A strain for producing PHA using waste oil and its construction method and application

Abstract

The present invention provides a strain for producing PHA using waste oil and fat, and a construction method and application thereof. The strain is based on Halomonas sp. LY03, overexpressing palace ‑ fadB ‑ fart ‑ fard Gene sequence; among them, palace ‑ fadB ‑ fart ‑ fard The gene sequence is shown in SEQ ID NO. 29. The recombinant strain of the present invention not only reduces the dependence on traditional carbon sources such as glucose, greatly reduces the production cost, but also promotes the recycling of resources by effectively converting waste oil into valuable biomaterials. The resource utilization of waste oil not only reduces the environmental pollution caused by the treatment of waste oil, but also can effectively alleviate the problem of resource shortage, and has significant environmental protection and economic benefits.

Description

Technical Field

[0001] The present invention relates to the field of synthetic biology, and in particular to a strain for producing PHA by utilizing waste oil and fat, and a construction method and application thereof. Background Art

[0002] "Biodegradable plastics" as innovative materials to replace traditional plastics have received widespread attention and are regarded as an important direction for the future development of the plastics industry. Among them, polyhydroxyalkanoates (PHA), as natural polymer materials synthesized by microorganisms, have good biodegradability and biocompatibility, and have become a research hotspot in the field of biomaterials. PHA can not only degrade in the natural environment, but also be decomposed into harmless small molecules by microorganisms, thereby reducing environmental pollution. Because it has both the thermal processing properties of plastics and the potential of biomedical materials, PHA materials can be used in the field of environmentally friendly packaging and as medical materials, such as degradable sutures and drug release carriers. The development of PHA has gone from the early poly 3-hydroxybutyrate (PHB) to the more flexible poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB). The continuous progress of this material has not only improved its industrial application value, but also provided more possibilities for its application in the medical field.

[0003] However, traditional fermentation processes rely on edible carbon sources such as glucose and xylose, which not only leads to high raw material costs, but also puts pressure on food resources, and easily produces by-products during the production process, causing environmental pollution. Therefore, how to find a more economical, efficient and environmentally friendly alternative carbon source has become a key issue in the development of PHA production technology.

[0004] Waste grease, as one of the main components of urban domestic waste, is the inedible residual grease produced during the processing, cooking and consumption of edible oil and meat. These waste greases not only have a wide range of sources and huge output, but are also difficult to collect and treat in a centralized manner. Improper disposal of waste grease can lead to serious environmental problems, such as decreased soil permeability and water pollution, and may even flow back into the edible oil market through illegal channels, endangering people's health. Therefore, converting waste grease into valuable biomaterials can not only reduce its pollution to the environment, but also achieve efficient utilization of resources.

[0005] In this context, synthetic biology, as a cutting-edge technology based on the design and optimization of biological systems, has shown great application potential. Through the fine modification of microbial metabolic pathways, synthetic biology can effectively transform non-renewable resources such as waste oils and fats into high value-added products. Summary of the invention

[0006] In view of the defects in the prior art, the present invention proposes a strain for producing PHA using waste oil and its construction method and application. The present invention screens different types of waste oil and deeply explores its activity in Halomonas sp. LY03

[0007] Application potential in the fermentation production of PHA (polyhydroxyalkanoates). Waste oils and fats are rich in a variety of fatty acids and trace nutrients, and can be used as a carbon source to replace traditional glucose. While promoting the growth of microorganisms, it can effectively reduce production costs and promote the efficient utilization of waste resources. In addition, the β-oxidation cycle pathway of the strain was enhanced through synthetic biology, and the production of PHB (poly 3-hydroxybutyrate) was further improved through genetic engineering regulation. By introducing the metabolic pathway for the de novo synthesis of 4-hydroxybutyric acid (4HB) and optimizing the pathway for the synthesis of 3-hydroxypropionic acid (3HB), a synergistic metabolic strain system was formed, which effectively increased the yield of the PHA copolymer P34HB, giving it a wider industrial application prospect with both environmental protection and economic benefits.

[0008] The present invention provides a strain for producing polyhydroxyalkanoate by utilizing waste oil, wherein the strain overexpresses fadA - fadB - fadE - fadD Gene sequence; among them, fadA - fadB - fadE - fadD The gene sequence is shown in SEQ ID NO.29;

[0009] The strain is any one of Halomonas sp. LY02, Halomonas sp. LY03, and Halomonas lutescens MDF-9; the deposit number of Halomonas sp. LY02 is GDMCC NO: 63381, the deposit number of Halomonas sp. LY03 is GDMCC NO: 63382, and the deposit number of Halomonas lutescens MDF-9 is GDMCC NO: 61850.

[0010] Furthermore, the strain also expresses any one or more of the following:

[0011] (i) 4hbD Gene, sucD Gene, ogdA Genes and orfZ Gene;

[0012] (ii) phaC Gene, phaA Genes andphaB Gene.

[0013] The present invention also provides a method for constructing the strain, comprising the following steps:

[0014] S1: In vitro expansion fadA - fadB - fadE - fadD The gene sequence is inserted into a vector to obtain a first vector plasmid;

[0015] S2: Introduce the first vector plasmid described in S1 into the strain. fadA - fadB - fadE - fadD The gene sequence is shown in SEQ ID NO.29.

[0016] The present invention also provides a method for constructing the strain, comprising the following steps:

[0017] In vitro expansion fadA - fadB - fadE - fadD The gene sequence is inserted into a vector to obtain a first vector plasmid, which is introduced into a strain;

[0018] Also include one or more of the following steps:

[0019] (i) In vitro expansion 4hbD - sucD - ogdA - orfZ The gene sequence is inserted into a vector to obtain a second vector plasmid, which is introduced into the strain;

[0020] (ii) In vitro expansion phaC-phaA-phaB The gene sequence is inserted into a vector to obtain a third vector plasmid, and the third vector plasmid is introduced into the strain.

