Engineered microorganisms

Abstract

The invention relates to the field of metabolic engineering, in particular to an engineered microorganism as well as a construction method and application thereof. A first gene and a second gene are simultaneously expressed in the engineered microorganism, the first gene is a perforin protein coding gene with a nucleotide sequence as shown in SEQ ID NO: 1, and the second gene is a coding gene of hydrolase protein with a nucleotide sequence as shown in SEQ ID NO: 2. The engineered microorganism can complete the extraction of intracellular target products, reduce or completely replace the addition of exogenous enzyme preparations, and significantly reduce the use cost of enzyme.

Description

Technical Field

[0001] This application relates to the field of metabolic engineering, specifically to an engineered microorganism, its construction method, and its uses. Background Technology

[0002] Engineered microorganisms have been increasingly used in the production of antibodies, peptides and proteins (such as recombinant human insulin, glutathione, and collagen peptides), lipids (such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and diglycerides (DAG),) and small molecules (such as coenzyme Q10, inositol, and guanylic acid). For these intracellular products (such as PHA), traditional extraction methods mainly include organic solvent extraction, chemical reagent extraction, physical methods, and enzymatic methods. Enzymatic extraction has become increasingly widely used due to its advantages, including mild conditions, minimal impact on the structure and properties of PHA, and higher quality of the obtained PHA.

[0003] Related technologies have mentioned methods for extracting PHA, including glucosidase hydrolysis, adding lysins to dissolve cell walls, and enzymatic treatment of the bacterial cells with specific enzymes. However, the enzymes used in these intracellular product extraction methods are not self-produced and must be purchased externally. This not only leads to unstable supply and high costs but also makes it difficult to adapt to changing production conditions and optimize with the production system. Alternatively, it may rely on physical or chemical methods such as ultrasound or alkali treatment, resulting in high energy consumption. Furthermore, the process involves multiple steps, making it complex, increasing the risk of introducing impurities, and making quality control more difficult.

[0004] Therefore, there is an urgent need for an engineered microorganism that can simply and effectively provide its own enzymes for extracting the target product produced intracellularly, and its applications. Summary of the Invention

[0005] The present invention aims to at least partially solve one of the technical problems in the related art.

[0006] Therefore, an embodiment of the first aspect of the present invention provides an engineered microorganism that simultaneously expresses a first gene and a second gene, wherein the first gene is a perforin protein encoding gene having the nucleotide sequence shown in SEQ ID NO:1, and the second gene is a hydrolase protein encoding gene having the nucleotide sequence shown in SEQ ID NO:2. The engineered microorganism of the present invention can utilize its own produced enzymes to extract intracellular target products, reducing or completely replacing the addition of exogenous enzyme preparations, and significantly reducing the cost of enzyme use.

[0007] In some embodiments, the engineered microorganisms also express a third gene for intracellular production of the target product.

[0008] The target products include polyhydroxyalkanoate (PHA), 3-hydroxybutyrate homopolymer (PHB), reduced coenzyme Q10, glutathione, recombinant human insulin, collagen, inositol, and docosahexaenoic acid (DHA).

[0009] In some embodiments, the target product includes PHA, inositol, and reduced coenzyme Q10. The engineered microorganisms of this invention can utilize self-produced enzymes to extract intracellular target products, and these self-produced enzymes exhibit high specificity for the extraction of products such as PHA, precisely targeting the intended substance and significantly improving product purity.

[0010] In some embodiments, the engineered microorganism comprises one or more copies of the first gene and the second gene.

[0011] In some embodiments, the engineered microorganism is derived from Halomonas bluephagenesis TD01.

[0012] In some embodiments, the engineered microorganism is derived from Halomonas bluephagenesis TD01.

[0013] In some embodiments, based on the fact that the third gene is a gene for producing PHA intracellularly, the engineered microorganism is derived from Halomonas bluephagenesis TD01.

[0014] Based on the fact that the third gene is for the intracellular production of reduced coenzyme Q10, the engineered microorganism is derived from *Rhodospirillum rubrum*, preferably *Rhodospirillum rubrum*. Microbial culture preservation number For ATCC11170 ;

[0015] Based on the fact that the third gene is used for intracellular production of inositol, the engineered microorganism is derived from Corynebacterium glutamicum, preferably with the strain preservation number NC_003450.

[0016] A second aspect of the present invention provides a method for constructing engineered microorganisms as described in any embodiment of the first aspect of the present invention, comprising:

[0017] A first gene and a second gene are inserted into the genome of the microorganism to enable its expression in the microorganism, wherein the first gene is a perforin protein encoding gene having a nucleotide sequence as shown in SEQ ID NO:1, and the second gene is a hydrolase protein encoding gene having a nucleotide sequence as shown in SEQ ID NO:2.

[0018] In some embodiments, the engineered microorganism further expresses a third gene for intracellular production of the target product, wherein the engineered microorganism is derived from *Halomonas bluephagenesis* TD01, based on the third gene being a gene for intracellular production of PHA; or wherein the engineered microorganism is derived from *Rhodospirillum rubrum*, preferably with the strain accession number ATCC11170, based on the third gene being a gene for intracellular production of reduced coenzyme Q10; or wherein the engineered microorganism is derived from *Corynebacterium glutamicum*, preferably with the strain accession number NC_003450, based on the third gene being a gene for intracellular production of inositol; or wherein the engineered microorganism is used to extract the target product from other microorganisms for intracellular production, wherein the engineered microorganism is derived from *Halomonas bluephagenesis* TD01.

[0019] The third aspect of the present invention provides the use of engineered microorganisms in the extraction of a target product, characterized in that it includes: using engineered microorganisms according to any embodiment of the first aspect of the present invention or engineered microorganisms prepared by the method according to any embodiment of the second aspect of the present invention to produce the target product by substrate fermentation.

