Recombinant halomonas capable of inducing protein self-degradation as well as construction method and application of recombinant halomonas

Abstract

The invention provides a recombinant halomonas capable of inducing protein self-degradation as well as a construction method and application of the recombinant halomonas. The recombinant halomonas does not express the SspB protein or the expressed SspB protein does not have functions or has reduced functions, and an SsrA tag is inserted into the C end of the to-be-degraded protein in the recombinant halomonas. The recombinant halomonas can degrade target protein in the middle and later stages of growth or the synthesis stage of products, and the method has the advantages of being easy to operate and small in influence on bacterial growth when used for achieving degradation of regulation and control protein.

Description

Technical Field

[0001] The present invention relates to the field of microbial engineering, and particularly relates to a recombinant Halomonas that induces self-degradation of proteins, a construction method thereof, and an application thereof. Background Art

[0002] Halomonas is a microorganism that can tolerate high salt and high pH environments, and is a precious scientific research material and production resource. At present, the next-generation industrial biotechnology based on halophilic bacteria has been relatively mature, and has strong market competitiveness and unique advantages by virtue of characteristics such as energy conservation, reduction of fresh water consumption, and cost reduction.

[0003] There are many essential genes in bacteria that cannot be directly knocked out because these genes play important roles in the early stage of bacterial growth. Some existing means of suppressing gene expression in the later stage, such as CRISPRi (Clustered regularly interspaced short palindromic repeats interference), ligate the dCas9 (dead CRISPR-associated protein 9) protein with a transcriptional repressor, such as KRAB (Kruppel associated box). Under the guidance of gRNA (guide RNA), the fusion protein dCas9-KRAB will bind to the target DNA targeted by the gRNA, inhibiting the transcription of DNA into RNA and suppressing the expression of the target gene. Another example is the siRNA (Small interfering RNA) technology. siRNA is a short double-stranded RNA. After entering the cytoplasm, it will undergo base complementary pairing with the target mRNA in the cell and cleave the target mRNA, so that the target mRNA cannot be normally translated into protein. However, these methods all regulate the expression of genes at the DNA (Deoxyribonucleic acid) or RNA (Ribonucleic acid) level and cannot regulate the already expressed proteins. If regulation is desired in the later stage, the interference of the proteins already expressed in the early stage cannot be avoided. In addition, both gRNA and siRNA may show off-target phenomena due to base mismatching.

[0004] Therefore, a means of regulating at the protein level in the middle and later stages of bacterial growth is needed, so that the target protein is degraded in the middle and later stages of bacterial growth, shortening the half-life of the target protein.

[0005] In many bacteria, there exists an ATP (Adenosine 5'-triphosphate) - dependent protease degradation system, including the Clp protease family, Lon protease, FtsH protease, and HslUV protease. Among them, the ClpXP protease in the Clp family is the most widely studied and applied. Its main function is to clear or degrade improperly synthesized, damaged, or useless proteins in the bacterial cell, maintaining the dynamic balance of intracellular proteins under normal metabolism and stress stimulation.

[0006] The working process of ClpXP protease is as follows: When protein synthesis on the ribosome stalls, tmRNA (Transfer - messenger RNA) is recruited to the ribosome. tmRNA can simultaneously perform the functions of mRNA (messenger RNA) and tRNA (transfer RNA), adding a degradation tag called ssrA to the C - terminus of the stalled - synthesized protein. Subsequently, with the assistance of the adaptor protein SspB, the target protein labeled with the ssrA tag is recognized by the ClpXP complex protein and rapidly degraded. Summary of the Invention

[0007] The present invention provides a method for inducing self - degradation of proteins in Halomonas to shorten the half - life of proteins. This method can regulate the protein level in bacteria at different stages of bacterial growth, especially in the middle and late stages of bacterial growth. Compared with the method of directly knocking out genes, it can achieve the regulation of essential genes in bacteria. And this method directly regulates at the protein level. Proteins are the bearers of life activities, and genes exercise corresponding functions only after transcription and translation to produce proteins. Therefore, this method is more direct than the regulation at the DNA or RNA level such as CRISPRi and siRNA.

[0008] Specifically, in this application, the SspB protein was first knocked out and verified. After knocking out the SspB protein, the growth and production of metabolites of the strain were not affected. The purpose of knocking out the SspB protein was to reduce the recognition accuracy of the clpXP protease for the ssrA sequence. In the presence of SspB, even if the ssrA sequence mutated, it would cause the rapid degradation of the protein. However, after knocking out SspB, the change in the ssrA sequence had a greater impact on the degradation effect. Further, the modified SsrA tag of this application was added to the C-terminus of the protein to be degraded, and the modified strain was cultured. The results showed that the protein to be degraded began to degrade in the middle and late stages of the strain growth. Therefore, this method was particularly suitable for the degradation of proteins that were essential in the early stage of strain growth but not needed in the later stage, such as proteins related to maintaining cell morphology and / or mitosis - MreB protein, FtsZ protein, or proteins related to metabolic flux regulation - GltA protein.

[0009] Since MreB protein, FtsZ protein, or GltA protein is essential in the early stage of strain growth, it is necessary to regulate their normal expression in the early stage, but degrade them in the middle and late stages. During the degradation experiments of MreB protein, FtsZ protein, and GltA protein, the inventors unexpectedly found that after knocking out the SspB protein and adding the SsrA tag to the C-terminus of MreB protein, FtsZ protein, or GltA protein, the MreB protein, FtsZ protein, or GltA protein began to degrade after culturing the modified bacteria for 12 - 24 hours, the dry cell weight and product yield increased, and the late degradation of MreB protein or FtsZ protein changed the morphology of the strain from rod-shaped to spherical, which made the separation of the strain easier.

[0010] In the first aspect of the present invention, a recombinant Halomonas for inducing self-degradation of proteins is provided.

[0011] The recombinant Halomonas does not express the SspB protein or the expressed SspB protein has no function or reduced function.

[0012] The amino acid sequence of the SspB protein contains SEQ ID NO: 2 or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with SEQ ID NO: 2, and has the same or similar function as it.

[0013] The coding gene sequence of the SspB protein contains SEQ ID NO: 3 or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with SEQ ID NO: 3, and has the same or similar function as it.

[0014] The non-expression of SspB protein or the expression of a non-functional or reduced-function SspB protein can be achieved by gene editing methods, such as knocking out or knocking down all or part of the SspB protein-encoding gene. Preferably, the CRISPR / Cas9 gene editing technology is used. For example, sgRNA targeting the SspB protein-encoding gene is introduced into Halomonas together with Cas9 to achieve targeted cleavage.

[0015] In a specific embodiment of the present invention, sgRNA targeting the sspB gene is used to knock out or knock down the sspB gene in Halomonas.

[0016] Preferably, the target site sequence of the sgRNA includes SEQ ID NO: 7 or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with SEQ ID NO: 7 and has the same or similar function as it.

[0017] In a specific embodiment of the present invention, a plasmid containing an sgRNA expression module and / or upstream and downstream homologous arms is introduced into Halomonas.