[0021] in, 4hbD - sucD - ogdA - orfZ The gene sequence is shown in SEQ ID NO.30, phaC-phaA-phaB The gene sequence is shown as SEQ ID NO.31.

[0022] The present invention also provides a method for producing polyhydroxyalkanoate using the strain, comprising the following steps: activating the strain, carrying out shake flask seed culture and then carrying out fermentation culture in a fermentation medium.

[0023] Furthermore, the culture medium used for the fermentation contains a carbon source, and the carbon source is waste oil, and the waste oil is any one or more of gutter oil, sewage oil, frying oil, waste rapeseed oil, Haitian waste soybean oil, crude glycerol, coffee grounds waste oil and waste palm oil; preferably, the waste oil is any one or more of Haitian waste soybean oil and crude glycerol.

[0024] Furthermore, the concentration of the Haitian waste soybean oil is 5-30 g / L, preferably 15-30 g / L.

[0025] Furthermore, the pH of the fermentation is 6.5-12, preferably 7-11.

[0026] Furthermore, the fermentation medium includes sodium chloride, and the concentration of the sodium chloride is 10-50 g / L, preferably 30-50 g / L.

[0027] The present invention also provides the use of the strain in fermentation production of polyhydroxyalkanoates;

[0028] Furthermore, waste oil is used as a carbon source in the fermentation medium used in the fermentation.

[0029] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0030] (1) The present invention successfully constructed an engineered strain with an enhanced β-oxidation cycle pathway, which can efficiently utilize waste oil as the sole carbon source for efficient production of PHB. The recombinant strain of the present invention not only reduces the dependence on traditional carbon sources such as glucose, significantly reducing production costs, but also promotes the recycling of resources by effectively converting waste oil into valuable biomaterials. The resource utilization of waste oil not only reduces the environmental pollution caused by the treatment of waste oil, but also effectively alleviates the problem of resource shortage, with significant environmental and economic benefits.

[0031] (2) The present invention further improves the utilization rate of waste oil by optimizing key parameters in the fermentation process, such as the initial concentration of the carbon source, the pH of the fermentation medium, and the salt concentration, ensuring that the engineered strain can metabolize efficiently under optimal conditions and improving the production efficiency of PHA.

[0032] (3) In the material system of P34HB copolymer, the increase of 4HB content can improve the toughness of the material. The present invention introduces a metabolic pathway for de novo synthesis of 4HB in engineered bacteria, thereby increasing the proportion of 4HB in the product P34HB and making its physical properties more suitable for various industrial application scenarios, laying a solid foundation for the development of sustainable materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0034] Figure 1 Producing a PHB pathway map for the metabolic pathways of the present invention;

[0035] Figure 2 Producing a P34HB pathway map for the metabolic pathway of the present invention;

[0036] Figure 3 pSEVA321 used in the construction process of Example 8 of the present invention fadABED Plasmid map;

[0037] Figure 4 In Example 8 of the present invention fadA-fadB-fadE-fadD Electropherogram of target fragment;

[0038] Figure 5 pSEVA321 used in the construction process of Example 9 of the present invention 4soo Plasmid map;

[0039] Figure 6 In Example 9 of the present invention 4hbD-sucD-ogdA-orfZ Electropherogram of target fragment;

[0040] Figure 7 pSEVA321 used in the construction process of Example 10 of the present invention phaCAB Plasmid map;

[0041] Figure 8 In Example 10 of the present invention phaC-phaA-phaB Electropherogram of target fragment. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0043] Halomonas sp. LY03 itself has a natural metabolic pathway for producing poly 3-hydroxybutyrate. The first part of the present invention found that by comparing the growth and fermentation of the strain in various waste oils, some types of waste oils, such as Haitian waste soybean oil, can effectively replace the traditional carbon source glucose as the main carbon source for fermentation and production of PHB. Waste oils are rich in various fatty acids, which mainly participate in the β-oxidation cycle to produce acetyl CoA. Among them, a part of acetyl CoA enters the tricarboxylic acid cycle (TCA) for cell growth, and the other part is used for the synthesis of PHB, which further promotes the strain's utilization of waste oils. Specific anabolic pathways such as Figure 1 .

[0044] The second part of the present invention adopts synthetic biology to overexpress the enzyme gene closely related to the β-oxidation cycle through genetic engineering: acyl-CoA synthetase ( fadD ), acyl-CoA dehydrogenase ( fadE ), enoyl-CoA hydratase ( fadB )、3-ketoacyl-CoA thiolase( fadA ), thereby strengthening the metabolic pathway of fatty acids and improving the metabolic efficiency of waste soybean oil, thereby increasing PHB production.

[0045] The third part of the present invention further improves the utilization rate of waste oil by optimizing key parameters in the fermentation process, such as the initial concentration of the carbon source, the pH of the fermentation medium, and the salt concentration, ensuring that the engineered strain can metabolize efficiently under optimal conditions and improving the production efficiency of PHA.

[0046] The fourth part of the present invention introduces a new metabolic pathway for de novo synthesis of 4-hydroxybutyrate (4HB) by overexpressing 4hbD, sucD, ogdA, orfZ Genes and overexpression phaC, phaA, phaB Genes can be used to increase the content of 4HB in P34HB and improve the performance of PHA materials. The synthesis of P34HB is as follows Figure 2 shown.

[0047] The Halomonas sp. LY02 strain used in the examples is derived from patent CN116925981A, Halomonas sp. LY03 is derived from patent CN116970538A, and Halomonas lutescens MDF-9 is derived from patent CN113801810A.