[0020] In some embodiments, the fermentation may also be carried out using other microorganisms for intracellular production of the desired product.

[0021] In some embodiments, based on the target product being PHA, the use further includes performing at least one of the following processes: centrifugation, pH adjustment, and surfactant addition.

[0022] In some embodiments, the substrate includes glucose, sucrose, peptone, and trace elements.

[0023] The advantages and technical effects brought about by the independent claims according to the embodiments of the present invention are as follows:

[0024] i) The engineered microorganisms of the present invention can use their own enzymes to extract intracellular target products, reduce or completely replace the addition of exogenous enzyme preparations, and significantly reduce the cost of enzyme use.

[0025] ii) The engineered microorganisms of the present invention can use self-produced enzymes to extract intracellular target products, and the self-produced enzymes have high specificity for the extraction of products such as PHA, can accurately act on target substances, and significantly improve product purity.

[0026] iii) The engineered microorganisms of this invention can use their own enzymes to extract intracellular target products. The process involves fewer steps and is simpler. It not only reduces the number of centrifugation cycles but also ensures that the product purity and other parameters meet the standards, saves more than 30% of water, and reduces the risk of introducing impurities, which helps with quality control. Detailed Implementation

[0027] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0028] Engineered microorganisms have been increasingly used in the production of antibodies, peptides and proteins (such as recombinant human insulin, glutathione, and collagen peptides), lipids (such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and diglycerides (DAG),) and small molecules (such as coenzyme Q10, inositol, and guanylic acid). For these intracellular products (such as PHA), traditional extraction methods mainly include organic solvent extraction, chemical reagent extraction, physical methods, and enzymatic methods. Enzymatic extraction has become increasingly widely used due to its advantages, including mild conditions, minimal impact on the structure and properties of PHA, and high-quality PHA. Furthermore, this production method does not use organic solvents, thus significantly reducing costs and posing virtually no harm to the environment or operators.

[0029] Related technologies have mentioned the use of enzymatic extraction in the process of extracting intracellular products. For example, JP2024037032A mentions that glucosidase hydrolysis is performed during the extraction of PHA, which helps to reduce the nitrogen content in powder products and improve product quality; JP2023108910A mentions adding lysin to dissolve the cell wall during the extraction process, followed by adding protease to remove surface proteins of PHA particles. Through certain extraction operations, it helps to obtain PHA products with lower water content; CN116323643A describes that by enzymatically treating the bacterial cells with specific enzymes, the aggregation of PHB copolymer particles during the manufacturing process can be inhibited, resulting in PHB copolymers with high bulk density; in addition, patents CN103740777A, CN102250179B, CN118745219A and CN115404246B respectively involve the extraction of reduced coenzyme Q10, recombinant human insulin, collagen and DHA through fermentation.

[0030] However, the enzymes used in the above-mentioned intracellular product extraction schemes are not produced in-house and must be purchased from external sources. This not only leads to unstable supply and high costs, but also makes it difficult to adapt to changing production conditions and to optimize the production system in synergy. Alternatively, they rely on physical or chemical methods such as ultrasound or alkali treatment, which consume a lot of energy. Furthermore, the process involves multiple steps, making the process complex, increasing the risk of introducing impurities, and making quality control more difficult.

[0031] Therefore, there is an urgent need for an engineered microorganism that can simply and effectively provide its own enzymes for extracting the target products produced intracellularly, and its applications, thereby optimizing the production process, further improving product quality, and conforming to the concept of green and environmentally friendly development.

[0032] This invention provides a self-producing enzyme strain (i.e., the engineered microorganism described herein). Utilizing this strain, enzymes with specific activities and selectivity can be efficiently synthesized during its own metabolism. These self-produced enzymes exhibit highly specific effects on the microbial cell structure containing the target product, accurately identifying and decomposing key components of the cell wall, cell membrane, and other impurities, enabling rapid and efficient release of the product from the cell. Compared to traditional methods, this not only significantly reduces enzyme usage costs but also substantially shortens extraction time and improves extraction efficiency, while minimizing adverse effects on the structure and properties of the target product (such as PHA), resulting in a product with high purity and superior quality. Furthermore, the entire extraction process is more environmentally friendly, reducing negative environmental impacts and providing a novel, efficient, and sustainable technological pathway for the large-scale industrial extraction of intracellular molecules such as PHA.

[0033] An embodiment of the first aspect of this invention provides an engineered microorganism that simultaneously expresses a first gene and a second gene, wherein the first gene is a perforin protein encoding gene having the nucleotide sequence shown in SEQ ID NO:1, and the second gene is a hydrolase protein encoding gene having the nucleotide sequence shown in SEQ ID NO:2. The engineered microorganism of this invention can utilize its own produced enzymes to extract intracellular target products, reducing or completely replacing the addition of exogenous enzyme preparations, and significantly reducing the cost of enzyme use.

[0034] In some embodiments, the perforin protein encoding gene has the nucleotide sequence shown in SEQ ID NO:1: (SEQ ID NO:1).

[0035] The amino acid sequence corresponding to the perforin protein encoding gene is shown in SEQ ID NO:3 below:

[0036] MAAPRISFSPSDILFGVLDRLFKDNATGKVLASRVAVVILLFIMAIVWYRGDSFFEYYKQSKYETYSEIIEKERTARFESVALEQLQIVHISSEADFSAVYSFRPKNLNYF VDIIAYEGKLPSTISEKSLGGYPVDKTMDEYTVHLNGRHYYSNSKFAFLPTKKPTPEINYMYSCPYFNLDNIYAGTITMYWYRNDHISNDRLESICAQAARILGRAK(SEQ ID N0:3).

