A chalcoatricus capable of producing polyhydroxyalkanoate by using various carbon sources such as ethanol and its application

Abstract

The application discloses cupriavidus capable of producing polyhydroxyalkanoate by using various carbon sources such as ethanol and application thereof. Cupriavidus The strain sp.ZWJ01 has a preservation number of CGMCC No.37462 in the China General Microbiological Culture Collection Center. The strain can grow and accumulate poly-3-hydroxybutyric acid ester by using various substances such as ethanol, volatile fatty acid and sugar as carbon sources; can grow by using mixed carbon sources of ethanol+propionic acid or ethanol+valeric acid and accumulate poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid ester) in cells; and can grow by using mixed carbon sources of ethanol+gamma-butyrolactone or ethanol+1,4-butanediol and accumulate poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) in cells, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and biotechnology, specifically relating to a copper-loving bacterium that can produce polyhydroxy fatty acid esters using carbon sources such as ethanol and its applications. Background Technology

[0002] Polyhydroxyalkanoates (PHAs) are linear polymers composed of hydroxy fatty acid monomers synthesized by microorganisms under conditions of nitrogen, phosphorus, and other nutrient limitation but carbon source surplus. They serve as intracellular storage substances for carbon and energy. PHAs are environmentally friendly materials that can be completely degraded by microorganisms in nature, without causing long-term environmental pollution. Compared to traditional plastics, PHAs exhibit excellent biodegradability and biocompatibility, making them suitable for use as biomedical materials and biodegradable packaging materials in the development and manufacture of various environmentally friendly chemical products.

[0003] Poly(3-hydroxybutyrate) (PHB) has a simple and regular structure, resulting in high hardness and strength. However, it has very low elongation at break and poor flexibility. Furthermore, PHB is highly susceptible to degradation when heated to temperatures above its melting point by more than 10°C. with Poly(3-hydroxyvalerate) (PHBV) is a copolymer of 3-hydroxybutyric acid and 3-hydroxyvalerate. With increasing 3-hydroxyvalerate monomer content, the regularity and symmetry of the polymer molecular chain are disrupted, and the crystallinity decreases accordingly, improving its brittleness. Simultaneously, the melting point and glass transition temperature of the material shift towards lower temperatures, thus improving thermal stability and broadening the temperature window for melt processing. with 4-Hydroxybutyrate (P34HB) is a copolymer of 3-hydroxybutyric acid (3-hydroxybutyric acid) and 4-hydroxybutyric acid (4-hydroxybutyric acid). The incorporation of 4-hydroxybutyric acid monomer increases the elongation at break and tensile strength of the P34HB copolyester. Furthermore, since the polymerized 4-hydroxybutyric acid monomer is unbranched, it improves the degradation performance of the P34HB copolyester. Generally, in the microbial synthesis of P34HB, the carbon sources used to produce the 3-hydroxybutyric acid monomer include conventional glucose, fructose, and sucrose, while the incorporation of the 4-hydroxybutyric acid monomer typically requires structurally similar carbon sources such as γ-butyrolactone or 1,4-butanediol. In the microbial synthesis of PHBV, the incorporation of the 3-hydroxyvalerate monomer requires the addition of structurally related odd-numbered carbon sources such as propionic acid or valerate.

[0004] Although PHA has received widespread attention in academia and industry as a material with many excellent properties, its production cost remains higher than that of traditional petrochemical plastics, especially since raw material costs account for the majority of the total cost. Therefore, it is necessary to select an economical and suitable carbon source to effectively support strain growth and PHA synthesis. Ethanol can be obtained through fermentation of various biomass feedstocks or produced by technologies such as CO2 catalysis and electroreduction, and in recent years it has been increasingly regarded as a potential substrate for microbial production of high-value-added chemicals. Compared to other carbon sources, ethanol requires only a few enzymatic reaction steps to be converted into acetyl-CoA, simultaneously generating NADH without additional ATP consumption. This means that metabolic processes using ethanol as a substrate have higher atom economy, energy utilization efficiency, and theoretical yield of the target chemical. Furthermore, as a neutral molecule, ethanol does not cause changes in the pH of the fermentation system during consumption.

[0005] Copper-loving bacteria ( Cupriavidus PHA (Proteobacteria) is a type of Gram-negative bacterium belonging to the class Beta-Proteobacteria. It is generally short rod-shaped, reproduces by binary fission, and possesses peritrichous flagella. Currently, many studies have used strains of the genus *P.* for PHA synthesis. For example, Beaulieu et al. used sugarcane molasses as a strain. Cupriavidus necator The carbon source for growth and PHB production reached a cell dry weight of 17.07 g / L after 75 hours of fermentation, with a PHB content of 44 wt% (Applied and Environmental Microbiology, 1995, 61(1): 165-169). Obruca et al. obtained the carbon source through random mutagenesis. Cupriavidus killer The H16 mutant strain EO1 accumulated 4.9 g / L of PHBV after culturing for 70 hours in a medium supplemented with 20 g / L waste frying oil and 5 g / L sodium propionate (World Journal of Microbiology and Biotechnology, 2013, 29: 2417-2428). In recent years, through metabolic engineering and screening of novel strains, microorganisms have made some progress in expanding their carbon source utilization and PHA synthesis capabilities. However, research on the synthesis of polyhydroxyalkanoates with different structures from multiple carbon sources such as ethanol and ethylene glycol by *Copper-loving bacteria* remains relatively limited, and there is still room for further optimization in terms of synergistic utilization of multiple substrates and polymer structure regulation. Summary of the Invention

[0006] The primary objective of this invention is to provide a copper-loving bacterium ZWJ01 ( Cupriavidussp. ZWJ01, a copper-loving bacterium, can utilize one or more of ethanol, ethylene glycol, glycerol, acetic acid, propionic acid, butyric acid, valeric acid, glucose, fructose, sucrose, xylose, γ-butyrolactone, and 1,4-butanediol as carbon sources to synthesize various polyhydroxy fatty acid esters with different structures, including poly-3-hydroxybutyrate, poly(3-hydroxybutyrate-... with 3-hydroxyvalerate) and poly(3-hydroxybutyrate- with 4-hydroxybutyrate (4-hydroxybutyrate) has significant industrial application value.

