Engineering modified halophilic microorganism, and construction method and use therefor

Abstract

Provided are an engineered halophilic microorganism and a construction method and use therefor. By means of knockout or knockdown of one or more than two genes on a transposon and encoding a transposase, a stable halophilic microorganism is obtained, which has good growth condition, can stably synthesize polyhydroxy alkanoates, and can effectively avoid spontaneous mutation of the strain.

Description

An engineered halophilic microorganism and its construction method and application Technical Field

[0001] The present invention relates to the technical field of microbial cultivation, in particular to an engineered halophilic microorganism and a construction method and application thereof. Background Art

[0002] Industrial microbial fermentation is an industrial production method that uses microorganisms for biological transformation. It is closely related to the microbial strain. Industrial microbial fermentation requires microorganisms to have high metabolic activity to convert substrates into the desired products. Therefore, the stability of the microbial traits directly affects their growth and the stability of the products. Taking halophilic microorganisms as an example, in the production of target products (such as polyhydroxyalkanoates, PHA) by microbial fermentation, open fermentation is adopted, which has the characteristics of high cell density and high yield. However, if the microbial traits are unstable, it will directly affect the product output, yield and the conversion rate of raw materials to final products, and even affect the quality of the product. In severe cases, in large-scale fermentation, the unstable traits will cause the death of a large number of microorganisms, which greatly increases the economic cost and risk of industrial biological production, which is very detrimental to the healthy development of the industry.

[0003] The stability of microorganisms is related to both the organism and its environment. Any organism has a certain error rate when replicating its genetic material, DNA. If these errors are not repaired in a timely manner, they will continue to accumulate during the bacterial propagation process, eventually causing changes in characteristics.

[0004] Transposons, also known as jumping genes, are mobile DNA sequences found widely in the genomes of plants, animals, and microorganisms. They can migrate within or between genomes and possess diverse structural and functional properties. Under the action of transposases, transposons can "transpose" from one genomic location to another, thereby causing genetic variation. Transposons encode transposases, which are primarily responsible for translocation and excision. Due to this property, they are currently commonly used in CRISPR-associated transposase systems. Patent CN116234918A discloses a composition or method for introducing one or more CRISPR-associated Tn7 transposases into a targeted nucleotide sequence to introduce one or more mutations.

[0005] However, the existence of transposons poses a potential threat to the stable expression of genes in the genome.

[0006] For example, the insertion of transposons into the host genome can cause gene recombination and interfere with gene expression (see Rebollo R, Romanish MT, Mager DL. Transposable elements: an abundant and natural source of regulatory sequences for host genes. Annu Rev Genet. 2012; 46: 21-42. doi: 10.1146 / annurev-genet-110711-155621.). The insertion of transposons can inactivate the receptor gene or cause changes such as DNA deletion and inversion, affecting the normal expression of the genome (see Yin Yang. Research on the transposition mechanism and related technologies in transposons [J]. Journal of Chifeng University (Natural Science Edition), 2015, 31(18): 190-191.).

[0007] Furthermore, the literature: Defensive Function of Transposable Elements in Bacteria (Catherine Fan et al., Synthetic Biology, 2019, 8, 2141-2151) also records that the designed gene pathway was introduced into the host for genetic modification to increase product yield. However, after 30 generations, the yield was similar to that before modification, which means that the presence of transposons may affect the stability of genetic modification. Summary of the Invention

[0008] In light of the above issues, the inventors of this application predicted the transposase sequence of a halophilic microorganism and attempted to knock out the transposase, thus avoiding the problem of large variability in properties between different batches of industrial microbial production. They unexpectedly obtained an industrial production chassis strain with stable production properties and increased cell dry weight and product yield. The specific technical solution is as follows:

[0009] In a first aspect, the present invention provides an engineered halophilic microorganism, wherein a transposon is inactivated or has reduced activity.

[0010] The inactivation or activity reduction includes inactivating or reducing the activity of transposons in halophilic microorganisms by gene editing.

[0011] For example, the transposon cannot bind to the transposase to function or the binding strength is reduced; or the transposon cannot express the transposase, or the expressed transposase is inactive or has reduced activity.

[0012] The transposase includes IS3 family transposase.

[0013] Preferably, one or more transposases in the halophilic microorganism are inactivated or have reduced activity. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 transposases in the halophilic microorganism are inactivated or have reduced activity.

[0014] Preferably, the amino acid sequence of the transposase includes one or more of SEQ ID NOs: 43-53.

[0015] Preferably, the nucleotide sequence encoding the transposase includes one or more of SEQ ID NOs: 1-21.

[0016] Preferably, the halophilic microorganism is obtained by knocking out or knocking down a gene encoding a transposase on a transposon. Preferably, one or more genes encoding a transposase are knocked out or knocked down, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 genes encoding a transposase are knocked out or knocked down.

[0017] Preferably, the knocked-out or knocked-down transposase gene includes but is not limited to a combination of two or more of tns1, tns2, tns3, tns4, tns5, tns6, tns7, tns8, tns9, tns10, tns11, tns12, tns13, tns14, tns15, tns16, tns17, tns18, tns19, tns20, and tns21.