[0037] In some embodiments, the hydrolase protein encoding gene has the nucleotide sequence shown in SEQ ID NO:2: ATGAACATCTTCGAAATGCTGCGCATCGACGAAGGCCTGCGCTTAAAAATTTACAAAGACACCGAAGGCTACTACACCATCGGCATCGGCCACCTGCTTACCAAAAGCCCCTCTCTGAATGCCGCCAAAAGCGAACTGGACAAAGCCATCGGCCGCAACTGCAACGGCGTTATTACCAAAGACGAAGCCGAAAAACTGTTCAACCAGGACGTGGACGCCGCCGTGCGTGGTATTCTTCGTAA TGCCAAACTGAAACCGGTGTACGACAGCCTGGACGCCGTTCGTCGTTGTGCCCTGATTAATATGGTGTTTCAGATGGGCGAAACCGGCGTGGCCGGTTTCACCAATAGCCTGCGTATGCTGCAACAG AAACGCTGGGACGAAGCCGCCGTTAACCTGGCCAAAAGCCGCTGGTATAACCAGACCCCGAACCGCGCTAAACGCGTGATTACTACTTTCCGCACTGGTACCTGGGACGCCTATAAAAACCTGT(SEQ ID NO:2).

[0038] The amino acid sequence corresponding to the gene encoding the hydrolase protein is shown in SEQ ID NO:4 below:

[0039] MNIFEMLRIDEGLRLKIYKDTEGYYTIGIGHLLTKSPSLNAAKSELDKAIGRNCNGVITKDEAEKLFNQDVDAAVRGILRNAKLKPVYDSLDAVRRCALINMVFQMGETGVAGFTNSLRMLQQKRWDEAAVNLAKSRWYNQTPNRAKRVITTFRTGTWDAYKNL (SEQ ID NO: 4).

[0040] In some embodiments, the engineered microorganisms also express a third gene for the intracellular production of a target product, wherein the target product includes polyhydroxyalkanoate (PHA), 3-hydroxybutyrate homopolymer (PHB), reduced coenzyme Q10, glutathione, recombinant human insulin, collagen, inositol, and docosahexaenoic acid (DHA).

[0041] In some embodiments, the target product includes PHA, inositol, and reduced coenzyme Q10. The engineered microorganisms of this invention can utilize self-produced enzymes to extract intracellular target products, and these self-produced enzymes exhibit high specificity for the extraction of products such as PHA, precisely targeting the intended substance and significantly improving product purity.

[0042] In some embodiments, the enzyme-producing strain may also possess the gene for producing the target product, i.e., a third gene. In other words, transforming a PHA-producing engineered bacterium with a plasmid containing the perforin-hydrolyzed enzyme gene sequence yields an enzyme-producing PHA engineered bacterium, which is then used in the subsequent extraction process of the fermentation product. In other embodiments, the enzyme-producing strain and the strain producing the target product may be two separate strains.

[0043] In some embodiments, the engineered microorganism comprises one or more copies of the first gene and the second gene.

[0044] In some embodiments, the engineered microorganism is derived from Halomonas.

[0045] In some embodiments, the engineered microorganism is co-cultured with a second microorganism derived from Halomonasbluephagenesis TD01, which contains a third gene expressing a target product for intracellular production. In this embodiment, the enzyme-producing strain and the product-producing strain are two separate strains.

[0046] In some embodiments, based on the fact that the third gene is a gene for producing PHA intracellularly, the engineered microorganism is derived from Halomonas bluephagenesis TD01 (specifically cited from patent application CN102120973A).

[0047] Based on the fact that the third gene is used to produce reduced coenzyme Q10 intracellularly, the engineered microorganism is derived from Rhodospirillum rubrum, preferably with the strain preservation number ATCC11170.

[0048] Based on the fact that the third gene is used for intracellular production of inositol, the engineered microorganism is derived from Corynebacterium glutamicum, preferably with the strain preservation number NC_003450.

[0049] A second aspect of the present invention provides a method for constructing engineered microorganisms as described in any embodiment of the first aspect of the present invention, comprising:

[0050] A first gene and a second gene are inserted into the genome of the microorganism to enable its expression in the microorganism, wherein the first gene is a perforin protein encoding gene having a nucleotide sequence as shown in SEQ ID NO:1, and the second gene is a hydrolase protein encoding gene having a nucleotide sequence as shown in SEQ ID NO:2.

[0051] In some embodiments,

[0052] Based on the fact that the third gene is used to produce PHA intracellularly, the engineered microorganism is derived from Halomonas bluephagenesis TD01 (this strain is specifically cited from patent application CN102120973A).

[0053] Based on the fact that the third gene is used to produce reduced coenzyme Q10 intracellularly, the engineered microorganism is derived from Rhodospirillum rubrum, preferably with the strain preservation number ATCC11170.

[0054] Based on the fact that the third gene is used for intracellular production of inositol, the engineered microorganism is derived from Corynebacterium glutamicum, preferably with the strain preservation number NC_003450; or based on the use of the engineered microorganism for extracting the target product from the intracellular production of other microorganisms, the engineered microorganism is derived from Halomonas bluephagenesis TD01.

[0055] It is understood that the first, second, and / or third genes described herein are transformed using plasmids to construct engineered microorganisms. This application does not intend to limit the specific knock-in steps and plasmids.

[0056] The third aspect of the present invention provides the use of engineered microorganisms in the extraction of a target product, characterized in that it includes: using engineered microorganisms according to any embodiment of the first aspect of the present invention or engineered microorganisms prepared by the method according to any embodiment of the second aspect of the present invention to produce the target product by substrate fermentation.