[0007] The second objective of this invention is to provide the application of the aforementioned copper-loving bacterium ZWJ01 in the synthesis of polyhydroxy fatty acid esters.

[0008] A third objective of the present invention is to provide a bacterial agent containing the above-mentioned strains.

[0009] A fourth objective of this invention is to provide the application of the above-mentioned microbial agent in the synthesis of polyhydroxy fatty acid esters.

[0010] The fifth objective of this invention is to provide a method for preparing polyhydroxy fatty acid esters using the aforementioned copper-loving bacterium ZWJ01 and / or bacterial agent.

[0011] Specifically, the copper-loving bacteria that produce polyhydroxy fatty acid esters provided by this invention ( Cupriavidus sp.) ZWJ01, with accession number CGMCC No. 37462.

[0012] This invention provides the application of the aforementioned copper-loving bacterium ZWJ01 in the synthesis of polyhydroxy fatty acid esters.

[0013] Furthermore, the polyhydroxy fatty acid ester is selected from poly-3-hydroxybutyrate, poly(3-hydroxybutyrate-... with 3-hydroxyvalerate) and poly(3-hydroxybutyrate- with One of the following: 4-hydroxybutyrate.

[0014] The bacterial agent provided by this invention includes the aforementioned copper-loving bacterium ZWJ01.

[0015] The preparation method of the bacterial agent includes the following steps: inoculating copper-loving bacteria ZWJ01 into a bacterial culture medium for cultivation, and the obtained bacterial solution is the bacterial agent.

[0016] The bacterial culture medium can be MS liquid medium or LB liquid medium.

[0017] In the preparation method of the bacterial agent, the culture conditions can be: 25-37℃, 100-300 rpm for 12-48h.

[0018] This invention provides the application of the above-mentioned microbial agent in the synthesis of polyhydroxy fatty acid esters.

[0019] The method for preparing polyhydroxy fatty acid esters using the above-mentioned copper-loving bacterium ZWJ01 and / or strain provided by the present invention includes: inoculating the copper-loving bacterium ZWJ01 and / or bacterial agent into a fermentation medium for fermentation culture to obtain the polyhydroxy fatty acid ester.

[0020] Preservation Instructions The strain provided by this invention has the following preservation date: January 19, 2026; preservation number: CGMCC No. 37462; classification and nomenclature: *Copper-loving Bacterium* (…). Cupriavidus sp.) ZWJ01; Name of depositary institution: China General Microbiological Culture Collection Center; Address: No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing.

[0021] The beneficial effects of this invention include: the copper-loving bacterium ZWJ01 provided by this invention can synthesize a variety of polyhydroxy fatty acid esters with different structures, including poly-3-hydroxybutyrate, poly(3-hydroxybutyrate-... with 3-hydroxyvalerate) and poly(3-hydroxybutyrate- with (4-Hydroxybutyrate). Compared with existing technologies, the copper-producing bacterium ZWJ01 can utilize multiple carbon sources such as ethanol, ethylene glycol, glycerol, acetic acid, propionic acid, butyric acid, valeric acid, glucose, fructose, sucrose, xylose, γ-butyrolactone, and 1,4-butanediol. In particular, when using ethanol as a carbon source, the copper-producing bacterium ZWJ01 in the fermentation broth can accumulate a large number of polyhydroxy fatty acid ester particles in its cells, indicating that it has a strong ability to utilize ethanol and synthesize PHA. Compared with existing copper-producing bacterium strains, this strain shows certain advantages in synthesizing polyhydroxy fatty acid esters with different structures using multiple carbon sources such as ethanol, thus providing a new strain for PHA production using ethanol as a carbon source. Attached Figure Description

[0022] Figure 1 The transmission electron microscopy observation results of copper-loving bacteria ZWJ01 in the fermentation broth obtained in step 2 of Example 2.

[0023] Figure 2 This is the result of optimizing the yeast powder concentration for the synthesis of polyhydroxy fatty acid esters by *Bacillus thuringiensis* ZWJ01 in Example 3.

[0024] Figure 3 The results show the optimized NaCl concentration for the synthesis of polyhydroxy fatty acid esters by *Bacillus thuringiensis* ZWJ01 in Example 4.

[0025] Figure 4 The fermentation temperature optimization results for the synthesis of polyhydroxy fatty acid esters by *Bacillus thuringiensis* ZWJ01 in Example 5 are shown. Detailed Implementation

[0026] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0027] According to one aspect of the present invention, a copper-loving bacterium capable of producing polyhydroxy fatty acid esters using multiple carbon sources is provided. Cupriavidus The strain (sp.) has the accession number CGMCC No. 37462.