[0018] Preferably, the upstream homologous arm includes the nucleotide sequence of positions 153-652 of SEQ ID NO: 1, or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with the nucleotide sequence of positions 153-652 of SEQ ID NO: 1 and has the same or similar function as it.

[0019] Preferably, the downstream homologous arm includes the nucleotide sequence of positions 653-1152 of SEQ ID NO: 1, or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with the nucleotide sequence of positions 653-1152 of SEQ ID NO: 1 and has the same or similar function as it.

[0020] Preferably, the sgRNA expression module includes the target site sequence of the sgRNA, preferably SEQ ID NO: 7.

[0021] Preferably, the sgRNA expression module includes the nucleotide sequence of positions 1-152 of SEQ ID NO: 1, or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with the nucleotide sequence of positions 1-152 of SEQ ID NO: 1 and has the same or similar function as it.

[0022] In a specific embodiment of the present invention, the plasmid containing the sgRNA expression module and / or upstream and downstream homologous arms contains SEQ ID NO: 1 or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with SEQ ID NO: 1, and has the same or similar function as it.

[0023] An SsrA tag is inserted at the C-terminus of the protein to be degraded in the recombinant Halomonas.

[0024] The SsrA tag can be wild-type or mutant-modified, and its sequence includes AANDENYAQGXXXX (SEQ ID NO: 4), where X can independently be selected from natural or unnatural amino acids. Preferably, the amino acid sequence of the SsrA tag includes one or more of SEQ ID NOs: 9-29, or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with one or more of SEQ ID NOs: 9-29, and has the same or similar function as it.

[0025] The insertion position of the SsrA tag is before the stop codon of the coding sequence of the protein to be degraded; preferably adjacent to the stop codon. That is, the insertion of the SsrA tag does not affect gene transcription and expression and does not regulate the protein at the gene level.

[0026] Preferably, the insertion of the SsrA tag can be achieved by gene editing methods, such as using the CRISPR / Cas9 gene editing technology. Preferably, the sgRNA targeting the coding gene of the protein to be degraded and Cas9 are co-introduced into Halomonas to achieve targeted cleavage, and the SsrA tag is added at the cleavage position.

[0027] In a specific embodiment of the present invention, a plasmid containing the sgRNA expression module and / or upstream and downstream homologous arms is introduced into Halomonas.

[0028] Preferably, the sgRNA for inserting the SsrA tag targets the C-terminal region of the protein to be degraded, preferably the stop codon of the coding sequence of the protein to be degraded.

[0029] Preferably, the upstream homologous arm is a nucleotide sequence located about 100-10000 mer (such as 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mer, etc.), preferably 400-600 mer, upstream of the stop codon of the protein coding gene to be degraded.

[0030] Preferably, the downstream homologous arm is located at the stop codon of the protein-encoding gene to be degraded or about 100 - 10,000 mer (such as 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mer, etc.) downstream thereof, preferably a nucleotide sequence of 400 - 600 mer.

[0031] The protein to be degraded is selected from proteins synthesized inappropriately intracellularly, damaged proteins, or proteins essential for microbial growth but with useless synthesis products. Preferably, the protein to be degraded is a protein maintaining cell morphology, a protein related to cell mitosis, or a protein regulating the metabolic flux of microorganisms.

[0032] More preferably, it is one, two or three of MreB protein, FtsZ protein or GltA protein.

[0033] When the protein to be degraded is MreB protein, the target site sequence of the sgRNA inserted with the SsrA tag includes SEQ ID NO: 8 or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with SEQ ID NO: 8, and has the same or similar function as it.

[0034] Preferably, the sequence of the sgRNA expression module includes the nucleotide sequence of positions 1 - 152 of SEQ ID NO: 6, or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with the nucleotide sequence of positions 1 - 152 of SEQ ID NO: 6, and has the same or similar function as it.

[0035] Preferably, the nucleotide sequence of the upstream homologous arm includes the nucleotide sequence of positions 153 - 652 of SEQ ID NO: 6, or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with the nucleotide sequence of positions 153 - 652 of SEQ ID NO: 6, and has the same or similar function as it.

[0036] Preferably, the nucleotide sequence of the downstream homologous arm includes the nucleotide sequence of positions 695 - 1194 of SEQ ID NO: 6, or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with the nucleotide sequence of positions 695 - 1194 of SEQ ID NO: 6, and has the same or similar function as it.

[0037] In a specific embodiment of the present invention, the plasmid containing the sgRNA expression module targeting mreB and / or the upstream and downstream homologous arms includes SEQ ID NO: 6, or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with SEQ ID NO: 6, and has the same or similar function as it.

[0038] When the protein to be degraded is FtsZ protein, the target site sequence of the sgRNA inserted with the SsrA tag includes SEQ ID NO: 30 or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with SEQ ID NO: 30, and has the same or similar function as it.

[0039] Preferably, the sequence of the sgRNA expression module includes the nucleotide sequence of positions 1-152 of SEQ ID NO: 31, or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with the nucleotide sequence of positions 1-152 of SEQ ID NO: 31, and has the same or similar function as it.

[0040] Preferably, the nucleotide sequence of the upstream homologous arm includes the nucleotide sequence of positions 153-652 of SEQ ID NO: 31, or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with the nucleotide sequence of positions 153-652 of SEQ ID NO: 31, and has the same or similar function as it.

[0041] Preferably, the nucleotide sequence of the downstream homologous arm includes the nucleotide sequence of positions 695-1194 of SEQ ID NO: 31, or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with the nucleotide sequence of positions 695-1194 of SEQ ID NO: 31, and has the same or similar function as it.

[0042] In a specific embodiment of the present invention, the plasmid containing the sgRNA expression module targeting ftsZ and / or the upstream and downstream homologous arms includes SEQ ID NO: 31, or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with SEQ ID NO: 31, and has the same or similar function as it.

[0043] When the protein to be degraded is GltA protein, the target site sequence of the sgRNA into which the SsrA tag is inserted includes SEQ ID NO: 32 or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with SEQ ID NO: 32, and has the same or similar function as it.

[0044] Preferably, the sequence of the sgRNA expression module includes the nucleotide sequence of positions 1-152 of SEQ ID NO: 5, or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with the nucleotide sequence of positions 1-152 of SEQ ID NO: 5, and has the same or similar function as it.

[0045] Preferably, the nucleotide sequence of the upstream homology arm includes the nucleotide sequence of positions 153-657 of SEQ ID NO: 5, or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with the nucleotide sequence of positions 153-657 of SEQ ID NO: 5, and has the same or similar function as it.

[0046] Preferably, the nucleotide sequence of the downstream homology arm includes the nucleotide sequence of positions 700-1199 of SEQ ID NO: 5, or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with the nucleotide sequence of positions 700-1199 of SEQ ID NO: 5, and has the same or similar function as it.

