 during the second subculture.

[0040] Subsequently, continuous subculturing was performed using this method, with culture conditions of 30℃ and 220 rpm. 10-20 g / L ethanol was added to the M9 medium used for subculturing. When the average growth rate of the strain no longer changed over multiple generations (0-24 h), the ethanol concentration in the medium was increased to increase the selection pressure.

[0041] Following the above method, the culture was passaged 28 times in 20 g / L ethanol medium, and the resulting mixed strain was named VYⅡ-28.

[0042] The mixed strain VYⅡ-28 was cultured in a 96-well plate with shaking for 48 hours at 30℃. The absorbance at 600 nm was measured every half hour. The experiment was performed in triplicate, with wild-type strain Vna as a control. Results are as follows: Figure 2 As shown.

[0043] Figure 2 The results showed that the mixed strain VYⅡ-28 could grow normally in 20 g / L ethanol basal salt medium, while the wild-type strain Vna did not grow.

[0044] A single colony of the mixed strain VYⅡ-28 was isolated by streaking on an LB3 agar plate and named VYⅡ-28D1. This strain VYⅡ-28D1 was deposited at the China Center for Type Culture Collection (CCTCC) on December 29, 2023, with accession number CCTCC M 20232715.

[0045] Example 2: Construction of 3-hydroxypropionic acid producing strain

[0046] This embodiment uses the pMB1 plasmid as a vector to simultaneously overexpress key enzymes in the 3-hydroxypropionic acid (3-HP) production pathway derived from *Chloroflexus aurantiacus* and *Corynebacterium glutamicum*, namely acetyl-CoA carboxylase and malonyl-CoA reductase, to construct an exogenous 3-HP synthesis pathway within sodium-dependent *Vibrio natriureticus*. The metabolic diagram of 3-HP production from ethanol in sodium-dependent *Vibrio natriureticus* is shown below. Figure 1 As shown. The specific construction process is as follows:

[0047] The acetyl-CoA carboxylase is encoded by the dtsR1 and accBC genes derived from Corynebacterium glutamicum, the sequences of which are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively. The malonyl-CoA reductase is encoded by the mutated mcr* gene derived from Chloroflexus aurantiacus, and the codons have been optimized. The mutated mcr* gene has mutations at three sites in the mcr gene, namely N940V, K1106W, and S1114R, the sequences of which are shown in SEQ ID No. 3.

[0048] Using a one-step cloning kit (Nanjing Novozymes), the acetyl-CoA carboxylase genes dtsR1 and accBC (SEQ ID No. 1 and SEQ ID No. 2) and the malonyl-CoA reductase gene mcr* (SEQ ID No. 3) were cloned into plasmid pMB1 to construct the recombinant plasmid pMB1-M*DA. Then, the recombinant plasmid pMB1-M*DA (production plasmid) was transformed into VYⅡ-28D1 using electroporation. The obtained genetically engineered bacteria were verified as correct by colony PCR. The colony PCR verification gel image is shown below. Figure 3 As shown, the theoretical size of the fragment is 7584 bp, which is consistent with the result in the image, proving that the 3-HP production plasmid has been successfully transformed into VYⅡ-28D1, and the resulting genetically engineered bacteria is named VYⅡ-28D1(M*DA).

[0049] Example 3: Shake-flask fermentation of genetically engineered bacteria

[0050] Single colonies of the genetically engineered bacterium VYⅡ-28D1(M*DA) obtained in Example 2 were picked from the plate and inoculated into test tubes containing 5 mL of LB3 medium and cultured overnight. Then, the colonies were transferred to Erlenmeyer flasks containing 50 mL of LB3 medium, with an inoculation volume of 1%. The culture conditions were 30°C and 220 rpm. After 12 hours of secondary seed culture, the culture was transferred to shake-flask fermentation medium (M9 medium) for further cultivation. The M9 medium contained 40 mg / L biotin, with an inoculation volume of 1%, and the culture conditions were 30°C and 220 rpm. IPTG was added at 0 h to a final concentration of 0.25 mM. During cultivation, the pH was maintained at 7.0 using 6 M NaOH or 3 M H2SO4. Antibiotics (zirconia at a final concentration of 200 mg / L) were added to both the seed culture and the fermentation medium.