[0048] Example 1 Growth of Halomonas sp.LY03 using different waste oils

[0049] (1) Seed solution preparation

[0050] ① Activation of bacterial strains: Take bacterial strains from the -80℃ refrigerator in the laboratory, use a pipette to pick up the bacterial liquid and streak it on a plate solid culture medium (yeast powder 5 g / L; tryptone 10 g / L; sodium chloride 60 g / L, pH 8.5), and culture at 37℃ for 24 h.

[0051] ② Primary seed culture: Pick a single colony and inoculate it into a 12 mL shaking tube (5 mL 60LB medium: yeast powder 5 g / L; tryptone 10 g / L; sodium chloride 60 g / L; pH 8.5), and culture the culture in a shaker at 37°C and 220 rpm for 12 hours.

[0052] ③ Secondary seed culture: Pipette 200 μL of the primary bacterial solution (1% inoculation volume) and inoculate it into a 150 mL conical flask (20 mL 60LB culture medium). Place the conical flask in a shaker at 37°C and 220 rpm for 12 h.

[0053] (2) Preparation of fermentation medium

[0054] Fermentation medium (50MM medium): Fermentation medium: waste oil 10 g / L, sodium chloride 50 g / L, yeast powder 1.2 g / L, urea 0.2~3 g / L, anhydrous magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 1.5~5.5 g / L, Fe(III)-NH4-Citrate 5 g / L, CaCl2·2H2O 2 g / L, HCl 12 mol / L, ZnSO4·7H2O 0.1 g / L, MnCl2·4H2O0.03 g / L, H2BO30.3 g / L, CoCl2·6H2O 0.2 g / L, CuSO4·5H2O 0.01 g / L, NiCl2·6H2O 0.02g / L, NaMoO4·2H2O 0.03 g / L, pH 9.0; 8 experimental groups were set up for waste oils, corresponding to waste cooking oils derived from vegetable oils and / or animal fats (gutter oil, sewage oil, frying oil), rapeseed oil refining by-products (waste rapeseed oil), Haitian soy sauce refining by-products (Haitian waste soybean oil), coffee oil refining by-products (coffee grounds waste oil), oily wastewater generated in the process of palm oil processing (waste palm oil), and crude glycerin, a by-product of biodiesel.

[0055] (3) Fermentation culture

[0056] Inoculate 5% of the seed solution (2.5 mL) into a 500 mL conical flask and culture it in a shaker at 37°C and 220 rpm for 48 h.

[0057] (4) Determination of cell dry weight and PHA content

[0058] Cell dry weight (CDW): Place 15-20 mL of fermented bacterial liquid in a 50 mL centrifuge tube, centrifuge at room temperature for 10 minutes at 9000 rpm, and discard the supernatant; add appropriate amount of deionized water, resuspend in an oscillator with oscillation conditions of 2500 rpm and 5 min to ensure sufficient resuspension, centrifuge under the same conditions, and discard the supernatant; seal the centrifuge tube with sealing film and prefreeze in a -80℃ refrigerator for 2 h; dry the centrifuge tube in a vacuum freeze dryer for 12-16 hours; weigh and calculate the cell dry weight (g / L).

[0059] Determination of PHA content: weigh 0.028~0.03 g of ground sample (standard product) into an esterification tube, then add 2 mL of esterification solution (containing methanol, 3% (v / v) concentrated sulfuric acid (98%, w / w) and 1 g / L benzoic acid) and 2 mL of chloroform, and esterify at 100°C for about 4 hours. After cooling to room temperature, add 1 mL of ddH2O, shake and mix thoroughly, and let stand to separate. After the aqueous phase and the organic phase are completely separated, take the chloroform layer (usually the lower layer) and filter it into a liquid phase bottle using a 0.22 μm organic filter membrane, and then use a GC-7800 gas chromatograph to determine the PHB content. Among them, a capillary column (Rtx-5 type, 30 m long, 0.25 mm inner diameter and 0.25 μm stationary phase) and a hydrogen flame ionization detector (FID). High-purity nitrogen is used as the carrier gas.

[0060] The test method is: the initial temperature is maintained at 80℃ for 1.5 min; in the first stage, the temperature is increased to 140℃ at a rate of 30℃ / min; in the second stage, it is increased to 240℃ at a rate of 40℃ / min, and this process takes 2 min; the total analysis time is 8 min; the injection temperature is 240℃ and the detector temperature is 250℃.

[0061] The injection volume was 1 μL, and the PHA was quantitatively analyzed by the external standard method, and the PHA yield was calculated based on the peak area.

[0062] (5) Fermentation results

[0063] The fermentation results using different waste oils as carbon sources are shown in Table 1:

[0064] Table 1 Fermentation results using different waste oils as carbon sources

[0065]

[0066] The results show that Halomonas sp. LY03 of the present invention can utilize different waste oils, such as gutter oil, swill oil, waste rapeseed oil, Haitian waste soybean oil, crude glycerol and waste palm oil. When the strain is cultured with different waste oils, it is found that when Haitian waste soybean oil is the only carbon source, the growth of the strain is the best, and its cell dry weight can reach up to 7.25 g / L; crude glycerol also has potential, and the cell dry weight can reach up to 4.39 g / L. Usually, when glucose is used as a carbon source, the cell dry weight of Halomonas can reach more than 10 g / L. The strain in the present invention can not only utilize most of the waste oils to produce PHB, but also when Haitian waste soybean oil is used as a carbon source, the cell dry weight is close to the cell dry weight when glucose is used as a carbon source.