[0028] In one specific embodiment, the plurality of carbon sources includes one or more of ethanol, ethylene glycol, glycerol, acetic acid, propionic acid, butyric acid, valeric acid, glucose, fructose, sucrose, xylose, γ-butyrolactone, and 1,4-butanediol.

[0029] According to another aspect of the invention, the use of the above-described strain in the production of polyhydroxyalkanoates is provided.

[0030] According to another aspect of the present invention, a method for producing polyhydroxyalkanoates is provided, the method comprising fermenting the strain according to the present invention (accession number CGMCC No. 37462) in a culture medium.

[0031] In one specific embodiment, the polyhydroxy fatty acid ester includes poly-3-hydroxybutyrate, poly(3-hydroxybutyrate-... with 3-hydroxyvalerate) and poly(3-hydroxybutyrate- with One or more of the following: 4-hydroxybutyrate.

[0032] In one specific embodiment, the fermentation medium includes one or more of ethanol, ethylene glycol, glycerol, acetic acid, propionic acid, butyric acid, valeric acid, glucose, fructose, sucrose, xylose, γ-butyrolactone, and 1,4-butanediol as a carbon source.

[0033] In one specific embodiment, the culture medium comprises MS liquid culture medium.

[0034] In a preferred embodiment, the yeast concentration in the culture medium is 0-5 g / L, preferably 4 g / L.

[0035] In a preferred embodiment, the NaCl concentration in the culture medium is 0-20 g / L, preferably 10 g / L.

[0036] In a preferred embodiment, the fermentation temperature is 25-37°C, preferably 30°C.

[0037] In a preferred embodiment, the culture medium contains one of the following combinations as a mixed carbon source: ethanol and propionic acid; ethanol and valeric acid; ethanol and γ-butyrolactone; ethanol and 1,4-butanediol.

[0038] In a preferred embodiment, the method is a production of poly(3-hydroxybutyric acid- with The method involves a medium containing ethanol and propionic acid or a mixture of ethanol and valerate as a carbon source.

[0039] In a preferred embodiment, the method is a production of poly(3-hydroxybutyric acid- with The method involves a culture medium containing a mixed carbon source consisting of ethanol and γ-butyrolactone or ethanol and 1,4-butanediol.

[0040] According to another aspect of the invention, a microbial agent is provided, which comprises the strain described in the invention (accession number CGMCC No. 37462).

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Two types of polyhydroxyalkanoate standards are available: poly(3-hydroxybutyric acid-... with 3-hydroxyvalerate (3-hydroxybutyrate), purchased from Sigma-Aldrich, with a 3-hydroxybutyrate monomer content of 90.8 mol% and a 3-hydroxyvalerate monomer content of 9.2 mol%. γ-Butyrolactone was used as a standard for 4-hydroxybutyrate monomer and purchased from Beijing Tongguang Fine Chemical Co., Ltd.

[0042] The freeze-drying method in the following examples is as follows: After fermenting and culturing the microorganisms, a certain volume of fermentation broth is taken, centrifuged at 10,000 rpm for 10 min, the supernatant is discarded, and the bacterial cells are resuspended in deionized water for washing; the bacterial cells are collected by centrifuging at 10,000 rpm for 10 min, and the centrifuge tube containing the washed bacterial cell precipitate is placed at -20℃ for 2 h, and then placed in a freeze dryer for freeze-drying for 10 h to obtain the freeze-dried product.

[0043] In the following examples, cell dry weight is measured as the cell dry weight per liter of fermentation broth. The unit of cell dry weight is g / L. Cell dry weight (CDW) = (weight of the freeze-dried centrifuge tube - weight of the original empty centrifuge tube) / volume of fermentation broth; the weight of the freeze-dried centrifuge tube and the weight of the original empty centrifuge tube are both in g; the volume of fermentation broth is in L.

[0044] The following examples illustrate a method for determining the polyhydroxyalkanoate (PHA) content in freeze-dried products: the freeze-dried products undergo an esterification reaction, and the content of the product after the esterification reaction is then calculated. Approximately 20 mg of poly(3-hydroxybutyric acid-) was taken... with The esterification reaction was carried out using the same method on a standard of 3-hydroxyvalerate and approximately 20 mg of γ-butyrolactone.

[0045] The specific steps of the esterification reaction are as follows: Take 30-40 mg of the freeze-dried product into an esterification tube, add 2 mL of chloroform and 2 mL of esterification solution (15 mL of concentrated sulfuric acid and 0.5 g of benzoic acid in 500 mL of methanol), mix well, cover and seal, and keep at 100℃ for 4 h; after cooling to room temperature, add 1 mL of deionized water, shake thoroughly with a vortex mixer, and let stand to separate the layers; after the chloroform phase and water are completely separated, take 1 μL of the chloroform phase for gas chromatography analysis.

[0046] Gas chromatography analysis parameters: HP 6890 gas chromatograph was used, the column was HP-5 capillary column, the column length was 30m, the inner diameter was 320 µm, the stationary phase was 25 nm thick phenylmethyl polysiloxane; the detector was flame ionization detector (FID); high-purity nitrogen was used as carrier gas, hydrogen as fuel gas, and air as combustion-supporting gas; the specific conditions were as follows: (1) Column temperature: 80℃ start, hold for 1.5 min; increase the temperature to 140℃ at a rate of 30℃ / min, hold for 0 min; increase the temperature to 220℃ at a rate of 40℃ / min, hold for 1 min. The total time was 6.5 min. (2) Column pressure: 10 psi start, hold for 1.5 min; increase the pressure to 20 psi at a rate of 2.5 psi / min, hold for 0.5 min. (3) Injection port: temperature was 200℃, split mode was used, split ratio was 30. (4) Detector: temperature 220℃, hydrogen flow rate 30 mL / min, air flow rate 400 mL / min.