[0018] Preferably, the knocked-out or knocked-down transposase genes include but are not limited to a combination of two or more of tns1, tns2, tns3, tns4, tns12, tns13, tns14, tns15, tns16, tns17, tns20, and tns21.

[0019] For example: the combination of TNS3 and TNS4; the combination of TNS5 and TNS9; the combination of TNS8 and TNS12; the combination of TNS19 and TNS21; the combination of TNS3, TNS4 and TNS5; the combination of TNS5, TNS9, TNS10 and TNS11; the combination of TNS8, TNS12, TNS13, TNS15, TNS19 and TNS21; the combination of TNS1, TNS8, TNS12 and TNS13; the combination of TNS1, TNS8, TNS12, TNS13, TNS15, TNS19 and TNS21; the combination of TNS1, TNS3, TNS4 and TNS13; the combination of TNS1, TNS3, TNS13 and TNS21; 1. The combination of TNS3 and TNS21; the combination of TNS8, TNS12, TNS13 and TNS1; the combination of TNS15, TNS19 and TNS21; the combination of TNS1, TNS3 and TNS5; the combination of TNS6 and TNS8; the combination of TNS2, TNS9 and TNS17; the combination of TNS3 and TNS19; the combination of TNS2, TNS9, TNS10, TNS11, TNS12, TNS13, TNS14 and TNS20; the combination of TNS1, TNS2, TNS3, TNS4, TNS12, TNS13, TNS14, TNS15, TNS16, TNS17, TNS20 and TNS21; the combination of TNS1-21; etc.

[0020] In a specific embodiment of the present invention, the amino acid sequence of the transposase on the Halomonas bluephagenesis knockout transposon is as follows:

[0021] (1) SEQ ID NO: 44;

[0022] (2) SEQ ID NO: 45;

[0023] (3) SEQ ID NO: 46;

[0024] (4) SEQ ID NO: 53;

[0025] (5) SEQ ID NO: 43 and 44;

[0026] (6) SEQ ID NOs: 46 and 48;

[0027] (7) SEQ ID NO: 43 and 53;

[0028] (8) SEQ ID NO: 45 and 46;

[0029] (9) SEQ ID NOs: 43, 44, and 45;

[0030] (10) SEQ ID NOs: 43, 46, 48, 49, and 53;

[0031] (11) SEQ ID NOs: 43, 46, and 48;

[0032] (12) SEQ ID NOs: 43, 49, and 53; or

[0033] (13) SEQ ID NO: 43-53;

[0034] The combination of amino acid sequences of the transposase on the Halomonas campaniensis knockout transposon includes SEQ ID NOs: 43 and 44.

[0035] Preferably, a transposon-targeting sgRNA is used to knock out or knock down genes encoding one or more transposases. Further preferably, the sgRNA targets one or more transposase genes. Even more preferably, the target site sequence of the sgRNA comprises a functional domain of a transposase-encoding gene, such as one or more exon regions, or before an exon region, or in an intron region, or in the start codon region.

[0036] In a specific embodiment of the present invention, the target site sequence of the sgRNA comprises one or more of SEQ ID NOs: 22-42.

[0037] In a specific embodiment of the present invention, the target site sequence combination of the sgRNA is as follows:

[0038] (1) SEQ ID NO: 24;

[0039] (2) SEQ ID NO: 26;

[0040] (3) SEQ ID NO: 29;

[0041] (4) SEQ ID NO: 40;

[0042] (5) SEQ ID NO: 24 and 25;

[0043] (6) SEQ ID NOs: 26 and 30;

[0044] (7) SEQ ID NO: 29 and 33;

[0045] (8) SEQ ID NO: 40 and 42;

[0046] (9) SEQ ID NOs: 24, 25, and 26;

[0047] (10) SEQ ID NOs: 26, 30, 31 and 32;

[0048] (11) SEQ ID NOs: 29, 33, 34, 36, 40, and 42;

[0049] (12) SEQ ID NOs: 22, 29, 33, 34, 36, 40, and 42;

[0050] (13) SEQ ID NOs: 29, 33, 34, and 22;

[0051] (14) SEQ ID NOs: 36, 40, and 42;

[0052] (15) SEQ ID NO: 22-42;

[0053] (16) SEQ ID NOs: 24, 22, 25, and 34;

[0054] (17) SEQ ID NO: 42, 24, 34 and 22; or

[0055] (18) SEQ ID NO: 24, 22 and 42.

[0056] Preferably, the halophilic microorganism refers to a microorganism that requires a certain salt concentration for growth and grows best in an environment with a certain salt concentration. It can be a halophilic bacteria, a halophilic archaea, and an algae. More specifically, the halophilic microorganism can be a halophilic bacteria, preferably including Halomonas.

[0057] The Halomonas bacteria include one or more of Halomonas bluephagenesis or its derivatives, Halomonas campaniensis or its derivatives, and Halomonas aydingkolgenesis or its derivatives.