[0067] Example 2 β-oxidation cycle pathway enhancement

[0068] fadA-fadB-fadE-fadD Gene cluster integration expression:

[0069] This example uses the integrase editing method to fadA-fadB-fadE-fadD The gene cluster was integrated into Halomonas p. LY03 which was modified with an integrase site, and the integration site strength was selected to be 3500 au.

[0070] (1) In vitro amplification of gene fragments

[0071] Overlap extension PCR fadA- fadB- fadE- fadD and pSEVA321 backbone; fadA- fadB- fadE- fadD and pSEVA321 backbone under the action of Gibson ligation system, fadA- fadB- fadE- fadD and pSEVA321 backbone to form a new plasmid and label it as pSEVA321 fadABED , with pSEVA321 fadABED As template, PCR was performed fadA-fadB-fadE-fadD Sequence, take part of the product to send to the biological company for sequencing, plasmid information such as Figure 3 shown.

[0072] The primer sequences (5'-3') for PCR amplification of the target gene are as follows:

[0073] Upstream fadA-F : See SEQ ID No.1;

[0074] Downstream fadA- R: see SEQ ID No. 2;

[0075] Upstream fadB- F: see SEQ ID No. 3;

[0076] Downstream fadB-R: see SEQ ID No.4;

[0077] Upstream fadE-F : See SEQ ID No.5;

[0078] Downstream fadE- R: see SEQ ID No.6;

[0079] Upstream fadD- F: see SEQ ID No.7;

[0080] Downstream fadD -R: see SEQ ID No.8;

[0081] Upstream 321-F: see SEQ ID No. 9;

[0082] Downstream 321-R: see SEQ ID No.10.

[0083] The amplification system and amplification procedure are shown in Table 2 and Table 3:

[0084] Table 2 Amplification system

[0085]

[0086] Table 3 Amplification program

[0087]

[0088] After the PCR reaction is completed, prepare agarose gel of corresponding concentration and perform electrophoresis to observe the size of DNA bands. Place the gel under ultraviolet light and quickly cut out the gel of the target DNA fragment, cutting off as much excess gel as possible.

[0089] (2) Gibson Assembly method connection

[0090] The concentration of the recovered DNA was tested, and the ratio of DNA addition was calculated according to the length and concentration of the target fragment and the pSEVA321 backbone, and the Gibson mixed enzyme was used for ligation. The Gibson Assembly ligation system and procedure are shown in Tables 4 and 5:

[0091] Table 4 Gibson Assembly connection system table

[0092]

[0093] Table 5 Gibson Assembly connection procedure

[0094]

[0095] After the reaction is completed, prepare agarose gel of corresponding concentration and perform electrophoresis to observe the size of DNA bands. Place the gel under ultraviolet light, quickly cut off the gel of the target DNA fragment, and cut off as much excess gel as possible.

[0096] (3) S17-1 Escherichia coli transformation

[0097] Step 1: Take out the prepared S17-1 E. coli competent cells from -80℃, thaw on ice, and wait for the bacterial block to melt after 5 minutes; Step 2: Add 5 μL of ligation product to the competent cells, gently flick the tube wall to mix the reaction solution (do not oscillate to mix). Note: The transformation volume of the ligation product should not exceed 1 / 10 of the volume of the competent cells used; Step 3: Ice bath on ice for 30 minutes, heat shock in a 42℃ water bath for 2 minutes, and immediately cool on ice for 2 minutes. Note: Shaking will reduce the transformation efficiency; Step 4: Add 400 μL LB medium (without antibiotics) to the centrifuge tube, mix well, and then put it in a 37℃ shaker at 200rpm for 60 minutes; Step 5: Centrifuge at 5000 rpm for 5 minutes to collect the bacteria, discard 350 μL of supernatant, keep 100 μL, gently blow to resuspend the bacterial block and apply it to the LB medium containing the corresponding antibiotics; Step 6: Invert the culture medium and culture it in a 37℃ incubator for 12~16 hours.

[0098] (4) Monoclonal colony positive verification

[0099] Colonies were picked out on the corresponding resistance LB plates and verified by colony PCR. PCR products with the correct band size were sent to a biological company for sequencing.

[0100] (5) Select a single colony with the correct sequence for expansion. After 12 to 16 hours, combine it with the modified Halomonas p. LY03 with an integrase site in a 20LB plate. After 8 hours, pick a small amount of the conjugated bacteria and spread it on a 60LB plate with the corresponding resistance. After 36 to 48 hours, perform colony verification. After successful verification, expand it again, combine it with the excision enzyme, and perform a second colony verification.

[0101] (6) Antibiotic withdrawal

[0102] In a deep-well plate 60LB, subculture for 3 to 7 generations at 37°C (one generation every 24 hours, plate verification starts from the 3rd generation), take a small amount of bacterial liquid after removing the antibiotics and add 60LB (about 10 5 ~10 6Dilute) and spread evenly on a 60LB plate. When subcultured, transfer to a new 60LB medium (without resistance) at a ratio of 0.02%. After a single colony grows on the plate, take a single colony and streak it on a 60LB plate and a 60LB plate with the corresponding resistance. After culturing for 12 hours, if it can grow on the 60LB plate but not on the 60LB plate with the corresponding resistance, it is considered to have successfully removed the resistance. Select the colonies that have successfully removed the resistance for PCR verification, and then sequence them after successful verification.

[0103] (7) Colony PCR verification

[0104] The results of colony PCR showed that the Halomonassp. LY03 strain in this example successfully integrated fadA- fadB- fadE- fadD Genes are verified by the size of the target product, such as Figure 4 As shown, the target fragment was 6534 bp, which was consistent with the expected results. The chassis strain with enhanced β-oxidation cycle pathway was named LY03-1.

[0105] Shake flask fermentation of chassis strains after strengthening the β-oxidation cycle pathway:

[0106] (1) Seed solution preparation

[0107] Please refer to Example 1 for the specific steps.