[0047] Using an Agilent microsyringe with an injection volume of 1 µL, the polymer was quantitatively analyzed using the internal standard method, and quantification was based on peak area. During gas chromatography detection, frozen stem cell samples and poly(3-hydroxybutyric acid-) with Both the 3-hydroxyvalerate (3-hydroxyvalerate) standard and the γ-butyrolactone standard were subjected to the above steps for esterification reaction and gas chromatography detection. Based on the peak positions of the samples and standards, the type of polyhydroxy fatty acid ester accumulated in the bacterial cells was determined.

[0048] The yield calculation for poly-3-hydroxybutyrate (PHB) is as follows: PHB yield = (PHB peak area in sample / internal standard peak area in sample) × [(internal standard peak area in standard / PHB peak area in standard) × (standard mass × 0.908)] / esterification mass of sample × cell dry weight Poly(3-hydroxybutyric acid- with The yield calculation for 3-hydroxyvalerate is as follows: PHBV yield = PHB yield + PHV yield, where: PHB yield = (PHB peak area in sample / internal standard peak area in sample) × [(internal standard peak area in standard / PHB peak area in standard) × (standard mass × 0.908)] / esterification mass of sample × cell dry weight PHV yield = (PHV peak area in sample / internal standard peak area in sample) × [(internal standard peak area in standard / PHV peak area in standard) × (standard mass × 0.092)] / esterification mass of sample × cell dry weight The calculation method for 3-hydroxyvalerate monomer content is: 3-hydroxyvalerate monomer content = (PHV yield in sample / 100) / [(PHV yield in sample / 100) + (PHB yield in sample / 86)], where: PHV yield in sample = (PHV peak area in sample / internal standard peak area in sample) × [(internal standard peak area in standard / PHV peak area in standard) × (standard mass × 0.092)] PHB yield in sample = (PHB peak area in sample / internal standard peak area in sample) × [(internal standard peak area in standard / PHB peak area in standard) × (standard mass × 0.908)] Poly(3-hydroxybutyric acid- with The yield calculation for 4-hydroxybutyrate (PHB) is as follows: P34HB yield = PHB yield + P4HB yield, where: PHB yield = (PHB peak area in sample / internal standard peak area in sample) × [(internal standard peak area in standard / PHB peak area in standard) × (standard mass × 0.908)] / esterification mass of sample × cell dry weight P4HB yield = (P4HB peak area in sample / internal standard peak area in sample) × [(internal standard peak area in standard / P4HB peak area in standard) × standard mass] / esterification mass of sample × cell dry weight The calculation method for 4-hydroxybutyric acid monomer content is: 4-hydroxybutyric acid monomer content = P4HB yield / P34HB yield Polymer content is defined as the ratio of polymer to cell dry weight, and polymer content = polymer yield / cell dry weight × 100%.

[0049] The culture media involved in the following examples are as follows: MS liquid culture medium: Dissolve 10 g of sodium chloride and 5 g of yeast powder in an appropriate amount of deionized water, and bring the volume to 1 L with deionized water to obtain the substrate; dissolve 492.5 g of disodium hydrogen phosphate dodecahydrate and 75 g of potassium dihydrogen phosphate in an appropriate amount of deionized water, and bring the volume to 1 L with deionized water to obtain component I; dissolve 100 g of ammonium sulfate and 20 g of magnesium sulfate heptahydrate in an appropriate amount of deionized water, and bring the volume to 1 L with deionized water to obtain component II; dissolve 5 g of ferric ammonium citrate and 2 g of calcium chloride dihydrate in an appropriate amount of 1 mol / L dilute hydrochloric acid, and bring the volume to 1 L with 1 mol / L dilute hydrochloric acid to obtain component III; dissolve 0.1 g of zinc sulfate heptahydrate, 0.03 g of manganese chloride tetrahydrate, 0.3 g of boric acid, 0.2 g of cobalt chloride hexahydrate, 0.01 g of copper sulfate pentahydrate, 0.02 g of nickel chloride hexahydrate, and 0.03 g of sodium molybdate dihydrate in an appropriate amount of 1 mol / L dihydrate hydrochloric acid to obtain component III. Component IV was obtained by diluting 1 mol / L hydrochloric acid to 1 L and then bringing the volume up to 1 L. Components III, IV, and deionized water were mixed in a ratio of 10:1:9, and the pH was adjusted to 4-5 with sodium hydroxide to obtain the trace element stock solution. After sterilization, components I, II, and the trace element stock solution were added to the substrate at a ratio of 1 / 50.

[0050] LB liquid medium: Dissolve 10 g of peptone, 5 g of yeast powder and 10 g of sodium chloride in an appropriate amount of deionized water, and then bring the volume up to 1 L with deionized water.

[0051] LB solid medium: Add 17.5 g of agar powder to 1 L of LB liquid medium, sterilize at 121℃ for 15 min, and allow to cool naturally.

[0052] MS liquid medium containing ethanol: When preparing the base of MS liquid medium, add 10 g of ethanol, and keep everything else unchanged.

[0053] MS liquid medium containing ethylene glycol: Replace 10 g of ethanol in the MS liquid medium containing ethanol with 10 g of ethylene glycol, and keep everything else unchanged.

