Enterococcus italicus producing fatty alcohol and its application

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to Enterococcus italicus for producing fatty alcohols and its applications. Aiming at the problems existing in the production of fatty alcohols by existing microorganisms through fermentation, such as low yield, slow strain improvement, cumbersome detection process, physiological toxicity, and unreasonable selection of metabolic pathways, the present invention provides a strain of Enterococcus italicus for producing fatty alcohols, with the preservation number of CGMCC NO.25136. This strain can simultaneously produce n-heptanol, 1-dodecanol, 1-pentadecanol, 1-hexadecanol, and 1-nonadecanol, providing a strain selection for the preparation of fatty alcohols by microorganisms, and facilitating the research on the synthesis pathway of higher fatty alcohols to improve the ability of engineered bacteria to synthesize fatty alcohols.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to Enterococcus italicus producing fatty alcohols and its applications. Background Art

[0002] Fatty alcohols refer to alcohols in which the hydroxyl group is connected to a fatty hydrocarbon group. Usually, those containing 1-2 carbon atoms are called low-carbon fatty alcohols or lower alcohols; those with 3-5 carbon atoms are medium-carbon fatty alcohols or intermediate alcohols; and those with 6 or more carbon atoms are high-carbon fatty alcohols or higher alcohols. Among fatty alcohols, C6-C11 alcohols can be used as plasticizers, which have advantages such as good compatibility, smooth surface, less pollution, and increased service life when used in plasticizers for PVC, nitrocellulose, nitrile rubber, etc.

[0003] According to the preparation raw materials, they can be divided into natural fatty alcohols and synthetic fatty alcohols. Fatty alcohols prepared from natural animal and plant oils and fats as raw materials or through microbial engineering transformation belong to natural fatty alcohols, and fatty alcohols obtained by refining from petroleum and synthesizing through cracking are called synthetic fatty alcohols.

[0004] Natural fatty alcohols can be used as raw materials to produce surfactants. Alcohol-based surfactants have strong detergency, good low-temperature washing effect, biodegradability, and are green and non-toxic. They are the most widely used surfactants. The product market has a large demand, which provides the production demand for fatty alcohols, especially natural fatty alcohols. They can be used as bases for oily cosmetics and pharmaceuticals (medical ointments), improving compatibility and dispersibility, and being mild and less irritating. Among higher alcohols, for example, C22 alcohol has an inhibitory effect on prostate tumors; C24 alcohol can enhance the function of nerve factors; C26 alcohol can reduce blood lipids; C28 alcohol has effects such as anti-fatigue, blood lipid reduction, and enhancement of sexual function; C30 alcohol is a plant growth regulator, and also has the effect of promoting animal growth and can be used to treat skin eczema and herpes.

[0005] Synthetic fatty alcohols have been gradually replaced by natural fatty alcohols due to energy and environmental problems. The production raw materials of natural fatty alcohols mainly include coconut oil, palm oil, beef tallow, etc., and most of them rely on imports, increasing production costs. The microbial preparation method has advantages such as renewable raw material resources and environmental friendliness, and has become a research hotspot.

[0006] For example, Patent CN101899411A discloses an engineered Escherichia coli for preparing medium-carbon fatty alcohols and a method for preparing medium-carbon fatty alcohols. By overexpressing thioesterase genes, acyl-CoA reductase genes, etc. in Escherichia coli, Escherichia coli is enabled to have the ability to produce medium-carbon fatty alcohols.

[0007] Also, CN102311966B discloses a construct for synthesizing fatty alcohols in cyanobacteria and a method for producing fatty alcohols in cyanobacteria. The fatty alcohols that can be produced are 1-hexadecanol and 1-octadecanol, and the total production is 137.63 μg / L.

[0008] However, at present, there are problems in the production of fatty alcohols by microbial fermentation, such as low yield, slow strain improvement, cumbersome detection process, physiological toxicity, and unreasonable selection of metabolic pathways, which seriously restrict the industrialization process of microbial production of fatty alcohols, especially higher fatty alcohols. Summary of the Invention

[0009] In view of the problems existing in the production of fatty alcohols by existing microbial fermentation, such as low yield, slow strain improvement, cumbersome detection process, physiological toxicity, and unreasonable selection of metabolic pathways, it is of great significance to find new strains that can produce various fatty alcohol compounds, especially higher fatty alcohols, and study related functional genes, metabolic pathways, etc. to promote bioengineering transformation.