[0057] In some embodiments, the fermentation may also be carried out using other microorganisms for intracellular production of the desired product.

[0058] In some embodiments, based on the target product being PHA, the use further includes performing at least one of the following processes: centrifugation, pH adjustment, and surfactant addition.

[0059] In some embodiments, the surfactant may be one or more of the following: anionic surfactants (sodium stearate, sodium lignosulfonate, sodium cocoyl sulfate, and potassium dodecyl phosphate, etc.); nonionic surfactants (alkoxylated alcohols, polysorbates, etc.), amphoteric surfactants, and cationic surfactants (quaternary ammonium salts, di-long-chain alkyl quaternary ammonium salts, etc.). The pH of the system is adjusted to 8.5, and 0.02-0.3% (wt) of nonionic surfactant (one or more of the following: fatty acid polyoxyethylene ester, fatty alcohol polyoxyethylene ether, polyoxyethylene alkylamine, polyoxyethylene alkylolamide, and polyglycerol fatty acid ester).

[0060] In some embodiments, the substrate includes glucose, sucrose, peptone, and trace elements.

[0061] In some embodiments, during fermentation, as PHA accumulates in the transformed strain containing the perforin-hydrolase gene sequence (i.e., SEQ ID NO:1 and SEQ ID NO:2), the PHA particles bind to the promoter and initiate the expression of the downstream perforin-hydrolase gene, leading to cell autolysis.

[0062] 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.

[0063] Example

[0064] Example 1: Obtaining engineered microorganisms

[0065] The target strain (e.g., Halomonas bluephagenesis TD01, described in patent application publication CN102120973A) was transformed with a plasmid containing the perforin-hydrolyzed enzyme gene sequence (i.e., SEQ ID NO:1 and SEQ ID NO:2). The promoter was PhaP1 derived from Halomonas. The transformed strain was then plated on antibiotic resistance plates to screen for positive clones. TD01 can produce PHA.

[0066] It should be noted that during this fermentation process, as PHA accumulates, PHA particles bind to the promoter and initiate the expression of downstream perforin-hydrolyzing enzyme genes, leading to cell autolysis. Specifically:

[0067] This embodiment uses TD01 as the starting strain. By constructing a plasmid and combining it with transformation, perforin-hydrolyzed enzyme gene sequences were inserted at sites G4, G7, GY13, and GY16 to finally obtain the engineered microorganism pAB-1. The specific steps are described below.

[0068] 1.1 Obtaining strain pAB-1 based on strain TD01

[0069] (1) The first knock-in plasmid pAB-1 was constructed on the backbone of plasmid pSEVA341 by homologous recombination.

[0070] (2) Plasmids pAB-1 and pQ08 were transformed into large-particle strain TD-01 of Halomonas bluephagenesis via conjugation transformation.

[0071] (3) The perforin-hydrolase gene sequence was successfully integrated into the genome of Halomonasbluephagenesis TD-01 by PCR and sequencing, and the pAB-1 strain was obtained.

[0072] (4) Subculture continuously in 60 LB medium and dilute 10 6The culture was spread onto 60 LB agar plates and grown for 36 h. Single colonies were picked and streaked onto spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates to identify the pAB-1 strain with CRISPR plasmid loss.

[0073] The perforin-hydrolase gene sequence was successfully transferred.

[0074] Example 2

[0075] In this embodiment, the fermentation broth obtained by fermenting the engineered microorganism (hereinafter referred to as the self-producing enzyme strain) obtained in Example 1 underwent additional centrifugation and pH adjustment to extract the intracellular target product PHA. The specific steps are as follows:

[0076] 2.1 Fermentation and Extraction

[0077] (1) Fermentation steps:

[0078] The pAB-1 strain obtained in Example 2.1 was expanded and cultured under the following conditions: 1% was transferred to LB medium (specifically composed of tryptone, yeast extract, and sodium chloride, etc.) and cultured for 16 h. Then, glucose, sucrose, peptone, and trace elements were added as substrates and fermented at 37°C and 220 rpm for 48 h.

[0079] (2) Centrifuge the fermentation broth using a rotor centrifuge. Set the centrifuge parameters to 3000-10000 rpm for 5-15 min.

[0080] (3) Resuspend the precipitate obtained by centrifugation in tap water to the volume of the raw material liquid.

[0081] (4) Heat the liquid to 40-60℃ and add 0.05-0.3%wt sodium stearate (or one or more of anionic surfactants such as sodium stearate, sodium lignosulfonate, sodium cocoyl sulfate and potassium dodecyl phosphate, etc.; nonionic surfactants such as alkoxylated alcohols, polysorbates, etc., zwitterionic surfactants and cationic surfactants such as quaternary ammonium salts, double long-chain alkyl quaternary ammonium salts, etc.). Adjust the pH of the system to 8.5 with HCl or NaOH, and add 0.02-0.3% (wt) nonionic surfactant fatty acid polyoxyethylene ester (or one or more of fatty acid polyoxyethylene ester, fatty alcohol polyoxyethylene ether, polyoxyethylene alkylamine, polyoxyethylene alkylolamide, polyglycerol fatty acid ester). Then keep it at 40-60℃ and stir for 1 hour.

[0082] (5) Adjust the pH of the system until pH = 8.0-11.0, and then keep it at 40-60℃ and stir for 1 hour.

[0083] (6) Centrifuge at 3000-10000 rpm for 5-10 min, collect the precipitate and resuspend it in water to the original volume; heat to 60℃, adjust pH to 8.0-10.0, add 0.02-0.3% (wt) of hydrolytic enzyme cellulase (or one or more of cellulase, pectinase, papain, lipase, nuclease); keep warm at 60℃ and stir for 1 h.