[0047] In a specific embodiment of the present invention, the plasmid containing the sgRNA expression module targeting gltA and / or the upstream and downstream homology arms includes SEQ ID NO: 5, or has a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more than 99.9% with SEQ ID NO: 5, and has the same or similar function as it.

[0048] Preferably, the plasmid for introduction (such as knocking out or knocking down sspB, or inserting the SsrA tag) can be transformed by Escherichia coli conjugation.

[0049] The recombinant Halomonas has also knocked out phaP1 and introduced phaP2-GFP.

[0050] Preferably, the recombinant Halomonas includes Halomonas bluephagenesis or its derivative strains (such as TD01ΔphaP1-phaP2-GFP, etc.), Halomonas campaniensis or its derivative strains, or Halomonas aydingkolgenesis or its derivative strains.

[0051] The recombinant Halomonas still maintains the function of producing PHA and can produce more PHA.

[0052] When the protein to be degraded is MreB protein, FtsZ protein or GltA protein, at the initial stage of culturing the recombinant Halomonas (such as the logarithmic phase or before it, such as the first 10 - 15 h), the MreB protein, FtsZ protein or GltA protein functions normally and there is no significant difference in the amount of the protein. When the recombinant Halomonas is continuously cultured to the middle and late stages (such as the stationary phase or the decline phase, such as 12 - 24 h), the MreB protein, FtsZ protein or GltA protein begins to degrade, and the degradation of the MreB protein and FtsZ protein makes the bacterial morphology change into a spherical shape. It shows that the recombinant Halomonas constructed in this application can achieve the degradation of the protein to be degraded in the middle and late stages of culturing.

[0053] The principle by which the recombinant Halomonas achieves the degradation of the protein to be degraded is as follows: adding an SsrA tag to the C-terminus of the protein to be degraded, so that the ClpXP protease in Halomonas can recognize and degrade the protein to be degraded. After knocking out the adaptor protein SspB, the delayed degradation of the protein to be degraded and the optimal degradation rate are achieved without affecting the growth and metabolism of the bacteria.

[0054] In the second aspect of the present invention, a method for constructing the above-mentioned recombinant Halomonas is provided, and the construction method includes knocking out or knocking down the sspB gene, and inserting an SsrA tag into the C-terminus of the protein to be degraded.

[0055] Preferably, the construction method includes introducing a plasmid for knocking out or knocking down the sspB gene, and a plasmid for inserting the SsrA tag into Halomonas.

[0056] The plasmid for knocking out or knocking down the sspB gene includes an sgRNA expression module targeting the sspB gene and / or upstream and downstream homologous arms. Preferably, the nucleotide sequence of the plasmid for knocking out or knocking down the sspB gene includes SEQ ID NO: 1.

[0057] The plasmid for inserting the SsrA tag includes an sgRNA expression module targeting the stop codon of the gene encoding the protein to be degraded and / or upstream and downstream homologous arms. Preferably, the nucleotide sequence of the plasmid for inserting the SsrA tag includes SEQ ID NO: 6, 31 or 5.

[0058] In a third aspect of the present invention, there is provided a method for producing PHA or a method for changing the morphology of Halomonas or regulating the metabolic flux of Halomonas, said method comprising culturing the above-mentioned recombinant Halomonas.

[0059] Said culturing can be carried out under conventional culture media and culture conditions in the prior art for culturing or fermentation.

[0060] Said culture medium can be solid, liquid or semi-solid.

[0061] Said culture medium comprises a carbon source, a nitrogen source, inorganic salts and other substances beneficial to the growth and metabolism of microorganisms.

[0062] Said culture medium can be LB or MM, etc.

[0063] The temperature of said culturing is any value in the range of 30 - 40 °C, such as 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C or 40 °C.

[0064] Said culturing can be an open culturing.

[0065] Said culturing includes seed culturing, shake flask culturing and / or fermenter culturing.

[0066] Said culturing can be batch fermentation, fed-batch fermentation or continuous fermentation, etc.

[0067] In a fourth aspect of the present invention, there is provided a method for inducing the self-degradation of proteins in Halomonas, said method comprising making Halomonas not express the SspB protein or the expressed SspB protein not have a function or have a reduced function, and inserting an SsrA tag at the C-terminus of the protein to be degraded in Halomonas.

[0068] In a fifth aspect of the present invention, there is provided a method for separating cells or extracting PHA, said method comprising centrifuging the fermentation broth obtained from the above-mentioned culturing.

[0069] In a sixth aspect of the present invention, there is provided a method for changing the morphology of Halomonas from rod-shaped to spherical, said method comprising making Halomonas not express the SspB protein or the expressed SspB protein not have a function or have a reduced function, and inserting an SsrA tag at the C-terminus of the protein to be degraded in Halomonas.

[0070] Preferably, the protein to be degraded is MreB protein and / or FtsZ protein.

[0071] Preferably, the transformation from rod-shaped to spherical occurs in the middle and late stages of culturing Halomonas.

[0072] The "PHA" in the present invention is a homopolymer or copolymer of monomers that make up PHA, and the monomers that make up PHA include, but are not limited to, one, two, or more of 2-hydroxypropionic acid, 3-hydroxybutyric acid, 4-hydroxybutyric acid, 3-hydroxypentanoic acid, 3-hydroxypropionic acid, 5-hydroxypentanoic acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 6-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, or 3-hydroxydodecanoic acid. More preferably, the PHA includes, but is not limited to, one or more of P3HP, PHB, P(HB-LA), PHV, P34HB, PHBV, PHBHHx, PHBHHp, PHO, PHN, PHD, P3HB4HB3HV, or P3HB4HB5HV.

[0073] The "natural amino acids" in the present invention are, for example, alanine, serine, asparagine, glutamic acid, threonine, phenylalanine, histidine, aspartic acid, methionine, leucine, isoleucine, cysteine, arginine, valine, proline, tryptophan, lysine, glycine, tyrosine, glutamine, and so on.

[0074] The "unnatural amino acids" in the present invention are compounds containing amino and carboxyl groups that are not naturally found in proteins. Preferably, the unnatural amino acids are any unnatural amino acids known in the art. More preferably, the unnatural amino acids include, but are not limited to, N-ethylaspartyl, hydroxylysine, 3-hydroxyproline, 2-aminobutyric acid, β-alanine, β-aminopropionic acid, 2-aminoadipic acid, 3-aminoadipic acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, allo-isoleucine, isodesmosine, 4-hydroxyproline, allo-hydroxylysine, 2-aminoisobutyric acid, N-methylglycine, N-methylisoleucine, 3-aminoisobutyric acid, 6-N-methyllysine, 2,4-diaminobutyric acid, N-methylvaline, ornithine, norleucine, norvaline, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, or 2-aminoheptanedioic acid, and so on. Of course, the "unnatural amino acids" described in this application can also be derivatives of natural amino acids after modification.

[0075] The "comprising" or "including" in the present invention is an open-ended expression. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still has the same or similar activity as the original sequence.

[0076] The Chinese full name or English full name corresponding to the English abbreviations in this application is shown in Table 1.