[0058] For example, one or a combination of two of Halomonas bluephagenesis TD01 (CGMCC No.4353), Halomonas campaniensis LS21 (CGMCC No.6593), Halomonas bluephagenesis TDH4AB (CGMCC No.22795), Halomonas aydingkolgenesis M1 (CGMCC No.19880), Halomonas bluephagenesis TDΔgabD2-D2, Halomonas bluephagenesis WZY254, Halomonas bluephagenesis WZY278, or derivatives thereof.

[0059] The production characteristics of the halophilic microorganism are stable, and the stability of the microbial metabolic production is improved by knocking out or knocking down one or more transposase genes.

[0060] The traits include, but are not limited to, growth rate, maximum achievable cell density, yield and productivity of various intracellular products, yield and productivity of various extracellular products, etc.

[0061] Preferably, after the transformation, the halophilic microorganism maintains substantially the same culture medium and culture method as before the knockout.

[0062] The second aspect of the present invention provides a method for constructing the above-mentioned halophilic microorganism, comprising inactivating or reducing the activity of the transposon in the halophilic microorganism by gene editing.

[0063] Preferably, the construction method comprises knocking out or knocking down the transposase gene in the genome of the halophilic microorganism.

[0064] Preferably, the construction method is a molecular biological method, preferably a gene editing technology, such as CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing nuclease or other molecular biology technology.

[0065] The construction method includes using a transposon-targeting sgRNA to knock out or knock down genes encoding one or more transposases. Preferably, the target site sequence of the sgRNA comprises one or more of SEQ ID NOs: 22-42.

[0066] The third aspect of the present invention provides a halophilic microorganism with stable properties obtained by the above construction method.

[0067] In a fourth aspect, the present invention provides a method for producing polyhydroxyalkanoates, wherein the method comprises culturing the above-mentioned halophilic microorganism or the halophilic microorganism obtained by the above-mentioned construction method.

[0068] The culture medium used in the method can be a solid culture medium, a liquid culture medium or a semi-solid culture medium.

[0069] The culture medium used in the method includes a carbon source. In the present invention, the carbon source is a nutrient that provides the carbon element required for the growth and reproduction of microorganisms, and can also be understood as a substrate for synthesizing products. In the present invention, "carbon source" is the source used by the halophilic microorganisms of the present invention to synthesize PHA, and therefore, it can be used interchangeably with "substrate" in this article. More preferably, the carbon source comes from glucose, gluconic acid, gluconate, gluconate or a combination thereof, and further preferably, the carbon source comes from glucose. The gluconate can be any one or more gluconates, as long as it can be used as a carbon source for the microorganisms involved in the present invention for polymer production, for example, sodium gluconate, potassium gluconate, calcium gluconate, etc.

[0070] More preferably, the concentration of the carbon source may be 1-100 g / L. Further preferably, the concentration of the carbon source may be any range or value within the above range, for example, about 1-90 g / L, about 1-80 g / L, about 1-70 g / L, or about 1-60 g / L; more preferably, the concentration may be in the range of about 3-60 g / L, about 3-50 g / L or about 3-40 g / L; further preferably, in the range of about 5-60 g / L, about 10-60 g / L, about 20-40 g / L, specifically about 10 g / L, about 15 g / L, about 20 g / L, about 25 g / L, about 30 g / L, about 35 g / L, and the like.

[0071] The culture medium also includes nitrogen sources, inorganic salts, trace elements, growth factors and other substances that are beneficial to bacterial growth and metabolism.

[0072] The nitrogen source may be an inorganic nitrogen source and / or an organic nitrogen source. Examples of the inorganic nitrogen source include one or more of nitrates, ammonium salts, nitrites, and ammonia. Examples of the organic nitrogen source include one or more of peanut meal, soybean meal, cottonseed meal, corn steep liquor, yeast powder, yeast extract, fish meal, silkworm pupa meal, peptone, tryptone, bran, and waste mycelium.

[0073] The inorganic salts are used to maintain the osmotic pressure required by the halophilic microorganisms, such as potassium salts and / or sodium salts.

[0074] The culture medium may also be appropriately supplemented with other substances such as antibiotics for resistance screening.

[0075] The culture medium can be a conventional formula in the prior art such as LB culture medium, MM culture medium, etc., or can be modified based on the conventional formula.

[0076] The general formula of LB medium includes: 4-6g / L yeast extract, 8-12g / L peptone, 8-12g / L sodium chloride, and the rest is distilled water (pH adjusted to 7.0-7.2); preferably: 5g / L yeast extract, 10g / L peptone, 10g / L sodium chloride, and the rest is distilled water (pH adjusted to 7.0-7.2).

[0077] The general formula of MM culture medium includes: 0.1‰-2‰ (NH4)2SO4 or urea, 0.1‰-1‰ MgSO4, 5‰-10‰ Na2HPO4·12H2O, 0.5‰-2‰ KH2PO4, 0.1% of other trace elements (Fe(III)-NH4-Citrate, CaCl2·2H2O, ZnSO4·7H2O, MnCl2·4H2O, H3BO3, CoCl2·6H2O, CuSO4·5H2O, NiCl2·6H2O, NaMoO4·2H2O trace (pH adjusted to about 9.0)).