[0084] (7) Use a 2um filter membrane for pressure filtration. Add water to the filter cake to resuspend it to its original volume and stir and wash for 0.5h. Filter the filter cake and dry it in an oven at 65℃.

[0085] It should be noted that adding additional hydrolytic enzymes refers to whether or not the hydrolytic enzymes are added after the (first) addition of surfactant. The step of adding hydrolytic enzymes before the second half of the process, before pressure filtration, is necessary and fixed.

[0086] 2.2 Product Testing

[0087] The particle size distribution before pressure filtration was tested according to GB / T 19077-2016, laser diffraction method. PHA purity was determined by gas chromatography according to Chapter 0521 of the Pharmacopoeia of the People's Republic of China (2020 Edition), Part IV. Nitrogen content and MFR were tested according to GB / T 30293, biomanufacturing of polyhydroxyalkanoates. Molecular weight was determined by gel permeation chromatography (GPC) for the determination of low molecular weight components in chemical polymers. The test results are shown in Table 2.

[0088] Example 3

[0089] (1) See step (1) of Example 2.1 for fermentation.

[0090] (2) Heat the liquid to 40-60℃, and add 0.05-0.3% wt of sodium stearate (or one or more of anionic surfactants such as sodium stearate, sodium lignosulfonate, sodium cocoyl sulfate and potassium dodecyl phosphate, etc.; nonionic surfactants such as alkoxylated alcohols, polysorbates, etc., zwitterionic surfactants and cationic surfactants such as quaternary ammonium salts, double long-chain alkyl quaternary ammonium salts, etc.) directly to the fermentation liquid. Adjust the pH of the system to 8.5 with HCl or NaOH, and add 0.02-0.3% (wt) of nonionic surfactant fatty acid polyoxyethylene ester (or one or more of fatty acid polyoxyethylene ester, fatty alcohol polyoxyethylene ether, polyoxyethylene alkylamine, polyoxyethylene alkylolamide, polyglycerol fatty acid ester). Then keep it at 40-60℃ and stir for 1 hour.

[0091] (3) Adjust the pH of the system until pH = 8.0-11.0, and then keep it at 40-60℃ and stir for 1 hour.

[0092] (4) Centrifuge at 3000-10000 rpm for 5-10 min, collect the precipitate and resuspend it in water to the original volume; heat to 60℃, adjust pH to 8.0-10.0, add 0.02-0.3% (wt) of hydrolytic enzyme (which can be one or more of cellulase, pectinase, papain, lipase, nuclease); keep warm at 60℃ and stir for 1 h.

[0093] (5) Use a 2um filter membrane for pressure filtration. Add water to the filter cake to resuspend it to its original volume and stir and wash for 0.5h. Filter the filter cake and dry it in an oven at 65℃.

[0094] (6) Refer to Example 2 for product testing. The test results are shown in Table 2.

[0095] Example 4

[0096] (1) See step (1) of Example 2.1 for fermentation.

[0097] (2) Centrifuge the fermentation broth using a rotor centrifuge. Set the centrifuge parameters to 3000-10000 rpm for 5-15 min.

[0098] (3) Resuspend the precipitate obtained by centrifugation in tap water to the volume of the raw material liquid.

[0099] (4) Heat the liquid to 40-60℃ and add 0.05-0.3%wt sodium stearate (or one or more of anionic surfactants such as sodium stearate, sodium lignosulfonate, sodium cocoyl sulfate and potassium dodecyl phosphate, etc.; nonionic surfactants such as alkoxylated alcohols, polysorbates, etc., zwitterionic surfactants and cationic surfactants such as quaternary ammonium salts, double long-chain alkyl quaternary ammonium salts, etc.). Adjust the pH of the system to 8.5 with HCl or NaOH, and add 0.02-0.3% (wt) nonionic surfactant fatty acid polyoxyethylene ester (or one or more of fatty acid polyoxyethylene ester, fatty alcohol polyoxyethylene ether, polyoxyethylene alkylamine, polyoxyethylene alkylolamide, polyglycerol fatty acid ester). Then keep it at 40-60℃ and stir for 1 hour.

[0100] (5) Adjust the pH of the system until pH = 8.0-11.0, and then keep it at 40-60℃ and stir for 1 hour.

[0101] (6) Without centrifugation, heat to 60℃, adjust pH to 8.0-10.0, add 0.02-0.3% (wt) hydrolytic enzyme (which can be one or more of cellulase, pectinase, papain, lipase, nuclease); keep warm at 60℃ and stir for 1 hour.

[0102] (7) Use a 2um filter membrane for pressure filtration. Add water to the filter cake to resuspend it to its original volume and stir and wash for 0.5h. Filter the filter cake and dry it in an oven at 65℃.

[0103] (8) Refer to Example 2 for product testing. The test results are shown in Table 2.

[0104] Example 5

[0105] (1) See step (1) of Example 2.1 for fermentation.

[0106] (2) Centrifuge the fermentation broth using a rotor centrifuge. Set the centrifuge parameters to 3000-10000 rpm for 5-15 min.

[0107] (3) Resuspend the precipitate obtained by centrifugation in tap water to the volume of the raw material liquid.