[0077] Table 1

[0078] Abbreviation in English Full Chinese Name or Full English Name SspB Stringent starvation protein B sspB Stringent starvation protein B encoding gene SsrA a small stable RNA ssrA a small stable RNA encoding gene PHA Polyhydroxyalkanoate MreB Actin-like protein mreB Actin-like protein encoding gene GFP Green fluorescent protein Description of the Drawings

[0079] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings, wherein:

[0080] Figure 1 : Degradation rate of GFP by different mutation tags at different time points.

[0081] Figure 2 : Fluorescence microscope images of recombinant bacteria TD01ΔsspBΔphaP1-phaP2-GFP and

[0082] TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA under different fields of view, where the first column is TD01ΔsspBΔphaP1-phaP2-GFP and the second column is TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA.

[0083] Figure 3 : Optical microscope images of recombinant bacteria TD01ΔsspBΔphaP1-phaP2-GFP and

[0084] TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA under different fields of view, where the first column is TD01ΔsspBΔphaP1-phaP2-GFP and the second column is TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA.

[0085] Figure 4 : Statistical analysis of the diameter of PHA granules in recombinant bacteria TD01ΔsspBΔphaP1-phaP2-GFP (control) and

[0086] TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA (mreB-ssrA21).

[0087] Figure 5 : Sedimentation of recombinant bacteria TD01ΔsspBΔphaP1-phaP2-GFP and TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA after centrifugation at 1000 rpm for 15 minutes, where the left three groups are TD01ΔsspBΔphaP1-phaP2-GFP and the right three groups are TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA.

[0088] Figure 6: Comparison of the dry cell weight and PHA content of recombinant bacterium TD01ΔsspBΔphaP1-phaP2-GFP (control) and

[0089] TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA (mreB-ssrA21).

[0090] Figure 7 : Optical microscope pictures of wild-type LS21 and LS21ΔsspB-ftsZ-ssrA under different fields of view. Among them, the first column is wild-type LS21, and the second column is LS21ΔsspB-ftsZ-ssrA.

[0091] Figure 8 : Sedimentation of wild-type LS21 and recombinant bacterium LS21ΔsspB-ftsZ-ssrA after centrifugation at 1000 rpm for 15 minutes. Among them, the left three groups are wild-type LS21, and the right three groups are recombinant bacterium LS21ΔsspB-ftsZ-ssrA.

[0092] Figure 9 : Dry cell weight and PHA content of recombinant bacterium LS21ΔsspB-ftsZ-ssrA21 (FtsZ-ssrA21) and LS21 (control) after 48 h of fermentation. Specific implementation manners

[0093] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0094] Sources of strains used in the embodiments:

[0095] Halomonas bluephagenesis TD01: Recorded 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 this bacterium from Tsinghua University.

[0096] Halomonas bluephagenesis TD01ΔphaP1-phaP2-GFP: described in LIU X, LID J, et al., Rapid Quantification of Polyhydroxyalkanoates Accumulated in Living Cells Based on Green Fluorescence Protein-Labeled Phasins: The qPHA Method [J]. Biomacromolecules, 2022.; The public can obtain this strain from Tsinghua University.

[0097] Halomonas campaniensis LS21: disclosed 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 the public can obtain this strain from Tsinghua University.

[0098] Halomonas aydingkolgenesis M1: described in the patent application publication number CN111593006A; the public can obtain this strain from Tsinghua University.

[0099] Gene editing methods used in the examples:

[0100] For example, to knockout sspB or insert an SsrA tag, the CRISPR / Cas9 gene editing technology is used. See the literature Qin et al., CRISPR / Cas9 editing genome of extremophile Halomonas spp. Metabolic Engineering. 47(2018)219 - 229.

[0101] Media used in the examples:

[0102] Formulation of LB60 medium:

[0103] 60 g / L sodium chloride, 5 g / L yeast extract, 10 g / L tryptone, 0.1 - 50 g / L carbon source combination.

[0104] Formulation of MM medium:

[0105] Urea 0.5 g / L; MgSO4 0.2 g / L; KH2PO4 1.5 g / L; and Fe(III)-NH4-Citrate, CaCl2·2H2O, ZnSO4·7H2O, MnCl2·4H2O, H3BO3, CoCl2·6H2O, CuSO4·5H2O, NiCl2·6H2O, NaMoO4·2H2O with a total of <0.1 g / L. And add 10 - 50 g / L of glucose as a carbon source, add 10 - 100 g / L of sodium chloride to provide the osmotic pressure environment required for the growth of halophilic bacteria; and adjust the pH to 8.0 - 9.0.

[0106] Detection methods used in the examples:

[0107] Method for detecting the content of polyhydroxyalkanoates (PHA) by gas chromatography:

[0108] Set the furnace temperature to 80 °C, the injector temperature to 200 °C, the detector temperature to 220 °C, the column head pressure to 0.25 Mpa, and the programmed temperature rise conditions are: stay at 80 °C for 1.5 minutes, rise to 140 °C at a rate of 30 °C / min, then rise to 220 °C at a rate of 40 °C / min and hold at this temperature for 0.5 minutes. The injection volume of the sample is 1 μL, and a micro syringe produced by Agilent is used.

[0109] Preparation of gas phase samples: Take 40 - 60 mg of the stem cells of the sample to be tested (take the bacterial liquid, centrifuge at 10000 rpm at room temperature for 10 minutes, wash the obtained cell precipitate once with water, and then dry it in ice to obtain stem cells, and the homopolymer is produced in the cells), add 2 mL of chloroform and 2 mL of esterification solution (3% (v / v) concentrated sulfuric acid and 1 g / L benzoic acid as an internal standard in pure methanol) to the esterification tube, seal it with a lid and heat it at 100 °C for 4 hours. After cooling, add 1 mL of distilled water, shake well and let it stand. After the chloroform phase and the water phase are completely separated, take 1 μL of the lower chloroform phase and inject it into a gas chromatograph (Hewlett Packard 6890 of HP company) for chromatographic analysis. Operate the gas chromatograph according to the instruction manual of the Hewlett Packard 6890 gas chromatograph of HP company.

[0110] Preparation of standard samples: Take 10 - 20 mg of the standard sample in the esterification tube, add 2 mL of chloroform and 2 mL of esterification solution, seal it with a lid and carry out esterification at 100 °C.

[0111] Result analysis: Taking the standard sample as a control, if the esterification sample (sample to be tested) of the cell to be tested has an obvious peak at the position of the standard sample, the mass of each monomer can be calculated according to the peak area, and then the molar ratio can be calculated according to the mass fraction of each monomer; the proportion (wt%) of the polymer contained in the cell dry weight can be calculated according to the amount of the sample added.

[0112] The described dry cell weight (DCW, g / L) is the ratio of the mass of the dried bacterial cells to the volume of the fermentation product.

[0113] The described PHA content (wt%) is the mass ratio of PHA to the dried bacterial cells.