[0108] (2) Fermentation broth preparation

[0109] Please refer to Example 1 for the specific steps.

[0110] (3) Fermentation culture

[0111] Please refer to Example 1 for the specific steps.

[0112] (4) Fermentation results

[0113] The fermentation results of the chassis strain LY03-1 after the β-oxidation cycle pathway was strengthened are shown in Table 6. Conversion rate = cell dry weight*PHA wt% / (initial amount of carbon source added-residual amount).

[0114] Table 6 Fermentation results of chassis strains after strengthening the β-oxidation cycle pathway

[0115]

[0116] The results in Table 6 show that the modified strain LY03-1 not only increased the cell dry weight but also increased PHB by strengthening the β-oxidation cycle pathway. When 10g / L Haitian waste soybean oil was used as the carbon source, the PHB yield increased from the original 37.63% to 40.12%; when 10g / L crude glycerol was used as the carbon source, PHB increased from the original 27.64% to 29.32%. This shows that by modifying the β-oxidation cycle pathway, the strain strengthened its use of fatty acids, improved the conversion rate, and effectively increased the PHB yield.

[0117] Example 3 Fermentation of modified strain LY03-1 in different concentrations of Haitian waste soybean oil

[0118] (1) Seed solution preparation: the same as the seed solution preparation steps in Example 1.

[0119] (2) Preparation of fermentation broth: Fermentation medium: Haitian waste soybean oil, sodium chloride 50 g / L, yeast powder 1.2 g / L, urea 0.2~3 g / L, anhydrous magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 1.5~5.5 g / L, Fe(III)-NH4-Citrate 5 g / L, CaCl2·2H2O 2 g / L, HCl 12 mol / L, ZnSO4·7H2O 0.1 g / L, MnCl2·4H2O 0.03 g / L, H2BO30.3g / L, CoCl2·6H2O 0.2 g / L, CuSO4·5H2O 0.01 g / L, NiCl2·6H2O 0.02 g / L, NaMoO4·2H2O0.03 g / L, pH 9.0; the concentration of Haitian waste soybean oil is set at 7 levels: 5, 7.5, 10, 15, 20, 25, and 30 g / L.

[0120] (3) Fermentation and culture: the same as the fermentation and culture step in Example 1.

[0121] (4) Fermentation results: The fermentation results of Haitian waste soybean oil with different concentrations are shown in Table 7:

[0122] Table 7 Fermentation results using different concentrations of Haitian waste soybean oil as carbon source

[0123]

[0124] The results show that the cell dry weight and PHA content of the modified strain LY03-1 are continuously increased when fermented in different concentrations of Haitian waste soybean oil. When the concentration of Haitian waste soybean oil reaches 30 g / L, the cell dry weight of the strain is close to the same level when fermented with glucose. In order to reduce costs, the present invention no longer continues to increase the fermentation concentration of Haitian waste soybean oil, and will subsequently ferment and synthesize PHB at this concentration.

[0125] Example 4 Growth of modified bacteria LY03-1 using Haitian waste soybean oil under different pH conditions

[0126] (1) Seed solution preparation

[0127] The same seed solution preparation steps as in Example 1.

[0128] (2) Fermentation broth preparation

[0129] Fermentation medium: Haitian waste soybean oil 30 g / L, sodium chloride 50 g / L, yeast powder 1.2 g / L, urea 0.2~3 g / L, anhydrous magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 1.5~5.5 g / L, Fe(III)-NH4-Citrate 5 g / L, CaCl2·2H2O2 g / L, HCl 12 mol / L, ZnSO4·7H2O 0.1 g / L, MnCl2·4H2O 0.03 g / L, H2BO30.3 g / L, CoCl2·6H2O 0.2 g / L, CuSO4·5H2O 0.01 g / L, NiCl2·6H2O 0.02 g / L, NaMoO4·2H2O 0.03g / L; the pH is set at 7 levels: 6.5, 7, 8, 9, 10, 11, 12.

[0130] (3) Fermentation culture

[0131] Inoculate 5% of the seed solution (2.5 mL) into a 500 mL conical flask and culture it in a shaker at 37°C and 220 rpm for 48 h.

[0132] (4) Fermentation results

[0133] The fermentation results under different pH conditions are shown in Table 8:

[0134] Table 8 Fermentation results under different pH conditions

[0135]

[0136] The results showed that the modified strain LY03-1 could grow well and synthesize PHB well with Haitian waste soybean oil as the only carbon source between pH 7 and 11. Among them, the cell dry weight and PHA content of the strain were the highest under the condition of pH 8, with the dry weight reaching 16.41 g / L and the PHB content reaching 68.82 wt%.

[0137] Example 5 Growth of modified bacteria LY03-1 using Haitian waste soybean oil under different salt concentration conditions

[0138] (1) Seed solution preparation: the same as the seed solution preparation steps in Example 1.

[0139] (2) Fermentation broth preparation

[0140] Fermentation medium: Haitian waste soybean oil 30 g / L, sodium chloride, yeast powder 1.2 g / L, urea 0.2-3 g / L, anhydrous magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 1.5-5.5 g / L, Fe(III)-NH4-Citrate 5 g / L, CaCl2·2H2O 2 g / L, HCl 12 mol / L, ZnSO4·7H2O 0.1 g / L, MnCl2·4H2O 0.03 g / L, H2BO3 0.3 g / L, CoCl2·6H2O 0.2 g / L, CuSO4·5H2O 0.01 g / L, NiCl2·6H2O 0.02 g / L, NaMoO4·2H2O 0.03 g / L, pH 8.0; the sodium chloride concentration was set at 5 levels: 10, 20, 30, 40, 50 g / L.