[0010] The present invention provides a strain of Enterococcus italicus that produces fatty alcohols, with the preservation number of CGMCC NO.25136. The preservation time is June 21, 2022, and the preservation center is the China General Microbiological Culture Collection Center CGMCC, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the postal code of 100101.

[0011] Among them, the 16S rDNA sequence of the above-mentioned Enterococcus italicus that produces fatty alcohols is shown as SEQ ID NO:1.

[0012] Among them, the growth conditions of the above-mentioned Enterococcus italicus that produces fatty alcohols are: the growth temperature is 10°C - 45°C, the tolerated pH is 9.6, and it can tolerate 5% NaCl. Preferably, the optimal temperature is 37°C.

[0013] Among them, the biological characteristics of the above-mentioned Enterococcus italicus that produces fatty alcohols are: Gram-positive facultative anaerobic cocci, paired or in chains, without flagella and not producing spores.

[0014] Among them, the above-mentioned Enterococcus italicus that produces fatty alcohols can simultaneously produce n-heptanol, 1-dodecanol (lauryl alcohol), 1-pentadecanol, 1-hexadecanol (cetyl alcohol), and 1-nonadecanol.

[0015] Among them, the contents of n-heptanol, 1-dodecanol (lauryl alcohol), 1-pentadecanol, 1-hexadecanol (cetyl alcohol), and 1-nonadecanol produced by the above-mentioned Enterococcus italicus that produces fatty alcohols are 1.087, 0.476, 0.578, 3.014, and 0.639 mg / L respectively, accounting for 92.2% of the total amount of its volatile alcohol substances.

[0016] The present invention also provides the application of the above-mentioned Enterococcus italicus that produces fatty alcohols in the production of fatty alcohols.

[0017] Among them, in the said application, Enterococcus italicus is statically cultured in a fermentation medium at 37°C for 36 h, and the pH value is 6.0. Preferably, the inoculation amount of Enterococcus italicus is 2% (×10 7 cfu / mL).

[0018] Among them, the composition of the fermentation medium is that each liter of the medium contains 16 g of glucose, 10 g of tryptone, 2.5 g of yeast powder, 5 g of beef extract, 3.85 g of bovine brain, 4.9 g of bovine heart, 0.5 g of Tween 80, 1 g of ammonium citrate, 2.5 g of sodium chloride, 2.5 g of anhydrous sodium acetate, 0.05 g of magnesium sulfate, 0.025 g of manganese sulfate, 1.25 g of disodium hydrogen phosphate, and 1 g of potassium hydrogen phosphate.

[0019] The present invention also provides the application of the above-mentioned Enterococcus italicus producing fatty alcohols in the brewing of Chinese liquor.

[0020] Beneficial effects:

[0021] The present invention isolates and purifies a strain of Enterococcus italicus from the Luzhou-flavor Chinese liquor pit mud sample, which can simultaneously produce fatty alcohols such as n-heptanol, 1-dodecanol (lauryl alcohol), 1-pentadecanol, 1-hexadecanol (cetyl alcohol), 1-nonadecanol, etc. The preservation number is CGMCC NO.25136. In the fermentation medium, the contents of n-heptanol, 1-dodecanol (lauryl alcohol), 1-pentadecanol, 1-hexadecanol (cetyl alcohol), and 1-nonadecanol produced by this strain are 1.087, 0.476, 0.578, 3.014, and 0.639 mg / L respectively, accounting for 92.2% of the total amount of its volatile alcohol substances. The strain of the present invention has not been reported in the existing strain resources, provides a strain selection for the preparation of fatty alcohols by microorganisms, and is conducive to promoting the research on the synthesis pathway of higher fatty alcohols to improve the ability of engineering bacteria to synthesize fatty alcohols.

[0022] The strain of Enterococcus italicus producing fatty alcohols provided by the present invention has a preservation number of CGMCC NO.25136. The preservation time is June 21, 2022. The preservation center is the China General Microbiological Culture Collection Center CGMCC, with the address of Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the postal code is 100101. The taxonomic name is Enterococcus italicus. Specific embodiments

[0023] Aiming at the deficiencies of the prior art, the present invention provides a strain of Enterococcus italicus capable of producing fatty alcohols, which has the ability to produce a variety of fatty alcohol compounds and mainly produces higher fatty alcohols, providing a new available microorganism for the biological preparation of fatty alcohols.

[0024] The strain of Enterococcus italicus capable of producing fatty alcohols has a preservation number of CGMCC NO. 25136. The preservation time is June 21, 2022, and the preservation center is the China General Microbiological Culture Collection Center CGMCC, with the address being Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101.