[0054] MS liquid medium containing glycerol: Replace 10 g of ethanol in the MS liquid medium containing ethanol with 10 g of glycerol, and keep everything else unchanged.

[0055] MS liquid medium containing acetic acid: Replace 10 g of ethanol in the MS liquid medium containing ethanol with 10 g of acetic acid, and keep everything else unchanged.

[0056] MS liquid medium containing propionic acid: Replace 10 g of ethanol in the MS liquid medium containing ethanol with 10 g of propionic acid, and keep everything else unchanged.

[0057] MS liquid medium containing butyric acid: Replace 10 g of ethanol in the MS liquid medium containing ethanol with 10 g of butyric acid, and keep everything else unchanged.

[0058] MS liquid medium containing valeric acid: Replace 10 g of ethanol in the MS liquid medium containing ethanol with 10 g of valeric acid, and keep everything else unchanged.

[0059] MS liquid medium containing glucose: Replace 10 g of ethanol in the MS liquid medium containing ethanol with 20 g of glucose, and keep everything else unchanged.

[0060] MS liquid medium containing fructose: Replace 10 g of ethanol in the MS liquid medium containing ethanol with 20 g of fructose, keeping everything else unchanged.

[0061] MS liquid medium containing sucrose: Replace 10 g of ethanol in the MS liquid medium containing ethanol with 20 g of sucrose, while keeping everything else unchanged.

[0062] MS liquid medium containing xylose: Replace 10 g of ethanol in the MS liquid medium containing ethanol with 20 g of xylose, keeping everything else unchanged.

[0063] MS liquid medium containing ethanol and yeast extract at different concentrations: The yeast extract concentration in the MS liquid medium containing ethanol was adjusted to 0 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L or 5 g / L, while other parameters remained unchanged.

[0064] MS liquid medium containing ethanol and 4 g / L yeast extract: Replace 5 g of yeast extract in the MS liquid medium containing ethanol with 4 g, and keep everything else unchanged.

[0065] MS liquid medium containing ethanol, 4 g / L yeast extract and different concentrations of NaCl: Replace 5 g of yeast extract in the MS liquid medium containing ethanol with 4 g, and adjust the concentration of sodium chloride to 0 g / L, 10 g / L and 20 g / L respectively, while keeping other parameters unchanged.

[0066] MS liquid medium containing ethanol, 4 g / L yeast extract and different concentrations of propionic acid: Replace 5 g of yeast extract in the MS liquid medium containing ethanol with 4 g, and add 2 g, 4 g, 6 g or 8 g of propionic acid respectively, while keeping everything else unchanged.

[0067] MS liquid medium containing ethanol, 4 g / L yeast extract and different concentrations of valeric acid: Replace 5 g of yeast extract in the MS liquid medium containing ethanol with 4 g, and add 2 g, 4 g, 6 g or 8 g of valeric acid respectively, while keeping everything else unchanged.

[0068] MS liquid medium containing ethanol, 4 g / L yeast extract and different concentrations of γ-butyrolactone: Replace 5 g of yeast extract in the MS liquid medium containing ethanol with 4 g, and add 2 g, 4 g, 6 g or 8 g of γ-butyrolactone respectively, while keeping everything else unchanged.

[0069] MS liquid medium containing ethanol, 4 g / L yeast extract and different concentrations of 1,4-butanediol: Replace 5 g of yeast extract in the MS liquid medium containing ethanol with 4 g, and add 2.5 g, 5 g, or 10 g of 1,4-butanediol respectively, while keeping everything else unchanged.

[0070] In the quantitative experiments described below, three replicates were performed, and the average value of the results was taken.

[0071] Example 1: Copper-loving bacteria Cupriavidus sp. Isolation, Identification and Preservation of ZWJ01 I. Isolation of bacterial strains 1. Add 1 g of soil sample (soil collected from near a tea garden in Pu'er City, Yunnan Province) to a conical flask containing 100 mL of sterile physiological saline, shake at 200 rpm for 1 h to obtain a water sample.

[0072] 2. Inoculate 1 mL of the water sample obtained in step 1 into MS liquid medium containing ethanol, and incubate at 30℃ and 200 rpm for 24 h to obtain the culture.

[0073] 3. Using a sterilized inoculation loop, take the culture obtained in step 2 and streak it onto LB solid medium, then incubate upside down for 24 h.

[0074] 4. After completing step 3, the single clones that can grow on LB solid medium will be isolated, cultured and purified. The isolated and purified strain will be named ZWJ01.

[0075] II. Identification of the strain 1. Morphological identification The ZWJ01 strain was inoculated onto LB solid medium and cultured at 37°C for 12 h. The colonies were observed visually and microscopically. The results showed that the ZWJ01 colonies were raised, white, round, with regular edges and a smooth surface. Microscopic observation revealed that the cells of the ZWJ01 strain were short rod-shaped and elongated oval.

[0076] 2. Molecular identification Genomic DNA was extracted from strain ZWJ01 and amplified by PCR using universal primers for 16S rRNA. The amplified product was sequenced, and the nucleotide sequence of the 16S rRNA of strain ZWJ01 was obtained as shown in SEQ ID NO:1. The 16S rRNA of strain ZWJ01 was analyzed using the EZBioCloud database (https: / / www.ezbiocloud.net / ). The results showed that strain ZWJ01 is related to the genus *Copper-bearing Bacteria* (…). Cupriavidus strains in the genus Cupriavidus yeoncheonensis The gene sequence similarity of DCY86 is high, at 98.57%.