[0108] (4) Heat the liquid to 40-60℃ and add 0.02-0.3% wt sodium stearate (or one or more of anionic surfactants such as sodium stearate, sodium lignosulfonate, sodium cocoyl sulfate and potassium dodecyl phosphate, etc.; nonionic surfactants such as alkoxylated alcohols, polysorbates, etc., zwitterionic surfactants and cationic surfactants such as quaternary ammonium salts, double long-chain alkyl quaternary ammonium salts, etc.). Adjust the pH of the system to 8.5 with HCl or NaOH, and add 0.02-0.3% (wt) nonionic surfactant fatty acid polyoxyethylene ester (or one or more of fatty acid polyoxyethylene ester, fatty alcohol polyoxyethylene ether, polyoxyethylene alkylamine, polyoxyethylene alkylolamide, polyglycerol fatty acid ester). Then keep it at 40-60℃ and stir for 1 hour.

[0109] (5) Without centrifugation, heat to 60℃, adjust pH to 8.0-10.0, add 0.02-0.3% (wt) of hydrolytic enzyme cellulase (which may also be one or more of cellulase, pectinase, papain, lipase, nuclease) and 0.4% (wt) of nonionic surfactant fatty alcohol polyoxyethylene ether (which may also be one or more of fatty alcohol polyoxyethylene ether, Triton X100, alkylphenol polyoxyethylene ether, polyoxyethylene fatty acid ester, Tween-80); keep warm at 60℃ and stir for 1 hour.

[0110] (6) Use a 2um filter membrane for pressure filtration. Add water to the filter cake to resuspend it to its original volume and stir and wash for 0.5h. Filter the filter cake and dry it in an oven at 65℃.

[0111] (7) Refer to Example 2 for product testing. The test results are shown in Table 2.

[0112] Example 6

[0113] (1) See step (1) of Example 2.1 for fermentation.

[0114] (2) Centrifuge the fermentation broth using a rotor centrifuge. Set the centrifuge parameters to 3000-10000 rpm for 5-15 min.

[0115] (3) Resuspend the precipitate obtained by centrifugation in tap water to the volume of the raw material liquid.

[0116] (4) Heat the liquid to 40-60℃ and add 0.02-0.3% wt sodium stearate (or one or more of anionic surfactants such as sodium stearate, sodium lignosulfonate, sodium cocoyl sulfate and potassium dodecyl phosphate, etc.; nonionic surfactants such as alkoxylated alcohols, polysorbates, etc., zwitterionic surfactants and cationic surfactants such as quaternary ammonium salts, double long-chain alkyl quaternary ammonium salts, etc.). Adjust the pH of the system to 8.5 with HCl or NaOH, and add 0.02-0.3% (wt) nonionic surfactant fatty acid polyoxyethylene ester (or one or more of fatty acid polyoxyethylene ester, fatty alcohol polyoxyethylene ether, polyoxyethylene alkylamine, polyoxyethylene alkylolamide, polyglycerol fatty acid ester). Then keep it at 40-60℃ and stir for 1 hour.

[0117] (5) Adjust the pH of the system until pH = 8.0-11.0, and then keep it at 40-60℃ and stir for 1 hour.

[0118] (6) Without centrifugation, heat to 60℃, adjust pH to 8.0-10.0, add 0.02-0.3% (wt) of hydrolytic enzyme cellulase (which may also be one or more of cellulase, pectinase, papain, lipase, nuclease) and 0.3% (wt) of anionic surfactant (which may be one or more of sodium stearate, sodium lignosulfonate, cocoyl alcohol sulfate, potassium ammonium dodecyl phosphate, sodium lauryl sulfate); keep warm at 60℃ and stir for 1 hour.

[0119] (7) Use a 2um filter membrane for pressure filtration. Add water to the filter cake to resuspend it to its original volume and stir and wash for 0.5h. Filter the filter cake and dry it in an oven at 65℃.

[0120] (8) Refer to Example 2 for product testing. The test results are shown in Table 2.

[0121] Example 7

[0122] (1) PHA fermentation was carried out using strains that do not produce their own enzymes (as control groups for Examples 2-6 and 8).

[0123] (2) Refer to steps (2)-(5) of Example 6.

[0124] (3) Centrifuge at 3000-10000 rpm for 5-10 min, collect the precipitate and resuspend it in water to the original volume; heat to 60℃, adjust pH to 8.0-10.0, add 0.02-0.3% (wt) of hydrolytic enzyme cellulase (or one or more of cellulase, pectinase, papain, lipase, nuclease), and keep warm at 60℃ with stirring for 1 h.

[0125] (4) Use a 2um filter membrane for pressure filtration. Add water to the filter cake to resuspend it to its original volume and stir and wash for 0.5h. Filter the filter cake and dry it in an oven at 65℃.

[0126] (5) Refer to Example 2.2 for product testing. The test results are shown in Table 2.

[0127] Example 8

[0128] (1) Extraction of PHA fermentation broth in a 100-liter system using a self-produced enzyme strain.

[0129] (2) Use a centrifuge at a speed of 7000-9500 rpm to separate the solid and liquid phases of the fermentation broth, and resuspend the heavy phase with tap water to the volume of the raw material broth.

[0130] (3) Heat the liquid to 40-60℃, add 0.05-0.3wt% sodium stearate (or one or more of anionic surfactants such as sodium stearate, sodium lignosulfonate, cocoyl alcohol sulfate and potassium dodecyl phosphate, etc.); nonionic surfactants such as alkoxylated alcohols, polysorbates, zwitterionic surfactants and cationic surfactants such as quaternary ammonium salts, double long-chain alkyl quaternary ammonium salts, etc.), adjust the pH of the system to 8.5, add 0.02-0.3% (wt) nonionic surfactant fatty acid polyoxyethylene ester (or one or more of fatty acid polyoxyethylene ester, fatty alcohol polyoxyethylene ether, polyoxyethylene alkylamine, polyoxyethylene alkylolamide, polyglycerol fatty acid ester), and then keep it at 40-60℃ and stir for 1 hour.