[0078] MM medium preferably includes: 0.1% (NH4)2SO4 or 0.2% urea, 0.02% MgSO4, 1.0% Na2HPO4·12H2O, 0.15% KH2PO4, no more than 0.1% of other trace elements (Fe(III)-NH4-Citrate, CaCl2·2H2O, ZnSO4·7H2O, MnCl2·4H2O, H3BO 3， CoCl2·6H2O, CuSO4·5H2O, NiCl2·6H2O, NaMoO4·2H2O (pH adjusted to about 9.0).

[0079] Substratum of the present invention is obtained by adding a carbon source and an inorganic salt on the basis of a basal medium on a basal medium, wherein the carbon source can be utilized by halophilic microorganisms to synthesize the desired product, and the inorganic salt is used to provide an osmotic pressure environment suitable for the growth of halophilic microorganisms. The basal medium refers to a medium comprising nutrients that can be used to support the growth of microorganisms of the present invention. The above-mentioned basal medium can be a medium conventionally used for microbial cultivation in this area, such as mineral culture medium, LB culture medium, MM culture medium or beef extract peptone, etc., or can be a medium improved according to the desired purpose on the basis of these culture media. That is to say, those skilled in the art can routinely select a suitable basal medium, as long as it can allow the growth of microorganisms, especially halophilic microorganisms.

[0080] In a specific embodiment of the present invention, the culture medium is MM basic medium, 10-50 g / L of carbon source, and 5-100 g / L of inorganic salts.

[0081] PHAs can be classified into homopolymers and copolymers based on their monomer composition. Based on the number of carbon atoms in the monomers, the PHAs of the present invention include, but are not limited to, short-chain PHAs (i.e., monomers containing C3-C5 hydroxy fatty acids) and medium-chain PHAs (i.e., monomers containing C6-C16 hydroxy fatty acids).

[0082] In some embodiments of the present invention, the PHA may be a homopolymer, including but not limited to polyhydroxypropionate, polyhydroxybutyrate, polyhydroxyvalerate, and the like, for example, poly-3-hydroxybutyrate (P3HB), poly-4-hydroxybutyrate (P4HB), poly-3-hydroxypropionate (P3HP), or poly-3-hydroxyvalerate (P3HV).

[0083] In some embodiments of the present invention, PHA may include but is not limited to copolymers such as dimers, trimers, etc. For example, the copolymer may be a copolymer of hydroxypropionate and hydroxybutyrate; a copolymer of hydroxypropionate and hydroxyvalerate; a copolymer of hydroxybutyrate and hydroxyvalerate; a copolymer of hydroxypropionate, hydroxybutyrate, and hydroxyvalerate, etc.

[0084] More specifically, in some embodiments of the present invention, the PHA may be poly 3-hydroxybutyrate-4-hydroxybutyrate copolyester (P3HB4HB), poly 3-hydroxybutyrate-3-hydroxyvalerate copolyester (P3HB3HV), or poly 3-hydroxybutyrate-4-hydroxybutyrate-3-hydroxyvalerate copolyester (P34HB3HV), or a combination thereof.

[0085] After knocking out the transposase gene in a halophilic microorganism using the present method, even after all transposase genes were knocked out, the halophilic microorganisms showed good growth, without experiencing the growth reduction seen when knocking out other metabolic-related genes, and product synthesis was also not reduced. When using halophilic microorganisms with knocked-out transposases for open fermentation, providing essentially the same culture medium components can more effectively prevent spontaneous mutations in the microorganisms and even slightly increase the yield of the halophilic microorganisms and products, such as PHA.

[0086] The English abbreviation and the full Chinese name of this application are compared in Table 1.

[0087] Table 1 Comparison of English abbreviations and Chinese full names

[0088] The above merely summarizes some aspects of the present invention and is not and should not be considered to limit the present invention in any aspect.

[0089] All patents and publications mentioned in this application are incorporated herein by reference in their entirety. Those skilled in the art will recognize that certain modifications may be made to the present invention without departing from the spirit or scope of the present invention. The following examples further illustrate the present invention and are not to be construed as limiting the scope of the present invention or the specific methods described herein. DETAILED DESCRIPTION

[0090] The present invention will be further explained below through specific examples. It should be understood that these examples are merely provided to illustrate and help understand the present invention, and are not intended to limit the present invention to these examples.

[0091] Unless otherwise specified, the experimental methods used in the examples are conventional methods.

[0092] Unless otherwise specified, the materials and reagents used in the examples can be obtained from commercial sources.

[0093] Halomonas bluephagenesis TD01 used in the examples is described in patent application publication number CN102120973A and the literature “Tan D, Xue Y, Aibaidula G, et al. Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01[J]. Bioresource Technology, 2011, 102: 8130-8136”; the public can obtain the bacteria from Tsinghua University.

[0094] Halomonas bluephagenesis TDΔgabD2-D2 used in the examples is a derivative strain of Halomonas bluephagenesis TD01, specifically disclosed in “Ye J, Hu D, Che X, Jiang X, Li T, Chen J, Zhang HM, Chen GQ. Engineering of Halomonas bluephagenesis for low cost production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose. Metab Eng. 2018 May; 47: 143-152”.