[0114] Example 1: Effect of sspB knockout on the growth of Halomonas

[0115] The verification results of this example show that knocking out sspB has no effect on the growth of bacteria.

[0116] The recombinant strain Halomonas bluephagenesis TD01ΔsspB was constructed by editing the genome using the CRISPR / Cas9 genome editing method. Specifically, the method for constructing the plasmid containing sgRNA (atagagagctcgggcgagat (SEQ ID NO: 7)) and the recombinant template was to insert DNA fragments such as the upstream and downstream homologous arms and the sgRNA expression module into the original expression plasmid pSEVA341 (containing kanamycin and spectinomycin resistance genes) by the Gibson method. The sequence of the inserted plasmid is SEQ ID NO: 1, and the arrangement is in turn the sgRNA expression module (nucleotides 1 - 152), the upstream homologous arm (nucleotides 153 - 652), and the downstream homologous arm (nucleotides 653 - 1152). The plasmid pSEVA341 expressing sgRNA and the recombinant template and the plasmid expressing Cas9 were transferred into Halomonas bluephagenesis TD01 (abbreviated as TD01) by conjugation transformation of Escherichia coli S17-1. Mutant strains with sspB gene knockout were screened by colony PCR using designed primers and confirmed by gene sequencing. Colony PCR is a routine operation. Then, the strain after successful genome editing was continuously passaged multiple times in liquid medium, and streaked on plates with spectinomycin resistance, chloramphenicol resistance, and no resistance to identify strains with CRISPR plasmid loss for the next round of genome editing. Finally, after confirmation by colony PCR and gene sequencing, it was confirmed that the endogenous sspB gene (amino acid sequence as shown in SEQ ID NO: 2, nucleotide sequence as shown in SEQ ID NO: 3) in the Halomonas bluephagenesis TD01 genome had been knocked out. The constructed recombinant strain was named TD01ΔsspB.

[0117] Nucleotide sequences containing the sgRNA expression module and the upstream and downstream homologous arms:

[0118]

[0119] Amino acid sequence of endogenous sspB:

[0120] MKSSRPYLARALYEWLLDNELTPYVVVDATQPGVEVPRQFVQNGQIVLNVAPTAV RDLFMENQAMGFNARFGGQPMQVMIPTPALIAIYARENGAGMVFGHEPELDAAEFDD AELDDNFSDSFDESSKPELTVTEPVPEKEGDVSDQSSKKAKKKPTLRVVK(SEQ ID NO: 2)

[0121] Nucleotide sequence of endogenous sspB:

[0122] Atgaaatcgagccgtccctatctcgcccgagctctctatgagtggttgttagataatgagttaacgccttatgtggtggttgatgctacccagcctggcgtggaagtgccacgtcagtttgttcaaaatgggcaaattgtgttgaatgtagcgccgaccgctgtgcgtgacctgttcatggaaaatcaggccatgggttttaacgctcgctttggcggccagcctatgcaagtaatgattccaacaccggcgcttattgcgatatacgctcgtgaaaatggtgctggtatggtatttgggcatgagccagagctggatgcggcagagtttgatgatgcagaactagacgataatttttcagactcttttgatgaatcgtctaagcccgaattaacggtgacagagccagtcccggaaaaagagggcgacgtgtctgatcagtcgtctaaaaaggctaagaaaaagccgactctgcgcgtagttaagtag(SEQ ID NO: 3)

[0123] The effect of gene knockout on bacteria was further determined by PHA production experiments. The knockout strain was first streaked on an LB60 plate to obtain a monoclonal strain, and then seed liquid was obtained by secondary LB60 transfer culture. The seed liquid was inoculated into MM medium at a 5% inoculation rate and cultured at 37°C and 200rpm for 48 hours. Finally, the dry weight of cells after freeze-drying and the PHA content were measured by gas chromatography. It can be proved that knocking out sspB has no effect on the growth of bacteria and the ability to produce PHA (see Table 2).

[0124] Table 2

[0125]

[0126]

[0127] Example 2: Adding a mutant SsrA tag to verify protein degradation

[0128] 1. Mutate the wild-type SsrA tag to construct a library

[0129] The wild-type SsrA tag from Halomonas bluephagenesis was mutated and the sequence was shown as AANDENYAQGXXXX (SEQ ID NO: 4), wherein XXXX is independently selected from natural or non-natural amino acids. The specific sequences obtained are shown in Table 3.

[0130] Table 3

[0131] Abbreviation of label Specific amino acid sequence Sequence number SsrA1 AANDENYAQGALAA SEQ ID NO: 9 SsrA2 AANDENYAQGADAC SEQ ID NO: 10 SsrA3 AANDENYAQGADAG SEQ ID NO: 11 SsrA4 AANDENYAQGADAN SEQ ID NO: 12 SsrA5 AANDENYAQGADAS SEQ ID NO: 13 SsrA6 AANDENYAQGADAT SEQ ID NO: 14 SsrA7 AANDENYAQGADAV SEQ ID NO: 15 SsrA8 AANDENYAQGADCS SEQ ID NO: 16 SsrA9 AANDENYAQGADKS SEQ ID NO: 17 SsrA10 AANDENYAQGADSA SEQ ID NO: 18 SsrA11 AANDENYAQGADSS SEQ ID NO: 19 SsrA12 AANDENYAQGADTS SEQ ID NO: 20 SsrA13 AANDENYAQGADVS SEQ ID NO: 21 SsrA14 AANDENYAQGAHHA SEQ ID NO: 22 SsrA15 AANDENYAQGAISS SEQ ID NO: 23 SsrA16 AANDENYAQGAISV SEQ ID NO: 24 SsrA17 AANDENYAQGALDD SEQ ID NO: 25 SsrA18 AANDENYAQGALGG SEQ ID NO: 26 SsrA19 AANDENYAQGALQS SEQ ID NO: 27 SsrA20 AANDENYAQGALWW SEQ ID NO: 28 SsrA21 AANDENYAQGAQSA SEQ ID NO: 29

[0132] 2. Degradation of green fluorescent protein (GFP) in bacteria by adding SsrA tag

[0133] The pSEVA321-GFP-ssrA series plasmids were constructed, which included the pSEVA series backbone, the gene encoding green fluorescent protein and the SsrA tag sequence fused to its C-terminus (each mutant SsrA tag in Table 3 was added respectively), and the plasmid was introduced into the TD01△sspB prepared in Example 1. The fluorescence intensity of GFP was detected at different time points to evaluate the degradation effect of GFP. The results showed that different sequences had different degradation rates. The degradation effects of some selected representatives are shown in the following table. Figure 1 shown.

[0134] 3. Compare the effects of the presence or absence of SspB on degradation.

[0135] The pSEVA321-GFP-ssrA21 plasmid was introduced into strain TD01 and TD01ΔsspB respectively, and the fluorescence was compared at 12 hours and 24 hours. It was observed that sspB could cause the rapid degradation of the tagged protein, which was not conducive to achieving the purpose of later degradation (see Table 4).