[0141] (3) Fermentation culture: refer to Example 4 for the specific steps.

[0142] (4) Fermentation results: The fermentation results under different salt concentration conditions are shown in Table 9:

[0143] Table 9 Fermentation results under different salt concentrations

[0144]

[0145] The results showed that the modified strain LY03-1 could grow well and synthesize PHB well when fermented with Haitian waste soybean oil as the only carbon source at 30-50 g / L NaCl. Among them, the strain had the highest cell dry weight and PHA content when fermented at 30 g / L NaCl.

[0146] Example 6 4L fermentation of modified bacteria LY03-1 using Haitian waste soybean oil

[0147] (1) Seed solution preparation

[0148] ① Bacteria activation: refer to Example 1 for the specific steps.

[0149] ② Primary seed culture: refer to Example 1 for specific steps.

[0150] ③ Secondary seed culture: Pipette 200 μL of the primary bacterial solution (1% inoculation volume) and inoculate it into a 150 mL conical flask (20 mL 60LB culture medium). Place the conical flask in a shaker at 37°C and 220 rpm for 12 h.

[0151] ④Third-stage seed culture: Pipette 2.5 mL of the first-stage bacterial solution (1% inoculation volume) and inoculate it into a 500 mL conical flask (250 mL 60LB medium). Place the conical flask in a shaker at 37°C and 220 rpm for 12 h.

[0152] (2) Preparation of fermentation medium

[0153] The fermentation medium used in the fermenter was obtained after the shake flask conditions were optimized and the process was optimized.

[0154] Haitian waste soybean oil 10 g / L, sodium chloride 30 g / L, yeast powder 8 g / L, urea 1.6 g / L, anhydrous magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 3.5 g / L, Fe(III)-NH4-Citrate 5 g / L, CaCl2·2H2O 2 g / L, HCl 12 mol / L, ZnSO4·7H2O 0.1 g / L, MnCl2·4H2O 0.03 g / L, H2BO3 0.3 g / L, CoCl2·6H2O 0.2 g / L, CuSO4·5H2O 0.01 g / L, NiCl2·6H2O 0.02 g / L, NaMoO4·2H2O 0.03 g / L, pH 8.0.

[0155] (3) Fermentation culture

[0156] Inoculate the third-grade seed liquid into the fermentation tank at a 10% inoculation rate.

[0157] Feed 1: Prepare 40 mL of mother solution containing 10.4 g urea and add 4 mL every hour.

[0158] Feed 2: Prepare 20 mL of mother solution containing 1.3 g urea and 1.5 g ammonium sulfate, and add 2 mL every hour.

[0159] The fermentation control conditions are as follows:

[0160] a. Samples are measured every 1 hour and OD needs to be measured 600 , prepare the control sample at the same time and add it via the control pipette.

[0161] b. Keep a sample every 4 hours, 35 mL each time, centrifuge and wash all the samples on the same day, and store them in a -80℃ refrigerator.

[0162] c. Control the amount of soybean oil added during the feed period. During 6 to 16 hours (adding feed one), control the soybean oil to 10 g / L. After 16 hours (adding feed two), control the soybean oil to 5 to 10 g / L.

[0163] d. When the foam is too high, close to 7L or so, add defoamer manually and record the volume.

[0164] e. Control the soybean oil conservatively, the flow rate should be slow rather than fast, and add it manually with a pipette every hour.

[0165] f. The fermentation volume is controlled at 5~5.1L, the excess is sucked out, the air flow is controlled at 3~4L / min, and the dissolved oxygen coupling speed is 30%.

[0166] g. The fermentation pH is controlled at 6.85, the NaCl concentration is 30g / L, the soybean oil is filtered with gauze to remove the precipitate, and urea is prepared into mother liquor. According to the control of soybean oil, urea is added every hour.

[0167] h. During the fermentation process, the Haitian waste soybean oil content and pH of the fermentation system were monitored in real time.

[0168] (4) Determination of cell dry weight and PHA content: The specific steps are as in Example 1.

[0169] (5) Fermentation results: The results of the 4L fermentation tank are shown in Table 10:

[0170] Table 10 4L fermentation tank results

[0171]

[0172] The results showed that in a 4L fermenter, the cell dry weight of the LY03-1 strain could reach 54.79g / L and the PHB content could reach 80.54%. Usually, when fermentation is expanded with glucose as the carbon source, the cell dry weight of Halomonas can reach between 85-88g / L and the PHB content can reach 75-80%. The above results show that when fermentation is expanded with Haitian waste soybean oil as the carbon source, the PHB production can reach the level when glucose is used as the carbon source.

[0173] Example 7 Construction of strains introducing 4HB metabolic pathway

[0174] 4hbD-sucD-ogdA-orfZ Gene expression

[0175] (1) Plasmid construction

[0176] Overlap extension PCR 4hbD-sucD-ogdA-orfZ The fragment and plasmid pSEVA321 backbone were connected by Gibson ligation system. 4hbD-sucD-ogdA-orfZ The fragment was recombined with the pSEVA321 backbone to form a new plasmid, named pSEVA321 4soo , with pSEVA321 4soo As template, PCR was performed 4hbD-sucD-ogdA-orfZ fragments, and part of the product was sent to a biological company for sequencing. 4soo Transfer to LY03 and LY03-1 chassis bacteria, refer to Example 8 for specific stepsfadA- fadB- fadE- fadD Gene integration expression), plasmid information such as Figure 5 shown.

[0177] The results showed that the LY03 and LY03-1 base plates of this example were successfully transformed with 4hbD-sucD-ogdA-orfZ Genes are verified by the size of the target product, such as Figure 6 As shown, the target fragment was 5063 bp, which was consistent with the expected result. The two strains were named LY03-2 and LY03-1+2, respectively.