[0095] Halomonas campaniensis LS21 used in the examples is a Gram-negative halophilic microorganism screened in our laboratory. It has excellent prospects for industrial production and application. It was published in the article "Jiang X, Yao Z, Chen G Q. Controlling cell volume for efficient PHB production by Halomonas [J]. Metabolic Engineering, 2017, 44: 30-37" and is available to the public from Tsinghua University. Halomonas bluephagenesis WZY278 used in the examples is a derivative strain of Halomonas bluephagenesis TD01. After a series of gene editing, its optimal salt concentration is significantly reduced, as specifically disclosed in the article "JI M, ZHENG T, WANG Z, et al. PHB production from food waste hydrolysates by Halomonas bluephagenesis Harboring PHB operon linked with an essential gene [J / OL]. Metabolic Engineering, 2023, 77: 12-20. DOI: 10.1016 / j.ymben.2023.03.003."

[0096] The gene editing performed by Halomonas used in this application, such as knocking out transposase, uses CRISPR / Cas9 technology, which can be found in the literature Qin et.al. CRISPR / Cas9 editing genome of extremophile Halomonas spp. Metabolic Engineering. 47 (2018) 219-229.

[0097] The specific formula of the MM medium for culturing halophilic microorganisms in the embodiment is as follows:

[0098] Urea 0.5g / L; MgSO4 0.2g / L; KH2PO4 1.5g / L; and a total of <0.1g / L of Fe(III)-NH4-Citrate, CaCl2·2H2O, ZnSO4·7H2O, MnCl2·4H2O, H3BO3, CoCl2·6H2O, CuSO4·5H2O, NiCl2·6H2O, and NaMoO4·2H2O.

[0099] 10-50 g / L of glucose is further added to the culture medium as a carbon source, and 5-100 g / L of sodium chloride is added to provide an osmotic pressure environment required for the growth of halophilic microorganisms; and the pH is adjusted to 8.0-9.0.

[0100] The specific formula of LB60 medium for culturing halophilic microorganisms in the embodiment is as follows:

[0101] Contains 60 g / L NaCl, 10 g / L peptone (purchased from OXIOD, UK, product catalog number LP0042), 5 g / L yeast extract (purchased from OXIOD, UK, product catalog number LP0021), and the balance is water.

[0102] Gas chromatography method for detecting polyhydroxyalkanoate (PHA) content:

[0103] The oven temperature was set at 80°C, the injector temperature at 200°C, the detector temperature at 220°C, and the column head pressure at 0.25 MPa. The temperature program was as follows: dwell at 80°C for 1.5 minutes, then increase the temperature to 140°C at a rate of 30°C / min, then increase the temperature to 220°C at a rate of 40°C / min and hold at this temperature for 0.5 minutes. The sample injection volume was 1 μL using an Agilent microinjector.

[0104] Gas phase sample preparation: 40-60 mg of dry cells of the sample to be tested (centrifuge the bacterial suspension at 10,000 rpm for 10 minutes at room temperature. Wash the resulting cell pellet once with water and then freeze-dry to obtain the dry cells. Homopolymers are produced in the cells). Add 2 mL of chloroform and 2 mL of esterification solution (3% (v / v) concentrated sulfuric acid in pure methanol and 1 g / L benzoic acid as an internal standard) to an esterification tube. Seal the tube and heat at 100°C for 4 hours. After cooling, add 1 mL of distilled water, shake thoroughly, and allow to stand. After the chloroform and aqueous phases have completely separated, remove 1 μL of the lower chloroform phase and inject it into a gas chromatograph (Hewlett Packard 6890) for chromatographic analysis. Operate the gas chromatograph according to the HP Hewlett Packard 6890 gas chromatograph manual.

[0105] Preparation of standard samples: Take 10-20 mg of standard sample in an esterification tube, add 2 mL of chloroform and 2 mL of esterification solution, seal the tube, and esterify at 100°C.

[0106] Result analysis: Using the standard sample as a control, if the esterified sample of the cells to be tested (test sample) has a clear peak at the standard sample, the mass of each monomer can be calculated based on the peak area, and then the molar ratio can be calculated based on the mass fraction of each monomer; the proportion of polymers in the dry weight of the cells can be calculated based on the amount of sample added (wt%).

[0107] The cell dry weight (CDW, g / L) is the ratio of the mass of the dry cells to the volume of the fermentation product.

[0108] The P3HB content (wt%) is the mass ratio of P3HB to the dry bacterial cells.

[0109] The P34HB content (wt%) is the mass ratio of P34HB to the dry bacterial cells.

[0110] The proportion of 4HB in P34HB (mol%) = the number of moles of 4HB ÷ (the number of moles of 3HB + the number of moles of 4HB) × 100%.

[0111] The target sequences of the transposases or hypothetical transposases and sgRNAs used for knockout described in this application are shown in Table 2, and the amino acid sequences corresponding to the transposases are shown in Table 3.