[0136] Table 4

[0137]

[0138] Example 3: Morphological engineering of bacteria by degrading MreB protein

[0139] Using Halomonas bluephagenesis TD01ΔphaP1-phaP2-GFP (abbreviated as TD01ΔphaP1-phaP2-GFP) as the starting strain, the SsrA21 tag was used to induce the degradation of MreB protein after 12 h. Specifically, the sspB gene on the genome of TD01ΔphaP1-phaP2-GFP was knocked out (according to the method of Example 1), and the recombinant strain was named TD01ΔsspBΔphaP1-phaP2-GFP. On the basis of the recombinant strain, a strain with automatic degradation of MreB protein in the later stage was constructed. Specifically, the coding sequence of SsrA21 (GCGgcaaatgacgaaaactacgctcaaggcGCGCAGAGCGCG (SEQ ID NO: 33)) was inserted before the stop codon of the endogenous mreB gene (adjacent to the stop codon), and the strain was named TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA.

[0140] The constructed recombinant bacteria TD01ΔsspBΔphaP1-phaP2-GFP and TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA were subjected to fermentation tests. Samples were taken for microscopic observation at 24 h of fermentation. The bacteria changed from rod-shaped to spherical, and the volume also increased. After 48 h, the fermentation ended. The bacteria were centrifuged at a low speed and analyzed by gas chromatography. The spherical bacteria were more easily centrifuged and precipitated, and the content of PHA (polyhydroxyalkanoates) also increased.

[0141] The specific implementation process is as follows:

[0142] (1) Construction of recombinant bacteria TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA

[0143] Editing the genome using the CRISPR genome editing method. Specifically, the method for constructing a plasmid containing sgRNA (tgtcgagcgactgatcgtag (SEQ ID NO: 8)) and a recombination template is to insert DNA fragments such as upstream and downstream homologous arms, sgRNA expression module, and SsrA21 tag into the original expression plasmid pSEVA341 (containing kanamycin and spectinomycin resistance genes) by the Gibson method. The sequence of the inserted plasmid is SEQ ID NO: 6, which is arranged in turn as the sgRNA expression module (nucleotides 1-152), upstream homologous arm (nucleotides 153-652), SsrA21 tag coding sequence (nucleotides 653-694), and downstream homologous arm (nucleotides 695-1194).

[0144]

[0145] The pSEVA341 plasmid expressing sgRNA and the recombinant template, and the pQ08 plasmid expressing Cas9 were transferred into the recombinant bacterium TD01ΔsspBΔphaP1-phaP2-GFP by Escherichia coli S17-1 conjugation transformation.

[0146] Colony PCR primers were designed to screen for mutants with the SsrA21 tag inserted, and it was confirmed by gene sequencing. Colony PCR is a routine operation.

[0147] Finally, after confirmation by colony PCR and gene sequencing, it was verified that the coding sequence of the SsrA21 tag of nucleotides 653-694 in SEQ ID NO: 6 was inserted before the stop codon of the mreB gene in the genome of the recombinant bacterium TD01ΔsspBΔphaP1-phaP2-GFP. The constructed recombinant bacterium was named TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA.

[0148] (2) Shake flask fermentation was used to verify the morphological changes and PHA particle size changes of the recombinant bacteria TD01ΔsspBΔphaP1-phaP2-GFP and TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA.

[0149] The specific steps for detecting the morphology and PHA particle size of the recombinant strains during shake flask fermentation are as follows:

[0150] The recombinant strains were inoculated into 20 mL of LB60 medium and cultured for 12-16 h, then transferred to a new 20 mL of LB60 medium at a volume ratio of 1% and cultured for another 8-12 h.

[0151] 2.5 mL of the seed culture was inoculated into a 500 mL conical flask containing 50 mL of PHA fermentation medium for shake flask experiments. Each experiment was set up with three parallels, the shaker temperature was 37 °C, and the rotation speed was 200 rpm. After culturing for 24 h, 3 μL of the sample was made into a temporary slide and observed by FV1200 Confocal / FLIM / FCS. The results are as Figure 2 and Figure 3 shown. The morphology of the recombinant bacterium TD01ΔsspBΔphaP1-phaP2-GFP was rod-shaped. On this basis, the recombinant bacterium TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA with the SsrA21 tag added at the end of the mreB gene had a spherical morphology and an increase in cell diameter. Some discrete small PHA particles were visible in the statistical chart, which were caused by the release of PHA particles in some bacteria during the slide preparation process.

[0152] Specifically, Figure 2 each small ball in represents a PHA particle. The size of the PHA particles was statistically analyzed using ImageJ software. It was found that the size of the PHA particles in the recombinant strain TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA was larger than that in TD01ΔsspBΔphaP1-phaP2-GFP, and there was a significant difference between the two, as shown in Figure 4 .

[0153] (3) Shake flask fermentation was used to verify the sedimentation degree of the recombinant strains TD01ΔsspBΔphaP1-phaP2-GFP and TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA during centrifugation.

[0154] The specific steps for detecting the sedimentation degree of the recombinant strains during centrifugation after shake flask fermentation are as follows:

[0155] The recombinant strains were inoculated into 20 mL of LB60 medium and cultured for 12 - 16 h, then transferred to a new 20 mL of LB60 medium at a volume ratio of 1% and cultured for another 8 - 12 h.

[0156] 2.5 mL of the seed culture was inoculated into a 500 mL conical flask containing 50 mL of PHA fermentation medium (MM medium) for shake flask experiments. Each experiment was set up with three parallels, the temperature of the shaker was 37 °C, and the rotation speed was 200 rpm. After culturing for 48 h, 35 mL of the sample was poured into a 50 mL centrifuge tube and centrifuged at 1000 revolutions per minute for 15 minutes. The results are shown in Figure 5 As shown, after centrifugation at 1000 revolutions per minute for 15 minutes, compared with TD01ΔsspBΔphaP1-phaP2-GFP, the recombinant strain TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA was more easily centrifuged, and the supernatant and precipitate could be clearly distinguished.

[0157] (4) Shake flask fermentation was used to verify the DCW (Dry cell weight) and PHA content of the recombinant strains TD01ΔsspBΔphaP1-phaP2-GFP and TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA.

[0158] The specific steps for detecting the PHA production capacity of the recombinant strains after shake flask fermentation are as follows:

[0159] The recombinant strains were inoculated into 20 mL of LB60 medium and cultured for 12 - 16 h, then transferred to a new 20 mL of LB60 medium at a volume ratio of 1% and cultured for another 8 - 12 h.

[0160] 2.5 mL of the seed bacterial solution was inoculated into a 500 mL conical flask containing 50 mL of PHA fermentation medium (MM medium) for a shake flask experiment. The temperature of the shaker was 37 °C and the rotation speed was 200 rpm. After culturing for 48 h, the PHA content was detected. The experiment was set up with three parallels and the results were averaged. The results are as Figure 6 shown. After 48 h of fermentation, the dry cell weight of the recombinant bacterium TD01ΔsspBΔphaP1-phaP2-GFP-mreB-ssrA was not significantly different from that of TD01ΔsspBΔphaP1-phaP2-GFP, but the PHA content increased from 64.36% to 69.34%, and the difference was significant.