[0077] Based on the above morphological and molecular biological analyses, strain ZWJ01 was identified as belonging to the genus *Copper-loving Bacteria*, and was named *Copper-loving Bacteria*. Cupriavidus sp. ZWJ01 (abbreviated as ZWJ01).

[0078] III. Preservation of bacterial strains Copper-loving bacteria Cupriavidus sp. ZWJ01 was deposited at the China General Microbiological Culture Collection Center on January 19, 2026, with accession number CGMCC No. 37462.

[0079] Example 2: Production of polyhydroxy fatty acid esters by *Bacillus thuringiensis* ZWJ01 using different carbon sources 1. Inoculate a single clone of ZWJ01 of *Bacillus thuringiensis* into 20 mL of LB liquid medium and culture at 37°C and 200 rpm for 16 h to obtain the seed culture of *Bacillus thuringiensis* ZWJ01.

[0080] 2. Inoculate 1.5 mL of the seed culture of *Z. WJ01* obtained in step 1 into a shake flask (250 mL) containing 30 mL of culture medium (MS liquid medium containing ethanol, MS liquid medium containing ethylene glycol, MS liquid medium containing glycerol, MS liquid medium containing acetic acid, MS liquid medium containing propionic acid, MS liquid medium containing butyric acid, MS liquid medium containing valeric acid, MS liquid medium containing glucose, MS liquid medium containing fructose, MS liquid medium containing sucrose, or MS liquid medium containing xylose) (5% inoculum). Incubate at 30℃ and 200 rpm for 48 h to obtain the fermentation broth.

[0081] The fermentation broth of *Bacillus cereus* ZWJ01 cultured in MS liquid medium containing ethanol was observed using transmission electron microscopy. The results are as follows: Figure 1 As shown (scale bar is 200 nm). The results indicate that when ethanol is used as the carbon source, the copper-loving bacterium ZWJ01 in the fermentation broth accumulated a large number of white polyhydroxy fatty acid ester particles in the cells.

[0082] 3. Place the fermentation broth in a centrifuge tube (50 mL), centrifuge at 10,000 rpm for 10 min, and collect precipitate 1; wash precipitate 1 twice with deionized water, centrifuge at 10,000 rpm for 10 min, and collect precipitate 2.

[0083] 4. Place the centrifuge tube containing the precipitate 2 collected in step 3 at -20°C for 2 hours, and then freeze-dry it in a freeze dryer for 10 hours to obtain the freeze-dried product.

[0084] Accurately weigh the centrifuge tubes before adding the fermentation broth and after lyophilization, and calculate the dry weight of the cells.

[0085] 5. The content of polyhydroxyalkanoates in the freeze-dried product was determined. The results are shown in Table 1.

[0086]

[0087] When propionic acid and valeric acid were used as carbon sources, the PHA type accumulated in *Bacillus cereus* ZWJ01 cells was PHBV; when other carbon sources were used, the PHA type accumulated in *Bacillus cereus* ZWJ01 cells was PHB. When cultured with glucose, fructose, and sucrose as carbon sources, cell dry weights exceeding 8.50 g / L were obtained. With fructose as the carbon source, a PHB yield of 3.46 g / L was obtained, while with glucose and xylose as carbon sources, the strain could grow but accumulated very little PHB intracellularly. When cultured with ethanol as the carbon source, *Bacillus cereus* ZWJ01 achieved the highest PHB yield, at 4.16 g / L. Cell growth was poor when ethylene glycol was used as the carbon source. With glycerol as the carbon source, the strain could grow, but the amount of PHB accumulated intracellularly was low, only 3.82 wt%. PHB yields were very low when acetic acid and butyric acid were used as carbon sources. When propionic acid and valeric acid were used as carbon sources, the copper-loving bacterium ZWJ01 showed strong tolerance to propionic acid, achieving a cell dry weight of 4.51 g / L and a PHBV yield of 1.26 g / L. However, the copper-loving bacterium ZWJ01 showed poor tolerance to valeric acid, with cells barely growing and a PHBV yield of only 0.07 g / L.

[0088] Example 3: Optimization of yeast powder concentration for the production of polyhydroxyalkanoates by *ZWJ01* (a type of *Copper-loving Bacterium*). To enable the copper-loving bacteria ZWJ01 to produce polyhydroxy fatty acid esters more effectively, the yeast powder concentration in the fermentation medium was optimized.

[0089] 1. Same as step 1 in Example 2.

[0090] 2. Inoculate 1.5 mL of the seed culture of ZWJ01 obtained in step 1 into a shake flask (250 mL) containing 30 mL of MS liquid medium (5% inoculum) containing ethanol and different concentrations of yeast powder, and incubate at 30℃ and 200 rpm for 48 h to obtain the fermentation broth.

[0091] 3. Same as step 3 in Example 2.

[0092] 4. Same as step 4 in Example 2.

[0093] 5. Same as step 5 in Example 2.

[0094] The results are as follows Figure 2 As shown, the polyhydroxyalkanoate (PHB) accumulated within the cells is a polyhydroxyalkanoate. With increasing yeast extract concentration, PHB production initially increases and then decreases. The cell dry weight, PHB production, and PHB content all reach their maximum values ​​when the yeast extract concentration in the culture medium is 4 g / L. At this point, the cell dry weight is 9.04 g / L, the PHB production is 5.07 g / L, and the PHB content accounts for 56.24% of the cell dry weight. Therefore, when the yeast extract concentration in the ethanol-containing MS liquid medium is 4 g / L, *Bacillus thuringiensis* ZWJ01 can utilize carbon sources more efficiently to produce polyhydroxyalkanoates.