[0051] During shake-flask fermentation, samples were taken at 12-hour intervals to determine ethanol consumption and 3-hydroxypropionic acid yield, as detailed below:

[0052] The sample was centrifuged at 12,000 rpm for 10 minutes to separate the bacterial cells and supernatant. The supernatant was filtered through a 0.22 μm microporous membrane, and the consumption of 3-hydroxypropionic acid and ethanol in the fermentation broth supernatant was monitored using a Shimadzu high-performance liquid chromatograph.

[0053] The chromatographic column was a BioRadAminex HPX-87 ion chromatography column (300 mm * 7.8 mm), equipped with a UV detector and a differential refractive index detector. The mobile phase was 5 mM H₂SO₄, the flow rate was 0.6 mL / min, and the column temperature was 65 °C.

[0054] The test results are shown in Table 2.

[0055] Table 2: Yield and 3-hydroxypropionic acid production rate of ethanol-evolving bacteria

[0056]

[0057] VYⅡ-28D1(M*DA) was fermented to produce 3-HP in M9 medium supplemented with 10 g / L ethanol and in medium supplemented with an additional 5 g / L YE. The fermentation period was 60 h, with samples taken every 12 h. Without YE, the OD value of the strain reached the end of fermentation. 600 The concentration of ethanol was 4.45, indicating that not all ethanol was consumed. The yield of 3-HP was 0.856 g / L, with a yield of 0.108 g / g. Adding yeast improved the growth of the strain, and the OD value at the end of fermentation was [not specified]. 600 The yield was 9.88, all ethanol was consumed, and both the yield and output increased. The 3-HP yield was 1.379 g / L, and the output was 0.135 g / g.

[0058]

[0059]

[0060]

[0061]

[0062]

Claims

1. An ethanol-utilizing sodium-dependent Vibrio strain, characterized in that, The strain is a sodium-dependent Vibrio ( V. natriegens ATCC 14048 was the starting strain, obtained through multiple generations of domestication and cultivation in M9 medium supplemented with ethanol to develop its ethanol utilization ability. Vibrio natriegens VYII-28D1, its accession number is CCTCC NO:M 20232715.

2. A strain for producing 3-hydroxypropionic acid, characterized in that, The production strain is derived from the ethanol-utilizing sodium-demanding Vibrio strain of claim 1. Corynebacterium glutamicum and Chloroflexus aurantiacus The key enzymes in the 3-hydroxypropionic acid production pathway are obtained by constructing an exogenous 3-HP synthetic pathway, namely acetyl-CoA carboxylase and malonyl-CoA reductase, which generate 3-hydroxypropionic acid from acetyl-CoA.

3. The 3-hydroxypropionic acid producing strain according to claim 2, characterized in that, The acetyl-CoA carboxylase is derived from... Corynebacterium glutamicum of dtsR1 and accBC Gene encoding, the dtsR1 and accBC The gene sequences are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively.

4. The 3-hydroxypropionic acid producing strain according to claim 2, characterized in that, The malonyl-CoA reductase is derived from... Chloroflexus aurantiacus And after mutation mcr *The gene is encoded, and its sequence is shown in SEQ ID No.

3.

5. The application of the 3-hydroxypropionic acid producing strain according to any one of claims 2 to 4, characterized in that, Used for fermentation production of 3-hydroxypropionic acid.

6. A method for producing 3-hydroxypropionic acid, characterized in that, Fermentation was carried out using the 3-hydroxypropionic acid producing strain according to any one of claims 2 to 4.

7. The production method according to claim 6, characterized in that, The fermentation medium was M9 medium supplemented with biotin, and ethanol was also added to the medium. The culture conditions were 30°C and 220 rpm. During the culture, IPTG was added as an inducer to induce the fermentation, and NaOH or H2SO4 was used to maintain the pH value at around 7.

0.

8. The production method according to claim 7, characterized in that, Yeast extract was also added to the M9 culture medium used for fermentation.

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