[0161] Example 4: Morphological engineering of bacteria by degrading FtsZ protein

[0162] Using Halomonas campaniensis LS21 (abbreviated as LS21) as the starting strain, the SsrA21 tag was used to induce the degradation of FtsZ protein after 12 h. Specifically, the sspB gene on the LS21 genome was knocked out (according to the method of Example 1) to construct a recombinant strain named LS21ΔsspB. On the basis of the recombinant strain, a strain with automatic degradation of FtsZ protein in the later stage was constructed. Specifically, the coding sequence of SsrA21 (GCGgcaaatgacgaaaactacgctcaaggcGCGCAGAGCGCG (SEQ ID NO: 33)) was inserted before the stop codon of the endogenous ftsZ gene (adjacent to the stop codon) to construct a strain named LS21ΔsspB-ftsZ-ssrA21.

[0163] The constructed recombinant bacteria LS21ΔsspB-ftsZ-ssrA21 and LS21 were subjected to fermentation tests. Samples were taken for microscopic observation at 24 h of fermentation, and the bacteria became slender from rod-shaped. After 48 h of fermentation, the fermentation was ended, and the bacteria were centrifuged at a low rotation speed and analyzed by gas chromatography. The slenderized bacteria were more easily centrifuged and precipitated, and the content of PHA (polyhydroxyalkanoates) also increased.

[0164] The specific implementation process is as follows:

[0165] (1) Construction of recombinant bacterium LS21ΔsspB-ftsZ-ssrA21

[0166] Editing the genome using the CRISPR genome editing method. Specifically, the method for constructing a plasmid containing sgRNA (acgtcgtcaggcagattaat (SEQ ID NO: 30)) and a recombination template is to insert DNA fragments such as upstream and downstream homologous arms, an sgRNA expression module, and an SsrA21 tag into the original expression plasmid pSEVA341 (containing kanamycin and spectinomycin resistance genes) by the Gibson method. The sequence of the inserted plasmid is SEQ ID NO: 31, arranged in sequence as an sgRNA expression module (nucleotides 1-152), an upstream homologous arm (nucleotides 153-652), an SsrA21 tag coding sequence (nucleotides 653-694), and a downstream homologous arm (nucleotides 695-1194).

[0167]

[0168] The pSEVA341 plasmid expressing sgRNA and the recombinant template and the pQ08 plasmid expressing Cas9 were transformed into the recombinant bacterium LS21ΔsspB by Escherichia coli S17-1 conjugation.

[0169] Mutant strains with the SsrA21 tag inserted were screened by designing primers through colony PCR and confirmed by gene sequencing. Colony PCR is a routine operation.

[0170] Finally, after confirmation by colony PCR and gene sequencing, it was verified that the coding sequence of the SsrA21 tag of nucleotides 653-694 in SEQ ID NO: 31 was inserted before the stop codon of the ftsZ gene in the genome of the recombinant bacterium LS21ΔsspB. The constructed recombinant bacterium was named LS21ΔsspB-ftsZ-ssrA21.

[0171] (2) Shake flask fermentation was used to verify the morphological changes and PHA content changes of the recombinant bacteria LS21ΔsspB-ftsZ-ssrA21 and LS21.

[0172] The specific steps for detecting the morphological changes during the shake flask fermentation of the recombinant strains are as follows:

[0173] The recombinant strains were inoculated into 20 mL of LB60 medium and cultured for 12-16 h, then transferred to a new 20 mL of LB60 medium at a volume ratio of 1% and cultured for another 8-12 h.

[0174] 2.5 mL of the seed bacterial solution was inoculated into a 500 mL conical flask containing 50 mL of PHA fermentation medium for a shake flask experiment. Each experiment was set with three parallels, the temperature of the shaker was 37 °C, and the rotation speed was 200 rpm. After culturing for 24 h, 3 μL of the sample was taken to make a temporary slide and observed by FV1200 Confocal / FLIM / FCS. The results are as Figure 7 shown. The morphology of the wild-type LS21 is rod-shaped. On this basis, the recombinant bacterium LS21ΔsspB-ftsZ-ssrA21 with the SsrA21 tag added to the end of the ftsZ gene has a more slender morphology and an obvious increase in the long axis of the cells. Some discrete PHA small particles can be seen in the statistical chart, which are caused by the release of PHA particles in some bacteria during the preparation of the slide due to being crushed.

[0175] (3) Shake flask fermentation was used to verify the sedimentation degree of the recombinant bacteria LS21ΔsspB-ftsZ-ssrA21 and LS21 during centrifugation.

[0176] The specific steps for detecting the sedimentation degree during centrifugation after the shake flask fermentation of the recombinant strains are as follows:

[0177] Inoculate the recombinant strain into 20 mL of LB60 medium, and after culturing for 12 - 16 h, transfer it to a new 20 mL of LB60 medium at a volume ratio of 1%, and continue culturing for 8 - 12 h.

[0178] Inoculate 2.5 mL of the seed culture into a 500 mL conical flask containing 50 mL of PHA fermentation medium (MM medium) for shake flask experiments. Set three parallels for each group of experiments, with the shaker temperature at 37 °C and the rotation speed at 200 rpm. After culturing for 48 h, take 35 mL of the sample and pour it into a 50 mL centrifuge tube, and centrifuge for 15 minutes at 1000 revolutions per minute. The results are as Figure 8 shown. After centrifuging for 20 minutes at 1000 revolutions per minute, compared with LS21, the recombinant strain LS21ΔsspB - ftsZ - ssrA21 is more easily centrifuged, and the supernatant and precipitate can be clearly distinguished.

[0179] (4) Verify the DCW (Dry cell weight) and PHA content of the recombinant strains LS21ΔsspB - ftsZ - ssrA21 and LS21 by shake flask fermentation.

[0180] The specific steps for detecting the PHA production capacity after shake flask fermentation of the recombinant strain are as follows:

[0181] Inoculate the recombinant strain into 20 mL of LB60 medium, and after culturing for 12 - 16 h, transfer it to a new 20 mL of LB60 medium at a volume ratio of 1%, and continue culturing for 8 - 12 h.

[0182] Inoculate 2.5 mL of the seed culture into a 500 mL conical flask containing 50 mL of PHA fermentation medium (MM medium) for shake flask experiments. The shaker temperature is 37 °C and the rotation speed is 200 rpm. After culturing for 48 h, detect the PHA content. Set three parallels for the experiment and take the average value of the results. The results are as Figure 9 shown. After 48 h of fermentation, the difference in the dry cell weight of the recombinant strains LS21ΔsspB - ftsZ - ssrA21 and LS21 is not significant, but the PHA content increases from 81.0% to 84.2%, and the difference is significant.