[0095] Example 4: Optimization of NaCl concentration for the production of polyhydroxyalkanoates by *Bacillus thuringiensis* ZWJ01 To enable the copper-loving bacterium ZWJ01 to produce polyhydroxy fatty acid esters more effectively, the NaCl concentration of the fermentation medium was optimized.

[0096] 1. Same as step 1 in Example 2.

[0097] 2. Inoculate 1.5 mL of the seed culture of ZWJ01 obtained in step 1 into a shake flask (250 mL) containing 30 mL of MS liquid medium (5% inoculum) containing ethanol, 4 g / L yeast powder and different concentrations of NaCl. Incubate at 30℃ and 200 rpm for 48 h to obtain the fermentation broth.

[0098] 3. Same as step 3 in Example 2.

[0099] 4. Same as step 4 in Example 2.

[0100] 5. Same as step 5 in Example 2.

[0101] The results are as follows Figure 3As shown, when the NaCl concentration in the culture medium was 10 g / L, the cell dry weight was 9.04 g / L, and the polyhydroxyalkanoate accumulated in the cells was PHB, with the highest yield of 5.07 g / L, accounting for 56.24% of the cell dry weight. Therefore, when the NaCl concentration in the culture medium was 10 g / L, *Bacillus thuringiensis* ZWJ01 could most effectively utilize the carbon source to produce polyhydroxyalkanoates.

[0102] Example 5: Temperature optimization for the production of polyhydroxyalkanoates by *Bacillus thuringiensis* ZWJ01 To enable the copper-loving bacteria ZWJ01 to produce polyhydroxy fatty acid esters more effectively, the fermentation temperature was optimized.

[0103] 1. Same as step 1 in Example 2.

[0104] 2. Inoculate 1.5 mL of the seed culture of ZWJ01 obtained in step 1 into a shake flask (250 mL) containing 30 mL of MS liquid medium (5% inoculum) containing ethanol and 4 g / L yeast powder. Incubate at 200 rpm for 48 h at 25℃, 30℃, or 37℃ to obtain the fermentation broth.

[0105] 3. Same as step 3 in Example 2.

[0106] 4. Same as step 4 in Example 2.

[0107] 5. Same as step 5 in Example 2.

[0108] The results are as follows Figure 4 As shown, the polyhydroxyalkanoate (PHB) accumulated within the cells is 30℃. Both cell dry weight and PHB yield reach their maximum values ​​at this fermentation temperature, with a cell dry weight of 9.04 g / L and a PHB yield of 5.07 g / L, representing 56.24% of the cell dry weight. Therefore, at a fermentation temperature of 30℃, *Bacillus thuringiensis* ZWJ01 can most effectively utilize carbon sources to produce polyhydroxyalkanoates.

[0109] Example 6: Production of polyhydroxy fatty acid esters by *Bacillus thuringiensis* ZWJ01 using ethanol and propionic acid. 1. Same as step 1 in Example 2.

[0110] 2. Inoculate 1.5 mL of the seed culture of ZWJ01 obtained in step 1 into a shake flask (250 mL) containing 30 mL of MS liquid medium (5% inoculum) containing ethanol, 4 g / L yeast powder and different concentrations of propionic acid, and incubate at 30℃ and 200 rpm for 48 h to obtain the fermentation broth.

[0111] 3. Same as step 3 in Example 2.

[0112] 4. Same as step 4 in Example 2.

[0113] 5. Same as step 5 in Example 2. The results are shown in Table 2.

[0114]

[0115] When ethanol and propionic acid are used as a mixed carbon source, PHBV can be obtained through fermentation. The cell dry weight decreases with increasing propionic acid concentration, and the PHBV yield and its proportion of cell dry weight also decrease with increasing propionic acid concentration. The 3-hydroxyvalerate monomer content increases with increasing propionic acid concentration, reaching a maximum of 43.39 mol%. The highest PHBV yield (3.18 g / L) is achieved with the addition of 2 g / L propionic acid, of which the 3-hydroxyvalerate monomer content is 5.57 mol.

[0116] Example 7: Production of polyhydroxy fatty acid esters by *Bacillus cereus* ZWJ01 using ethanol and valeric acid. 1. Same as step 1 in Example 2.

[0117] 2. Inoculate 1.5 mL of the seed culture of ZWJ01 obtained in step 1 into a shake flask (250 mL) containing 30 mL of MS liquid medium (5% inoculum) containing ethanol, 4 g / L yeast powder and different concentrations of valeric acid. Incubate at 30℃ and 200 rpm for 48 h to obtain the fermentation broth.

[0118] 3. Same as step 3 in Example 2.

[0119] 4. Same as step 4 in Example 2.

[0120] 5. Same as step 5 in Example 2. The results are shown in Table 3.

[0121]

[0122] PHBV can be obtained through fermentation using ethanol and valeric acid as a mixed carbon source. With increasing valeric acid concentration, cell dry weight, PHBV yield, and PHBV content all decrease, reaching their maximum when using 10 g / L ethanol and 2 g / L valeric acid as the mixed carbon source: cell dry weight 6.27 g / L, PHBV yield 2.21 g / L, and PHBV accounting for 35.35 wt% of cell dry weight. The content of 3HV monomer in PHBV reaches its maximum at 42.74 mol% when using 10 g / L ethanol and 6 g / L valeric acid as the mixed carbon source. Furthermore, cell growth is poor when valeric acid concentration exceeds 2 g / L, indicating that *Copper-loving Bacterium* ZWJ01 has poor tolerance to valeric acid.