[0131] (4) Adjust the pH of the system to 8.0-11.0, keep warm at 40-60℃ and stir for 1 hour.

[0132] (5) Use a centrifuge at 7000-9500 rpm to separate the solid and liquid phases of the fermentation broth; add water to the heavy phase to resuspend it to the original volume; raise the temperature to 60℃, adjust the pH to 8.0-10.0, and add 0.02-0.3% (wt) of hydrolytic enzyme cellulase (or one or more of cellulase, pectinase, papain, lipase, and nuclease); keep warm at 60℃ and stir for 1 hour.

[0133] (6) Collect the product by centrifugation or pressure filtration, and obtain the target product by spray drying or double cone drying.

[0134] (7) Refer to Example 2 for product testing. The test results are shown in Table 2.

[0135] Example 9

[0136] (1) Use non-self-producing enzyme strains to carry out reduced coenzyme Q10 fermentation (taking Rhodospirillum rubrum as an example: clone the key enzyme gene into the bacteria and enhance its expression to obtain a high-yield strain of reduced coenzyme Q10, in which the key enzyme gene is the 2,3-dimethoxy-5-methyl-6-decylisopentenylbenzoquinone synthase gene). Then, use a rotor centrifuge to centrifuge the fermentation broth. The centrifuge parameters are set to 3000-10000 rpm for 5-15 min. The centrifuged precipitate is resuspended in tap water to the volume of the raw material broth.

[0137] (2) Adjust the pH of the solution to 3-6 with citric acid, add 0.02-0.3% (wt) of hydrolytic enzyme cellulase (or one or more of cellulase, pectinase, papain, lipase, nuclease, etc.), and keep warm at 20-45℃ and stir for 2 hours to fully enzymatically hydrolyze and break the cell wall.

[0138] (3) Centrifuge at 3000-8000 rpm for 5 min, and spray dry the precipitate to obtain the crude product.

[0139] (4) Add ethanol to make it account for 75%-95% of the total mass of the system, and keep it at 20-45℃ and stir for 1 hour.

[0140] (5) Add hexane for extraction three times, combine the hexane and perform vacuum distillation. After the organic solvent is completely removed, the reduced coenzyme Q10 dry powder is obtained.

[0141] The results showed that the purity reached 95.67%, the nitrogen content was 0.1753%, and the content of reduced coenzyme Q10 was 0.45 mg / g (wet bacterial weight).

[0142] Example 10

[0143] 1. Reduced coenzyme Q10 fermentation was carried out using a self-produced enzyme strain (hodospirillum rubrum was used as an example, but unlike Example 9, this strain was transformed with a plasmid containing the perforin-hydrolase gene). The fermentation broth was then centrifuged using a rotor centrifuge with centrifuge parameters set to 3000-10000 rpm for 5-15 min. The centrifuged precipitate was resuspended in tap water to the original volume of the raw material broth.

[0144] (2) Adjust the pH of the solution to 3-6 with citric acid, and keep it at 20-45℃ for 2 hours with stirring to allow for full enzymatic hydrolysis and cell wall breaking.

[0145] (3) Centrifuge at 3000-8000 rpm for 5 min, and spray dry the precipitate to obtain the crude product.

[0146] (4) Add ethanol to make it account for 75%-95% of the total mass of the system, and keep it at 20-45℃ and stir for 1 hour.

[0147] (5) Add hexane for extraction three times, combine the hexane and perform vacuum distillation. After the organic solvent is completely removed, the reduced coenzyme Q10 dry powder is obtained.

[0148] The results showed that the purity reached 98.65%, the nitrogen content was 0.0243%, and the content of reduced coenzyme Q10 was 0.55 mg / g (wet bacterial weight).

[0149] Example 11

[0150] (1) Inositol fermentation was carried out using non-self-producing enzyme strains (Corynebacterium glutamicum was used as an example), and then the fermentation broth was centrifuged using a rotor centrifuge. The centrifuge parameters were set to 3000-10000 rpm for 5-15 min. The centrifuged precipitate was resuspended in water to the volume of the raw material broth.

[0151] (2) Adjust the pH to 3-6, add 0.02-0.3% (wt) of hydrolytic enzyme (which can be one or more of cellulase, pectinase, papain, lipase, nuclease), and keep warm at 60-80℃ for 2 hours to fully enzymatically hydrolyze and break the cell wall.

[0152] (3) Add chloromethyl polystyrene resin and alkali to the cell wall breaking liquid and react for 1-5 hours. After the reaction is completed, filter the resin and wash it with water and alcohol.

[0153] (4) The resin with glutathione is uniformly dispersed in a solvent, and trifluoroacetic acid and triethylsilane are added to react for 1-5 hours. After the reaction is completed, the mixture is filtered, the filter cake is washed with water 1-3 times, the filtrates are combined, and the solvent is removed under reduced pressure to obtain crude glutathione.

[0154] (5) The crude glutathione is redissolved in ethanol, filtered, and the filter cake is washed with ethanol and dried to obtain the pure glutathione.

[0155] The results showed that the purity was 91.8% and the nitrogen content was 0.1665% as determined by LC-MS.

[0156] Example 12

[0157] (1) Inositol fermentation was carried out using a self-produced enzyme strain (Corynebacterium glutamicum was used as an example, but unlike Example 11, this strain was transformed with a plasmid containing the perforin-hydrolase gene). The fermentation broth was then centrifuged in a rotor centrifuge with centrifuge parameters set to 3000-10000 rpm for 5-15 min. The centrifuged precipitate was resuspended in water to the volume of the raw material broth.

[0158] (2) Adjust the pH to 3-6, keep warm at 60-80℃ and stir for 2 hours to allow for full enzymatic hydrolysis and cell wall breaking.