[0025] Among them, the 16S rDNA sequence of the above-mentioned Enterococcus italicus capable of producing fatty alcohols is as shown in SEQ ID NO: 1.

[0026] The 16S rDNA sequence of Enterococcus italicus capable of producing fatty alcohols is as shown in SEQ ID NO: 1:

[0027]

[0028] Among them, the growth conditions of the Enterococcus italicus producing fatty alcohols are as follows: the growth temperature is 10°C - 45°C, the optimum temperature is 37°C, the tolerated pH is 9.6, and the tolerance to 5% NaCl is also considered.

[0029] Among them, the biological characteristics of the Enterococcus italicus producing fatty alcohols are as follows: Gram-positive facultative anaerobic cocci, paired or in chains, without flagella and not producing spores.

[0030] The present invention provides a method for screening and identifying Enterococcus italicus producing fatty alcohols, which includes the following steps: After the Luzhou-flavor liquor pit mud sample is dispersed, separated, purified by streaking, and the strains are preserved, using the ability to produce fatty alcohols as the screening criterion, Enterococcus italicus capable of producing various fatty alcohols is obtained.

[0031] Among them, after the sample is dispersed by shaking, the suspension is taken and diluted, and diluents of multiple gradients are spread on the solid medium plate for cultivation.

[0032] Among them, single colonies with different colony morphologies are picked from the plate with growing colonies, and all single colonies are purified by secondary streaking.

[0033] Among them, each purified strain is cultured in a liquid medium in the form of single colonies.

[0034] Among them, different strains are fermented to obtain fermentation broth, the target compounds are extracted and enriched by headspace solid-phase microextraction, and then chromatographic detection and analysis are carried out by GC-MS to screen and obtain Enterococcus italicus capable of producing various fatty alcohols by comparing volatile substances.

[0035] The present invention also provides the application of the Enterococcus italicus zqw24 in the production of fatty alcohols.

[0036] Specifically, in the application, Enterococcus italicus is statically cultured in a fermentation medium at 37°C for 36 h, the pH value is 6.0, and the inoculation amount of Enterococcus italicus is 2% (×10 7 cfu / mL).

[0037] More specifically, the composition of the fermentation medium is that each liter of the medium contains 16 g of glucose, 10 g of tryptone, 2.5 g of yeast extract, 5 g of beef extract, 3.85 g of bovine brain, 4.9 g of bovine heart, 0.5 g of Tween 80, 1 g of ammonium citrate, 2.5 g of sodium chloride, 2.5 g of anhydrous sodium acetate, 0.05 g of magnesium sulfate, 0.025 g of manganese sulfate, 1.25 g of disodium hydrogen phosphate, and 1 g of potassium hydrogen phosphate.

[0038] Using the above fermentation process, the contents of n-heptanol, 1-dodecanol (lauryl alcohol), 1-pentadecanol, 1-hexadecanol (cetyl alcohol), and 1-nonadecanol produced by this strain were 1.087, 0.476, 0.578, 3.014, and 0.639 mg / L respectively, accounting for 92.2% of the total amount of its volatile alcohol substances.

[0039] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those without specific technical or conditions noted in the embodiments, the techniques or conditions described in the literature in this field or according to the product specifications are followed. Those reagents or instruments without the manufacturer noted are all conventional products that can be obtained through commercial purchases.

[0040] The medium formulations involved in the embodiments:

[0041] MRS liquid medium: Glucose 20 g / L, peptone 10 g / L, yeast extract 4 g / L, beef extract powder 5 g / L, Tween 80 1 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, dipotassium hydrogen phosphate 1 g / L, pH 6.0, autoclaved at 115 °C for 20 minutes.

[0042] MRS solid medium: Add 15 g / L of agar to the MRS liquid medium and autoclave at 115 °C for 20 minutes.

[0043] Fermentation medium: Glucose 16 g / L, tryptone 10 g / L, yeast extract 2.5 g / L, beef extract 5 g / L, bovine brain 3.85 g / L, bovine heart 4.9 g / L, Tween 80 0.5 g / L, ammonium citrate 1 g / L, sodium chloride 2.5 g / L, anhydrous sodium acetate 2.5 g / L, magnesium sulfate 0.05 g / L, manganese sulfate 0.025 g / L, disodium hydrogen phosphate 1.25 g / L, dipotassium hydrogen phosphate 1 g / L, pH 6.0, autoclaved at 115 °C for 20 minutes.