[0178] PCR amplification 4hbD-sucD-ogdA-orfZ The primer sequences (5'-3') of the fragment and pSEVA321 plasmid backbone are as follows:

[0179] Upstream 4hbD -F: see SEQ ID No.11;

[0180] Downstream 4hbD -R: see SEQ ID No.12;

[0181] Upstream sucD -F: see SEQ ID No.13;

[0182] Downstream sucD -R: see SEQ ID No.14;

[0183] Upstream ogdA -F: see SEQ ID No.15;

[0184] Downstream ogdA -R: see SEQ ID No.16;

[0185] Upstream orfZ -F: see SEQ ID No.17;

[0186] Downstream orfZ -R: see SEQ ID No.18;

[0187] Upstream 321-F: see SEQ ID No. 19;

[0188] Downstream 321-R: see SEQ ID No.20.

[0189] (2) Shake flask fermentation of strains with 4HB metabolic pathway

[0190] Seed solution preparation:

[0191] Please refer to Example 1 for the specific steps.

[0192] Fermentation broth preparation:

[0193] Haitian waste soybean oil / crude glycerol 30 g / L, sodium chloride 30 g / L, yeast powder 1.2 g / L, urea 0.2~3 g / L, anhydrous magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 1.5~5.5 g / L, Fe(III)-NH4-Citrate 5 g / L, CaCl2·2H2O 2 g / L, HCl 12 mol / L, ZnSO4·7H2O 0.1 g / L, MnCl2·4H2O 0.03 g / L, H2BO30.3 g / L, CoCl2·6H2O0.2 g / L, CuSO4·5H2O 0.01 g / L, NiCl2·6H2O 0.02 g / L, NaMoO4·2H2O 0.03 g / L, pH 8.0.

[0194] (3) Fermentation culture

[0195] Please refer to Example 1 for the specific steps.

[0196] (4) Fermentation results:

[0197] The fermentation results of the strains introduced into the 4HB metabolic pathway are shown in Table 11:

[0198] Table 11 Fermentation results of strains introduced with 4HB metabolic pathway

[0199]

[0200] The results showed that based on the wild strain and the modified strain LY03-1, the modified strains LY03-2 and LY03-1+2 introduced with the 4HB metabolic pathway could use waste oil to synthesize the polymer P34HB. After strengthening the β-oxidation cycle, the 4HB metabolic pathway was introduced to obtain the strain LY03-1+2 with a synergistic metabolic pathway, which could further utilize waste oil, not only increasing the cell dry weight, but also promoting the increase in the production of P34HB.

[0201] Example 8 3HB metabolic pathway enhancement

[0202] phaC- phaA- phaB Gene expression

[0203] Plasmid construction:

[0204] Overlap extension PCR phaC- phaA- phaB and pSEVA341 backbone; phaC- phaA- phaB and pSEVA341 backbone under the action of Gibson ligation system, phaC- phaA- phaB and pSEVA341 backbone to form a new plasmid and label it as pSEVA341 phaCAB , with pSEVA341 phaCAB Get the template phaC- phaA- phaBSequence, take part of the product to send to the biological company for sequencing (pSEVA341 phaCAB Transferred into LY03-2 and LY03-1+2 respectively. For specific steps, refer to Example 8. fadA- fadB- fadE- fadD Gene integration expression), plasmid information such as Figure 7 shown.

[0205] The results showed that the strains LY03-2 and LY03-1+2 were successfully transformed with phaC- phaA- phaB Genes are verified by the size of the target product, such as Figure 8 As shown, the target fragment was 4923 bp, which was in line with the expected results. The two metabolic pathway synergistic strains were named LY03-2+3 and LY03-1+2+3.

[0206] The primer sequences (5'-3') for PCR amplification of the target gene are as follows:

[0207] Upstream phaC -F : See SEQ ID No.21;

[0208] Downstream phaC - R: see SEQ ID No. 22;

[0209] Upstream phaA - F: see SEQ ID No. 23;

[0210] Downstream phaA -R: see SEQ ID No.24;

[0211] Upstream phaB - F: see SEQ ID No. 25;

[0212] Downstream phaB -R: see SEQ ID No.26;

[0213] Upstream 341-F: see SEQ ID No. 27;

[0214] Downstream 341-R: see SEQ ID No.28.

[0215] Shake flask fermentation of strains with enhanced 3HB metabolic pathway

[0216] (1) Seed solution preparation

[0217] Please refer to Example 1 for the specific steps.

[0218] (2) Fermentation broth preparation

[0219] Please refer to Example 7 for the specific steps.

[0220] (3) Fermentation culture

[0221] Please refer to Example 1 for the specific steps.

[0222] (4) Fermentation results

[0223] The fermentation results of the strains with enhanced 3HB metabolic pathway are shown in Table 12:

[0224] Table 12 Fermentation results of strains with enhanced 3HB metabolic pathway

[0225]

[0226] The results showed that the synergistic metabolic engineering bacteria LY03-2+3 and LY03-1+2+3 that strengthened the 3HB metabolic pathway could increase the proportion of PHA to a certain extent. Among them, the strain LY03-1+2+3 further promoted the increase of PHA production. When Haitian waste soybean oil was used as the carbon source, the cell dry weight reached 17.61g / L and the PHA content was as high as 71.03%; when crude glycerol was used as the carbon source, the cell dry weight reached 14.81g / L and the PHA content reached 63.78%. In the P34HB copolymer material, the toughness increases with the increase of 4HB content, so the engineered strain LY03-1+2+3 has strong application potential in the field of industrial materials.