[0112] Table 2: Transposase sequences and target sequences of sgRNA used to knock out transposase (underlined upstream and downstream sequences)

[0113] Table 3: Amino acid sequence of transposase

[0114] Example 1: Knockout of a single transposase in strain Halomonas bluephagenesis TD01 for P3HB production fermentation

[0115] In this embodiment, the inventors attempted to use a halophilic microorganism in which a single transposase gene was knocked out from the Halomonas bluephagenesis TD01 genome to carry out a P3HB production fermentation experiment. At the same time, the wild-type Halomonas bluephagenesis TD01 was used as a control strain for fermentation culture, and the aforementioned MM medium was used as the basal medium, and inorganic salts and glucose were added. A shake flask experiment was used. Three parallel samples were set up for each group of experiments. The corresponding molar concentration of sodium chloride used in the fermentation process was 0.171 mol / L, the amount of glucose added was 40 g / L, the fermentation temperature was 37 ° C, and the shake flask speed was 200 rpm. The experimental results are shown in Table 4.

[0116] Table 4: Comparison of shake flask culture results of Halomonas bluephagenesis before and after knockout of transposase gene

[0117] The values ​​of dry weight and P3HB content are expressed as xx±error, where the smaller the error, the more stable the strain growth and product synthesis.

[0118] The results showed that compared to the wild-type Halomonas bluephagenesis TD01 strain culture experiment 1-1, when a single transposase gene was knocked out, the growth and product synthesis stability of Halomonas bluephagenesis TD01 strain were significantly improved. This suggests that knocking out a single transposase gene can achieve more stable production.

[0119] Example 2: Combined knockout of two transposases in strain Halomonas bluephagenesis TD01 for P3HB production and fermentation

[0120] In this experiment, Halomonas bluephagenesis TD01 was used as a control fermentation strain. The aforementioned MM medium was used as the basal medium, supplemented with inorganic salts and glucose. A shake flask experiment was performed. Three replicates were run in each experiment, and the results were averaged. The corresponding sodium chloride molarity used during the fermentation process was 0.171 mol / L, the glucose addition amount was 40 g / L, the fermentation temperature was 37°C, and the shake flask speed was 200 rpm. The experimental results are shown in Table 5.

[0121] Table 5: Shake flask results of Halomonas bluephagenesis culture by combined knockout of two transposases

[0122] In this experiment, a combination of knockouts was performed on the strain's transposase genes, achieving the knockout of two transposase genes. The results in Table 5 demonstrate that when two transposase genes in the genome were knocked out, the growth and product synthesis stability of Halomonas bluephagenesis TD01 bacteria were significantly improved. Thus, it is possible to achieve more stable production by knocking out two transposase genes in combination.

[0123] Example 3: Knockout of two or more transposase genes in strain Halomonas bluephagenesis TD01 for stable P3HB production and fermentation

[0124] In this experiment, Halomonas bluephagenesis TD01 was used as the fermentation strain for fermentation culture, the aforementioned MM medium was used as the basal medium, and inorganic salts and glucose were added. The difference between this experiment and Example 2 is that a larger number of transposase genes were knocked out. A shake flask experiment was used. Three parallel samples were set up for each group of experiments, and the results were averaged. The corresponding molar concentration of sodium chloride used in the fermentation process was 0.171 mol / L, the amount of glucose added was 40 g / L, the fermentation temperature was 37 ° C, and the shake flask speed was 200 rpm. The experimental results are shown in Table 6.

[0125] Table 6: Shake flask results of combined knockout of three or more transposase genes Halomonas bluephagenesis

[0126] In this experiment, a combination of knockouts of three or more transposase genes were performed on the strain. The results in Table 6 demonstrate that when multiple transposase genes across the genome were knocked out, the growth and product synthesis stability of Halomonas bluephagenesis TD01 bacteria were significantly improved. Thus, it is possible to achieve more stable P3HB production by knocking out three or more transposase genes in combination.

[0127] Example 4: Stable P34HB production and fermentation by knocking out the transposase gene in the strain Halomonas bluephagenesis TDΔgabD2-D2

[0128] In this experiment, Halomonas bluephagenesis TD△gabD2-D2 was used as a fermentation strain for fermentation culture. The strain has the ability to produce P34HB using glucose. The aforementioned MM medium was used as the basal medium, and inorganic salts and glucose were added. A shake flask experiment was used. Three parallel samples were set up for each group of experiments, and the results were averaged. The molar concentration of sodium chloride corresponding to the inorganic salt used in the fermentation process was 0.171 mol / L, the amount of glucose added was 40 g / L, the fermentation temperature was 37 ° C, and the shake flask speed was 200 rpm. In this experiment, the product produced by the strain fermentation was poly 3-hydroxybutyric acid-4-hydroxybutyric acid copolyester (P3HB4HB), and the results are shown in Table 7.

[0129] Table 7: Shake flask results of transposase knockout culture of Halomonas bluephagenesis TDΔgabD2-D2

[0130] Results showed that when Halomonas bluephagenesis TD△gabD2-D2, in which the transposase gene was knocked out, produced P34HB, the bacteria grew well, with PHA yields and 4HB ratios comparable to those of the non-knockout strain. This suggests that knocking out three or more transposase genes in combination could lead to more stable P3HB4HB production.