[0044] Example 1: Isolation and purification of the strain

[0045] (1) Isolation method: Take 20 g of Luzhou Laojiao strong-flavor liquor pit mud sample and put it into a conical flask containing 180 mL of sterile distilled water. Oscillate in a constant-temperature shaker for 10 min to fully disperse and mix the sample. Take 1 mL of the sample suspension, and using the serial dilution method, dilute it to 10 -2 ~10 -7 , and draw 100 μL from each gradient of the dilution solution, evenly coat it on the MRS solid medium plate, prepare two plates in parallel, invert them, place them in an incubator at 37 °C for 36 - 48 h, and observe in time.

[0046] (2) Streak purification: Take out the petri dish with colonies grown, pick single colonies with different colony morphologies, and perform secondary streaking until all single colonies are purified.

[0047] (3) Strain preservation: Pick the single colonies of each strain after purification into 5 mL of MRS liquid medium, place it in a static culture at 37 °C for 24 h, pipette 1 mL of the bacterial solution into a preservation tube, add 0.5 mL of 60% sterile glycerol solution, resuspend, and store at -80 °C.

[0048] Example 2: Detection of fatty alcohol production capacity

[0049] (1) Prepare the bacterial solution to be tested:

[0050] After the glycerol preservation tubes of the screened strains are dissolved, inoculate them into MRS liquid medium respectively, and place them in a static culture at 37 °C for 24 h. After three generations of activation, inoculate them into 50 mL of MRS liquid medium at an inoculation amount of 2% (volume ratio) and culture for 24 h to obtain the bacterial solution to be tested.

[0051] (2) HS-SPME / GC-MS method for detecting fatty alcohols:

[0052] Using headspace solid-phase microextraction / gas chromatography-mass spectrometry technology: Take the supernatant, add it to a headspace vial, add saturated NaCl solution, keep the prepared sample at 60 °C for heat preservation and equilibration for 5 min, then extract with a 50 / 30 μm DVB / CAR / PDMS extraction head at 60 °C for 50 min. After the extraction is completed, desorb at 250 °C in the GC injection port for 5 min. The compound retrieval results are matched with the NIST standard spectral library, and the compound is confirmed as the target compound when the similarity reaches more than 80%.

[0053] GC-MS detection chromatographic conditions:

[0054] Gas chromatography conditions: HP-INNOWAX chromatographic column (60 m × 0.25 mm × 0.25 μm); temperature programming: initial temperature 40 °C, hold for 5 min, rise to 100 °C at 4 °C / min, then rise to 230 °C at 6 °C / min,

[0055] Hold for 10 min, the carrier gas is high-purity helium (1.0 mL / min); the injection port temperature is 250 °C, without splitting.

[0056] Mass spectrometry conditions: Electron ionization source, electron energy 70 eV; electron multiplier voltage 350 V; ion source temperature 230 °C; transfer line temperature 250 °C; mass range 40 - 450 m / z.

[0057] Using the above detection method, a lactic acid bacterium capable of producing multiple fatty alcohols was screened from the isolated strains, and this strain was selected for further study.

[0058] Example 3: Molecular Identification of the Strain

[0059] After the target strain was expanded and cultured, fresh bacterial liquid in the logarithmic growth phase was taken, the cells were collected by centrifugation, and genomic DNA was extracted using a bacterial genomic DNA extraction kit. The universal primers 27F / 1541R for lactic acid bacteria were used to amplify the full-length 16S rDNA sequence, as follows:

[0060] SEQ ID NO:2: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′)

[0061] SEQ ID NO:3: 1541R (5′-AAGGAGGTGATCCAGCC-3′)

[0062] ① Reaction system (50 μl)

[0063]

[0064] ② Reaction program

[0065]

[0066] The PCR products were separated and examined by 1.0% agarose gel electrophoresis at a voltage of about 11 V / cm for 20 min.

[0067] The purification of the PCR products was carried out according to the instructions of the small-scale gel recovery PCR product purification kit of Shanghai Sangon Biotech Co., Ltd., and the sequencing was completed by Shanghai Sangon Biotech Co., Ltd.

[0068] The gene sequence of the 16S rDNA fragment obtained by sequencing was aligned by BLAST of NCBI to determine the species information of the strain, and its sequence is shown as SEQ ID NO:1. It was identified as Enterococcus italicus and named Enterococcus italicus zqw24.

[0069] Example 4: Physiological and Biochemical Characteristics of the Strain

[0070] Enterococcus italicus zqw24 was inoculated on MRS solid medium and cultured statically at 37°C for 2 days, and then the colony and cell morphology of the strain were observed.