[0159] (3) Add chloromethyl polystyrene resin and alkali to the cell wall breaking liquid and react for 1-5 hours. After the reaction is completed, filter the resin and wash it with water and alcohol.

[0160] (4) The resin with glutathione is uniformly dispersed in a solvent, and trifluoroacetic acid and triethylsilane are added to react for 1-5 hours. After the reaction is completed, the mixture is filtered, the filter cake is washed with water 1-3 times, the filtrates are combined, and the solvent is removed under reduced pressure to obtain crude glutathione.

[0161] (5) The crude glutathione is redissolved in ethanol, filtered, and the filter cake is washed with ethanol and dried to obtain the pure glutathione.

[0162] The results showed that the purity was 99.8% and the nitrogen content was 0.0325% as determined by LC-MS.

[0163] The conditions in Examples 2-12 are listed in Table 1. The product determination results of Examples 2-8 are shown in Table 2.

[0164] Table 1

[0165]

[0166] Table 2

[0167]

[0168] As can be seen from the above examples, the PHA products produced and extracted by each example using the self-producing enzyme strain (Examples 2-5 and 8) can all be filtered through a 2µm filter membrane with a filtration loss of <0.5%; in the example using a non-self-producing enzyme strain (Example 7), the content of substances with a particle size of less than 1µm before filtration is about 2.471%, and the filtration loss is more serious.

[0169] Furthermore, in the relevant examples using self-produced enzyme strains for extraction, the PHA product had a high purity, reaching over 98%, while having a low nitrogen content, meeting market demand; in the relevant examples using non-self-produced enzyme strains for extraction, the PHA product had a lower purity and a higher nitrogen content, resulting in poorer quality.

[0170] In addition, the melt index of the PHA products in each embodiment was measured at 190°C, and the melt index of all of them met the application requirements; the melt index of Example 3 was slightly different from that of the other embodiments, which is presumably related to the reduction of centrifugation steps in its process.

[0171] In summary, the molecular weights of the PHA products in each embodiment are similar, proving that the use of self-produced enzyme strains does not affect the molecular weight of PHA.

[0172] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0173] In this invention, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0174] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An engineered microorganism, characterized in that, The engineered microorganisms simultaneously express the first gene and the second gene. The first gene is a perforin protein encoding gene having the nucleotide sequence shown in SEQ ID NO:1, and the second gene is a hydrolase protein encoding gene having the nucleotide sequence shown in SEQ ID NO:

2.

2. The engineered microorganism according to claim 1, characterized in that, The engineered microorganisms also express a third gene for the intracellular production of the target product. The target products include polyhydroxyalkanoate (PHA), 3-hydroxybutyrate homopolymer (PHB), reduced coenzyme Q10, glutathione, recombinant human insulin, collagen, inositol, and docosahexaenoic acid (DHA). Preferably, the target product includes PHA, inositol, and reduced coenzyme Q10.

3. The engineered microorganism according to claim 1, characterized in that, The engineered microorganism contains one or more copies of the first gene and the second gene.

4. The engineered microorganism according to claim 1, characterized in that, The engineered microorganisms mentioned above are derived from Halomonas bacteria. Preferably, the engineered microorganism is co-cultured with a second microorganism containing a third gene for intracellular production of the target product, the second microorganism being derived from Halomonas bluephagenesis TD01.

5. The engineered microorganism according to claim 2, characterized in that, in Based on the fact that the third gene is used to produce PHA intracellularly, the engineered microorganism is derived from Halomonas bluephagenesis TD01. Based on the fact that the third gene is used to produce reduced coenzyme Q10 intracellularly, the engineered microorganism is derived from Rhodospirillum rubrum, preferably with the strain preservation number ATCC11170. Based on the fact that the third gene is used for intracellular production of inositol, the engineered microorganism is derived from Corynebacterium glutamicum, preferably with the strain preservation number NC_003450.

6. A method for constructing engineered microorganisms as described in any one of claims 1 to 5, characterized in that, include: A first gene and a second gene are inserted into the genome of the microorganism to enable its expression in the microorganism, wherein the first gene is a perforin protein encoding gene having a nucleotide sequence as shown in SEQ ID NO:1, and the second gene is a hydrolase protein encoding gene having a nucleotide sequence as shown in SEQ ID NO:

2.

7. The method according to claim 6, characterized in that, The engineered microorganisms also express a third gene for intracellular production of the target product. Based on the fact that the third gene is used to produce PHA intracellularly, the engineered microorganism is derived from Halomonas bluephagenesis TD01. Based on the fact that the third gene is used to produce reduced coenzyme Q10 intracellularly, the engineered microorganism is derived from Rhodospirillum rubrum, preferably with the strain preservation number ATCC11170. Based on the fact that the third gene is for the intracellular production of inositol, the engineered microorganism is derived from Corynebacterium glutamicum, preferably with the strain preservation number NC_003450; or The engineered microorganisms are used to extract intracellular products from other microorganisms, and the engineered microorganisms are derived from Halomonas bluephagenesis TD01.

8. The use of engineered microorganisms in the extraction of target products, characterized in that, include: The target product is produced by substrate fermentation using the engineered microorganisms according to any one of claims 1 to 6, or the engineered microorganisms prepared according to claim 6 or 7. Optionally, the fermentation may also include the use of other microorganisms for intracellular production of the desired product.

9. The use according to claim 8, characterized in that, Based on the fact that the target product is PHA, the use also includes performing at least one of the following processes: centrifugation, pH adjustment and surfactant addition.

10. The method according to claim 9, characterized in that, The substrates include glucose, sucrose, peptone, and trace elements.

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