[0227] Example 9: Other strains synthesizing PHB using different waste oils

[0228] (1) Seed solution preparation

[0229] Please refer to Example 1 for the specific steps.

[0230] (2) Preparation of fermentation medium

[0231] Please refer to Example 1 for the specific steps.

[0232] (3) Fermentation culture

[0233] Please refer to Example 1 for the specific steps.

[0234] (4) Determination of cell dry weight and PHA content

[0235] Please refer to Example 1 for the specific steps.

[0236] (5) Fermentation results

[0237] The fermentation results of other strains using different waste oils to synthesize PHB are shown in Tables 13 and 14:

[0238] Table 13 Synthesis of PHB using different waste oils and fats by MDF-9

[0239]

[0240] Table 14 Synthesis of PHB by LY02 using different waste oils

[0241]

[0242] The results showed that the strains Halomonaslutescens MDF-9 and Halomonas ssp . LY02 can utilize different waste oils, such as gutter oil, sewage oil, waste rapeseed oil, Haitian waste soybean oil, crude glycerol and waste palm oil. When these two strains were cultured with different waste oils, it was found that when Haitian waste soybean oil was the only carbon source, the strain grew best, and its cell dry weight could reach more than 6 g / L; followed by crude glycerol, and the cell dry weight could reach 3~4g / L.

[0243] Based on the above embodiments, the present application discloses a strain for producing PHA using waste oil and its construction method and application. The method includes: on the premise that the strain can produce PHB by fermentation, waste oil from different sources is screened, and an engineered strain that effectively increases the yield of PHB is constructed, and then the fermentation conditions are optimized. In addition, on the basis of the optimization, an engineered strain that can produce P34HB using waste oil is constructed. The construction method of the engineered strain includes the following steps: S1: constructing a β-oxidation cycle pathway overexpression plasmid ( fadA- fadB- fadE- fadD ), the gene was integrated into Halomonas p. LY03, the β-oxidation cycle pathway was successfully enhanced in Halomonas p. LY03, and the LY03-1 strain was obtained; S2: Construction of 4HB pathway plasmid ( 4hbD-sucD-ogdA-orfZ ), the plasmid was introduced into LY03 and LY03-1, and the de novo synthesis of 4HB metabolic pathway was successfully introduced into LY03 and LY03-1 to obtain LY03-2 and LY03-1+2 strains; S2: Construction of 3HB pathway overexpression plasmid ( phaC- phaA-phaB ), the plasmid was introduced into LY03-2 and LY03-1+2, and the 3HB metabolic pathway was successfully enhanced in LY03-2 and LY03-1+2 to obtain LY03-2+3 and LY03-1+2+3 strains.

[0244] The present invention obtains an effective carbon source that can replace glucose for strain growth and PHB production by comparing the growth of strains in different waste oils, which greatly reduces the production cost. At the same time, the synthetic biology technology is used to strengthen the β-oxidation cycle pathway, so as to enhance the utilization efficiency of fatty acids by the transformed strain, so as to achieve the purpose of increasing cell dry weight and PHB production, and effectively improve the cell dry weight of the transformed strain by optimizing the fermentation conditions; in addition, on the basis of the transformed strain LY03-1, a metabolic pathway for de novo synthesis of 4HB is introduced, and other metabolic pathways are optimized to increase the efficiency of the transformed strain LY03-1 in synthesizing P34HB. Studies have shown that on the basis of the wild strain itself having the ability to synthesize poly 3-hydroxybutyrate, strengthening the β-oxidation cycle pathway can effectively enhance the utilization of fatty acids by the strain, improve the PHB yield, and by introducing a metabolic pathway for de novo synthesis of 4HB, it is found that the transformed strain can use waste oil for the synthesis of P34HB. In addition, by combining the de novo synthesis of 4HB pathway with the optimized pathway, a synergistic metabolic pathway strain is constructed, which can further increase the yield of P34HB.

[0245] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain for producing polyhydroxyalkanoates using waste oils, characterized in that: The strain is overexpressed by strain Halomonas sp. LY03 fadA - fadB - fadE - fadD Gene sequence, 4hD - sucD - oGD - f Gene sequences and phaC-phaA-phaB Gene sequence obtained; The deposit number of the Halomonas sp. LY03 is GDMCC NO: 63382; Among them, the fadA - fadB - fadE - fadD The gene sequence is shown in SEQ ID NO.29, 4hD - sucD - oGD - f The gene sequence is shown in SEQ ID NO.

30. phaC-phaA-phaB The gene sequence is shown as SEQ ID NO.

31.

2. The method for constructing the strain according to claim 1, characterized in that: The steps include: In vitro expansion fadA - fadB - fadE - fadD The gene sequence is inserted into a vector to obtain a first vector plasmid, which is introduced into the Halomonas sp. LY03; The construction method further comprises the following steps: (i) In vitro expansion 4hD - sucD - oGD - f The gene sequence is inserted into a vector to obtain a second vector plasmid, which is introduced into the Halomonas sp. LY03; (ii) In vitro expansion phaC-phaA-phaB The gene sequence is inserted into a vector to obtain a third vector plasmid, and the third vector plasmid is introduced into the Halomonas sp. LY03.

3. A method for producing polyhydroxyalkanoates using the strain according to claim 1, characterized in that: The method comprises the following steps: activating the strain, carrying out shake flask seed culture, and then fermenting the strain in a fermentation medium; the fermentation medium contains a carbon source, and the carbon source is Haitian waste soybean oil or crude glycerol; The concentration of the Haitian waste soybean oil is 20-30 g / L; The pH of the fermentation is 7-9; The fermentation medium includes sodium chloride, and the concentration of the sodium chloride is 30-50 g / L.

4. Use of the strain according to claim 1 in the fermentation production of polyhydroxyalkanoates.

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