[0071] Determination of the suitable growth temperature: Take Enterococcus italicus zqw24 in the logarithmic growth phase and inoculate it into MRS liquid medium at an inoculation amount of 2%. After culturing at 5, 10, 15, 20, 30, 37, 45, 50°C for 18 h, measure the OD of the bacterial liquid600 Value;

[0072] pH value tolerance determination: Use 2M hydrochloric acid and 1M sodium hydroxide to adjust the pH of MRS liquid medium to 2.0, 3.0, 5.0, 6.0, 8.0, 9.0, 9.6, and 10.5 respectively. Take Enterococcus italicus zqw24 in the logarithmic growth phase and inoculate it at an inoculation amount of 2%. Mix well, and measure the OD of the bacterial liquid after culturing at 37°C for 18h. 600 Value;

[0073] NaCl tolerance ability detection: Take Enterococcus italicus zqw24 in the logarithmic growth phase and inoculate it at an inoculation amount of 2% into MRS liquid medium with NaCl mass fractions of 0%, 2%, 4%, 5%, 7%, 8%, and 9% (w / v) respectively. Mix well, and measure the OD of the bacterial liquid after culturing at 37°C for 18h. 600 Value.

[0074] The experimental results show that the growth conditions of Enterococcus italicus zqw24 are as follows: the growth temperature is 10°C - 45°C, the optimum temperature is 37°C, the tolerated pH is 9.6, and the tolerated NaCl is 5%.

[0075] The biological characteristics of Enterococcus italicus zqw24 are: Gram-positive facultative anaerobic cocci, paired or in chains, without flagella, and non-spore-forming.

[0076] Example 5: Fermentation culture of Enterococcus italicus

[0077] After dissolving the glycerol preservation tube of Enterococcus italicus zqw24, inoculate it into MRS liquid medium, and culture it statically at 37°C for 24h. After activating three generations, inoculate it into 50 mL of fermentation medium at an inoculation amount of 2% (volume ratio) and culture it for 36h. After the fermentation is completed, obtain the bacterial liquid, centrifuge it at 10000 rpm for 5 min, and take the supernatant. The detection method is the same as the GC-MS detection method in Example 2. Using 2-octanol at 0.822 mg / mL as the internal standard and the original medium as the blank control, calculate the content of each volatile substance.

[0078] The detection results of fatty alcohol content are shown in Table 1. Enterococcus italicus zqw24 can produce n-heptanol, 1-dodecanol (lauryl alcohol), 1-pentadecanol, 1-hexadecanol (cetyl alcohol), and 1-nonadecanol, with contents of 1.087, 0.476, 0.578, 3.014, and 0.639 mg / L respectively, accounting for 92.2% of the total amount of its volatile alcohol substances.

[0079] Table 1 Detection results of fatty alcohols in the fermentation broth of Enterococcus italicus zqw24

[0080] Fatty alcohol Content mg / L n-Heptanol 1.087 1-Dodecanol (Lauryl alcohol) 0.476 1-Pentadecanol 0.578 1-Hexadecanol (Cetyl alcohol) 3.014 1-Nonadecanol 0.639

Claims

1. Enterococcus italicus strain for producing fatty alcohol, characterized in that: The preservation number is CGMCC NO. 25136.

2. Application of the Enterococcus italicus for producing fatty alcohol described in claim 1 in the production of fatty alcohol.

3. According to the application described in claim 2, characterized in that: In the said application, Enterococcus italicus is statically cultured in a fermentation medium at 37 °C for 36 h, and the pH value is 6.

0.

4. According to the application described in claim 3, characterized in that: The inoculation amount of the said Enterococcus italicus is 2%.

5. According to the application described in claim 3 or 4, characterized in that: The composition of the said fermentation medium is that each liter of the medium contains 16 g of glucose, 10 g of tryptone, 2.5 g of yeast powder, 5 g of beef extract, 3.85 g of bovine brain, 4.9 g of bovine heart, 0.5 g of Tween 80, 1 g of ammonium citrate, 2.5 g of sodium chloride, 2.5 g of anhydrous sodium acetate, 0.05 g of magnesium sulfate, 0.025 g of manganese sulfate, 1.25 g of disodium hydrogen phosphate, and 1 g of potassium hydrogen phosphate.

6. Application of the Enterococcus italicus for producing fatty alcohol described in any one of claims 1 - 5 in the brewing of Chinese liquor.

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