[0183] Example 5: Regulating the metabolic flux of bacteria by degrading the GltA protein

[0184] Using Halomonas aydingkolgenesis M1 (abbreviated as M1) as the starting strain, the SsrA7 tag was used to induce partial degradation of the GltA protein at 12 h. Specifically, the sspB gene on the M1 genome was knocked out (according to the method of Example 1), and the recombinant strain was named M1ΔsspB. On the basis of the recombinant strain, a strain with automatic degradation of partial GltA protein at a later stage was constructed. Specifically, the coding sequence of SsrA7 (GCGgcaaatgacgaaaactacgctcaaggcGCGGATGCGGTG (SEQ ID NO: 34)) was inserted before the stop codon of the endogenous gltA gene (adjacent to the stop codon), and the strain was named M1ΔsspB-gltA-ssrA7.

[0185] The constructed recombinant bacteria M1ΔsspB-gltA-ssrA7 and M1 were subjected to fermentation tests. After 48 h, the fermentation was completed, centrifuged at a low rotation speed and analyzed by gas chromatography. The DCM (Dry cell weight) of the strain with partial degradation of the GltA protein at a later stage increased by 1.7 g / L, and the PHA (polyhydroxyalkanoates) content increased from 75.3% to 83.2%.

[0186] The specific implementation process is as follows:

[0187] (1) Construction of recombinant bacteria M1ΔsspB-gltA-ssrA7

[0188] The CRISPR genome editing method was used to edit the genome. Specifically, the plasmid construction method containing sgRNA (cgactatcctaaaaaataac (SEQ ID NO: 32)) and the recombinant template was to insert DNA fragments such as upstream and downstream homologous arms, sgRNA expression module, and SsrA7 tag into the original expression plasmid pSEVA341 (containing kanamycin and spectinomycin resistance genes) by the Gibson method. The inserted plasmid sequence was SEQ ID NO: 5, and the arrangement was in turn the sgRNA expression module (nucleotides 1-152), upstream homologous arm (nucleotides 153-657), SsrA7 tag coding sequence (nucleotides 658-699), and downstream homologous arm (nucleotides 700-1199).

[0189]

[0190] The pSEVA341 plasmid expressing sgRNA and the recombinant template, and the pQ08 plasmid expressing Cas9 were transferred into the recombinant bacterium M1ΔsspB by Escherichia coli S17-1 conjugation transformation.

[0191] Colony PCR was used to design primers to screen for mutant strains with the SsrA7 tag inserted, and it was confirmed by gene sequencing. Colony PCR is a routine operation.

[0192] Finally, after confirmation by colony PCR and gene sequencing, it was verified that the SsrA7 tag coding sequence of nucleotides 658-699 in SEQ ID NO: 5 was inserted before the stop codon of the gltA gene in the genome of the recombinant bacterium M1ΔsspB-gltA-ssrA7. The constructed recombinant bacterium was named M1ΔsspB-gltA-ssrA7.

[0193] (2) Shake flask fermentation was used to verify the DCW (Dry cell weight) and PHA content of the recombinant bacteria M1ΔsspB-gltA-ssrA7 and M1.

[0194] The specific steps for detecting the PHA production capacity of the recombinant strain after shake flask fermentation are as follows:

[0195] The recombinant strain was inoculated into 20 mL of LB60 medium, and after culturing for 12-16 h, it was transferred to a new 20 mL of LB60 medium at a volume ratio of 1% and cultured for another 8-12 h.

[0196] 2.5 mL of the seed bacterial solution was inoculated into a 500 mL conical flask containing 50 mL of PHA fermentation medium (MM medium) for shake flask experiments. The temperature of the shaker was 37 °C and the rotation speed was 200 rpm. After culturing for 48 h, the PHA content was detected. The experiment was set up with three parallels and the results were averaged. As shown in Table 5, after 48 h of fermentation, compared with M1, the dry cell weight of the recombinant bacterium M1ΔsspB-gltA-ssrA7 increased from 10.13 to 11.82, and the PHA content increased from 75.3% to 83.2%, both showing significant differences.

[0197] Table 5

[0198] Group Cell dry weight (g / L) PHA content (%) M1 10.13±0.2 75.3±0.2 M1ΔsspB - gltA - ssrA7 11.82±0.3 83.2±0.4

[0199] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0200] In addition, it should be noted that, in the above specific embodiments, the various specific technical features described can be combined in any appropriate manner without contradiction. To avoid unnecessary repetition, the present invention will not separately describe various possible combination manners.

Claims

1. A recombinant Halomonas that induces self-degradation of proteins, characterized in that, The recombinant Halomonas does not express the SspB protein, or the expressed SspB protein is non-functional or has reduced function, and an SsrA tag is inserted at the C-terminus of the protein to be degraded in the recombinant Halomonas.

2. The recombinant Halomonas according to claim 1, characterized in that, Knock out or knockdown the sspB gene in Halomonas using an sgRNA targeting the sspB gene; Preferably, the target site sequence of the sgRNA includes SEQ ID NO:

7.

3. The recombinant Halomonas according to claim 1, characterized in that, The insertion position of the SsrA tag is before the stop codon of the coding sequence of the protein to be degraded; preferably adjacent to the stop codon.

4. The recombinant Halomonas according to claim 1 or 3, characterized in that, The amino acid sequence of the SsrA tag includes one or more of SEQ ID NOs: 9-29.

5. The recombinant Halomonas according to claim 1, characterized in that, The protein to be degraded is selected from proteins synthesized inappropriately intracellularly, damaged proteins, or proteins essential for microbial growth but with useless synthesis products; Preferably, the protein to be degraded is a protein maintaining cell morphology, a protein related to cell mitosis, or a protein regulating the metabolic flux of microorganisms; More preferably, it is one, two, or three of the MreB protein, FtsZ protein, or GltA protein.

6. The recombinant Halomonas according to any one of claims 1-5, characterized in that, The recombinant Halomonas includes Halomonas bluephagenesis or its derivative, Halomonas campaniensis or its derivative, or Halomonas aydingkolgenesis or its derivative.

7. A method for constructing the recombinant Halomonas according to any one of claims 1-6, characterized in that, The construction method includes knocking out or knocking down the sspB gene and inserting an SsrA tag at the C-terminus of the protein to be degraded.

8. The construction method according to claim 7, characterized in that, The construction method includes introducing a plasmid for knocking out or knocking down the sspB gene and a plasmid for inserting the SsrA tag into Halomonas; Preferably, the nucleotide sequence of the plasmid for knocking out or knocking down the sspB gene includes SEQ ID NO: 1; Preferably, the nucleotide sequence of the plasmid for inserting the SsrA tag includes SEQ ID NOs: 6, 31, or 5.

9. A method for producing PHA or a method for changing the morphology of Halomonas, characterized in that, The method includes culturing the recombinant Halomonas according to any one of claims 1-6.

10. A method for inducing self-degradation of proteins in Halomonas, characterized in that, The method includes making Halomonas not express the SspB protein, or the expressed SspB protein is non-functional or has reduced function, and inserting an SsrA tag at the C-terminus of the protein to be degraded in Halomonas.

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