[0131] Example 5: Knockout of the transposase gene in Halomonas campaniensis LS21 for P3HB fermentation

[0132] The aforementioned MM medium was used as the basal medium, supplemented with inorganic salts and glucose. Shake flask experiments were performed. Three replicates were performed for each experiment, and the results were averaged. During fermentation, the corresponding sodium chloride molar concentration for the inorganic salts used was 0.171 mol / L, the glucose addition amount was 40 g / L, the fermentation temperature was 37°C, and the shake flask speed was 200 rpm. The experimental results are shown in Table 8.

[0133] Table 8: Comparison of shake flask results of Halomonas campaniensis LS21 before and after transposase knockout

[0134] In the strain Halomonas campaniensis LS21, knocking out either a single or all transposases did not affect P3HB production. Such knockouts also reduced the instability of microbial traits caused by transposition. Thus, a combination of knockouts of transposase genes could be used to achieve more stable P3HB production.

[0135] Example 6: P3HB fermentation experiment of Halomonas bluephagenesis TD01 with transposase knockout

[0136] Halomonas bluephagenesis TD01 and a transposase-knockout strain were used as fermentation strains for fermentation culture. The aforementioned MM medium was used as the basal medium, supplemented with 20 g / L glucose and 10 g / L sodium chloride, and fermentation tank experiments were conducted. The fermentation tank used was a 7L fermenter, the fermentation broth volume was 3L, the pH was set at 9.0, and the temperature was maintained at 37°C. A major advantage of halophilic microbial fermentation is that it can be open fermented without additional sterilization. At the same time, the fermentation process does not require the addition of various air filters and complex aseptic operations to reduce the possibility of contamination, greatly saving production costs and manpower and material resources. Conventional open fermentation relies on a high-salt, high-pH environment to inhibit the growth of other bacteria. The results are shown in Table 9.

[0137] Table 9: Results of P3HB fermentation experiments with transposase knockout Halomonas bluephagenesis TD01

[0138] For strain TD01, knocking out either one or all of the transposases did not affect P3HB production in a 7-L fermenter-scale production experiment. Such knockouts also reduced the instability of microbial traits caused by transposition. Thus, it is possible to achieve more stable P3HB production by knocking out transposase genes in combination.

[0139] In this experiment, it was unexpectedly discovered that more PHA could be obtained when the transposase gene knockout strain was fermented. In comparison, the dry weight of the fermentation system was lower when the control strain was fermented. This has great industrial value and significance for large-scale open fermentation production of PHA.

[0140] Example 7: Fermentation experiment of Halomonas campaniensis LS21 with transposase knockout

[0141] Halomonas campaniensis LS21 is a halophilic microorganism that shares the advantages of open fermentation described in Example 6. Fermentation experiments were conducted using Halomonas campaniensis LS21 as the engineered strain using the aforementioned MM medium as the basal medium supplemented with 20 g / L glucose and 10 g / L sodium chloride. This fermentation was performed in a 7 L fermentor, with a 3 L fermentation broth volume, a pH of 8.5, and a temperature of 37°C. A control group was run under the same experimental conditions, except that no transposase gene was deleted. The results are shown in Table 10.

[0142] Table 10: P3HB fermentation results of Halomonas campaniensis LS21 with transposase knockout

[0143] As shown in Table 10, knocking out either a single or all transposases in Halomonas campaniensis LS21 did not affect P3HB production in a 7-L fermenter-scale production experiment. Such knockouts also reduced the problem of microbial trait instability caused by transposition. Thus, it is possible to achieve more stable P3HB production by knocking out transposase genes in combination.

[0144] Example 8: Knockout of the transposase gene in the strain Halomonas bluephagenesis WZY278 for P3HB production and fermentation

[0145] In this experiment, Halomonas bluephagenesis WZY278 was used as the fermentation strain. The aforementioned MM medium was used as the basal medium, supplemented with inorganic salts and glucose. A shake flask experiment was performed. Three replicates were run for each experiment, and the results were averaged. The molar concentration of sodium chloride corresponding to the inorganic salts used during fermentation was 0.684 mol / L, the amount of glucose added was 40 g / L, the fermentation temperature was 37°C, and the shake flask speed was 200 rpm. The experimental results are shown in Table 11.

[0146] Table 11: Comparison of shake flask results of transposase knockout culture of Halomonas bluephagenesis WZY278

[0147] As shown in Table 11, knocking out either a single or all transposases in Halomonas bluephagenesis WZY278 did not affect P3HB production in shake flask production experiments. Such knockouts also reduced the problem of microbial trait instability caused by transposition. Thus, it is possible to achieve more stable P3HB production by knocking out transposase genes in combination.

[0148] Example 9: P3HB fermentation experiment of Halomonas bluephagenesis WZY278 with transposase knockout

[0149] In this experiment, Halomonas bluephagenesis WZY278 with transposase knocked out was used as a fermentation strain for fermentation culture. This strain was the same as the strain in Example 8.