[0123] Example 8: Production of polyhydroxy fatty acid esters by *Bacillus cereus* ZWJ01 using ethanol and γ-butyrolactone 1. Same as step 1 in Example 2.

[0124] 2. Inoculate 1.5 mL of the seed culture of ZWJ01 obtained in step 1 into a shake flask (250 mL) containing 30 mL of MS liquid medium (5% inoculum) containing ethanol, 4 g / L yeast powder and different concentrations of γ-butyrolactone, and incubate at 30℃ and 200 rpm for 48 h to obtain the fermentation broth.

[0125] 3. Same as step 3 in Example 2.

[0126] 4. Same as step 4 in Example 2.

[0127] 5. Same as step 5 in Example 2. The results are shown in Table 4.

[0128]

[0129] P34HB was obtained through fermentation using ethanol and γ-butyrolactone as a mixed carbon source. With increasing γ-butyrolactone concentration, cell dry weight initially increased and then decreased, while the yield and content of P34HB continuously declined. The yield and content of P34HB reached their maximum when using 10 g / L ethanol and 2 g / L γ-butyrolactone as the mixed carbon source, with a cell dry weight of 5.51 g / L, a P34HB yield of 2.97 g / L, and P34HB accounting for 53.99% of the cell dry weight. The 4-hydroxybutyric acid monomer content increased with increasing γ-butyrolactone concentration, reaching its highest level of 6.88 mol at the addition of 8 g / L γ-butyrolactone.

[0130] Example 9: Production of polyhydroxy fatty acid esters by *Bacillus cereus* ZWJ01 using ethanol and 1,4-butanediol. 1. Same as step 1 in Example 2.

[0131] 2. Inoculate 1.5 mL of the seed culture of ZWJ01 obtained in step 1 into a shake flask (250 mL) containing 30 mL of MS liquid medium (5% inoculum) containing ethanol, 4 g / L yeast powder and different concentrations of 1,4-butanediol. Incubate at 30℃ and 200 rpm for 48 h to obtain the fermentation broth.

[0132] 3. Same as step 3 in Example 2.

[0133] 4. Same as step 4 in Example 2.

[0134] 5. Same as step 5 in Example 2. The results are shown in Table 5.

[0135]

[0136] Poly(P34HB) can be obtained through fermentation using ethanol and 1,4-butanediol as a mixed carbon source. With increasing 1,4-butanediol concentration, both cell dry weight and P34HB yield decreased, reaching their maximum values ​​when using 10 g / L ethanol and 2.5 g / L 1,4-butanediol as the mixed carbon source. At this concentration, the cell dry weight was 8.35 g / L, and the P34HB yield was 4.77 g / L. The 4HB monomer content in P34HB also reached its maximum at 1.94 mol%. When using 10 g / L ethanol and 5 g / L 1,4-butanediol as the mixed carbon source, the P34HB content reached its maximum of 73.31 wt%.

[0137] In conclusion, copper-loving bacteria Cupriavidus sp. ZWJ01 possesses the unique property of producing various types of polyhydroxy fatty acid esters using multiple carbon sources (especially ethanol), including poly-3-hydroxybutyrate, poly(3-hydroxybutyrate-... with 3-hydroxyvalerate) and poly(3-hydroxybutyrate- with 4-hydroxybutyrate (4-hydroxybutyrate) has good application prospects.

[0138] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A copper-loving bacterium capable of producing polyhydroxy fatty acid esters using multiple carbon sources ( Cupriavidus The strain (sp.) has the accession number CGMCC No. 37462.

2. The application of the strain according to claim 1 in the production of polyhydroxyalkanoates.

3. The application according to claim 2, wherein the polyhydroxy fatty acid ester comprises poly-3-hydroxybutyrate, poly(3-hydroxybutyrate-... co 3-hydroxyvalerate) and poly(3-hydroxybutyrate- co One or more of the following: 4-hydroxybutyrate.

4. A method for producing polyhydroxyalkanoates, the method comprising fermenting the strain according to claim 1 in a culture medium.

5. The method according to claim 4, wherein the polyhydroxy fatty acid ester comprises poly-3-hydroxybutyrate, poly(3-hydroxybutyrate-... co 3-hydroxyvalerate) and poly(3-hydroxybutyrate- co One or more of the following: 4-hydroxybutyrate.

6. The method according to claim 4 or 5, wherein the fermentation medium comprises one or more of ethanol, ethylene glycol, glycerol, acetic acid, propionic acid, butyric acid, valeric acid, glucose, fructose, sucrose, xylose, γ-butyrolactone and 1,4-butanediol as a carbon source.

7. The method according to any one of claims 4 to 6, wherein the culture medium comprises MS liquid culture medium.

8. The method according to any one of claims 4 to 7, wherein: 1) The yeast extract concentration in the culture medium is 0-5 g / L, preferably 4 g / L; 2) The NaCl concentration in the culture medium is 0-20 g / L, preferably 10 g / L; 3) The fermentation temperature is 25-37℃, preferably 30℃.

9. The method according to any one of claims 4 to 8, wherein the culture medium comprises a combination selected from the following as a mixed carbon source: ethanol and propionic acid; ethanol and valeric acid; ethanol and γ-butyrolactone; ethanol and 1,4-butanediol.

10. A microbial agent comprising the strain according to claim 1.

Images