[0150] The aforementioned MM medium was used as the basal medium, and inorganic salts and glucose were added (same as in Example 8). A shake flask experiment was used. Three parallel samples were set for each group of experiments, and the results were averaged. In this experiment, the amount of glucose added was 40 g / L, the fermentation temperature was 37 ° C, and the shake flask speed was 200 rpm. This fermentation used a 7 L fermentor tank, the fermentation liquid volume was 3 L, the pH was set at 8.5, and the temperature was maintained at 37 ° C. The control group was carried out under the same experimental conditions, except that no transposase gene was knocked out in the control group. The results are shown in Table 12.

[0151] Table 12: P3HB fermentation results of Halomonas bluephagenesis WZY278 with transposase knockout

[0152] Results showed that knocking out either a single or all transposases in Halomonas bluephagenesis WZY278 did not affect P3HB production in a 7-L fermenter-scale production experiment. Furthermore, such knockouts reduced the instability of microbial traits caused by transposition. This suggests that combined knockout of transposase genes could lead to more stable P3HB production.

[0153] Example 10: Mutation rate measurement experiment of Halomonas bluephagenesis WZY278 with transposase knockout

[0154] In this experiment, Halomonas bluephagenesis WZY278 with transposase knocked out was used as a fermentation strain for fermentation culture. This strain was the same as the experimental strain 8-4 in Example 8.

[0155] A rifampicin resistance experiment was used to determine the mutation rate of the strain. The transposase-knockout Halomonas bluephagenesis WZY278 and the original Halomonas bluephagenesis WZY278 strain were inoculated into LB60 medium and cultured overnight at 37°C, 200 rpm. The strains were then transferred to LB60 medium at a concentration of 1% and continued to be cultured until the OD value reached 1. The strains were then diluted and plated onto LB60 plates containing rifampicin (100 mg / L) or without the antibiotic. The number of colonies under different conditions was counted, and the mutation rate was calculated using the formula: (number of colonies on the resistant plate * dilution factor) / (number of colonies on the non-resistant plate * dilution factor).

[0156] Table 13: Mutation rate results of Halomonas bluephagenesis WZY278 with transposase knockout

[0157] The results showed that in Halomonas bluephagenesis WZY278, when the transposases were knocked out individually or completely, the mutation rate at different time points during the culture process was significantly lower than that of the control group. Therefore, this bacterium can be used to achieve more stable P3HB production.

[0158] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. An engineered halophilic microorganism, characterized in that: The transposon is inactivated or has reduced activity in the halophilic microorganism.

2. The halophilic microorganism according to claim 1, characterized in that The inactivation or reduced activity of a transposon includes: the transposon cannot bind to a transposase to function or the binding strength is reduced; or the transposon cannot express a transposase or the expressed transposase is inactivated or the activity is reduced.

3. The halophilic microorganism according to claim 1, characterized in that The inactivation or activity reduction includes inactivating or reducing the activity of the transposon in the halophilic microorganism by gene editing.

4. The halophilic microorganism according to claim 1, characterized in that The halophilic microorganisms include the genus Halomonas.

5. The halophilic microorganism according to any one of claims 1 to 4, characterized in that One or more transposases in the halophilic microorganism are inactivated or have reduced activity.

6. The halophilic microorganism according to claim 2 or 5, characterized in that The transposase includes IS3 family transposase.

7. The halophilic microorganism according to any one of claims 2 to 6, characterized in that The amino acid sequence of the transposase includes one or more of SEQ ID NOs: 43-53.

8. The halophilic microorganism according to any one of claims 2 to 7, characterized in that The nucleotide sequence encoding the transposase includes one or more of SEQ ID NOs: 1-21.

9. The halophilic microorganism according to any one of claims 1 to 8, characterized in that The halophilic microorganism is prepared by knocking out or knocking down the gene encoding the transposase on the transposon.

10. The halophilic microorganism according to claim 9, characterized in that The knocked-out or knocked-down transposase gene includes a combination of two or more of tns1, tns2, tns3, tns4, tns5, tns6, tns7, tns8, tns9, tns10, tns11, tns12, tns13, tns14, tns15, tns16, tns17, tns18, tns19, tns20, and tns21.

11. The halophilic microorganism according to claim 9 or 10, characterized in that Use sgRNA targeting a transposon to knock out or knock down the gene encoding one or more transposases.

12. The halophilic microorganism according to claim 11, characterized in that The target site sequence of the sgRNA comprises one or more of SEQ ID NOs: 22-42.

13. The halophilic microorganism according to claim 4, characterized in that The Halomonas genus includes one or more of Halomonas bluephagenesis or its derivatives, Halomonas campaniensis or its derivatives, and Halomonas aydingkolgenesis or its derivatives.

14. A method for constructing a halophilic microorganism according to any one of claims 1 to 13, characterized in that: This includes inactivating or reducing the activity of transposons in halophilic microorganisms through gene editing.

15. The construction method according to claim 14, characterized in that: The construction method includes using sgRNA targeting a transposon to knock out or knock down a gene encoding one or more transposases.

16. The construction method according to claim 15, characterized in that: The target site sequence of the sgRNA comprises one or more of SEQ ID NOs: 22-42.

17. A method for producing polyhydroxyalkanoate, characterized in that: The method comprises culturing the halophilic microorganism according to any one of claims 1 to 13.

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