Recombinant halomonas for producing gamma-aminobutyric acid and application of recombinant halomonas

Abstract

The invention relates to the technical field of engineering modification of microorganisms, in particular to recombinant halomonas and application thereof in production of gamma-aminobutyric acid. The invention provides a recombinant halomonas, the recombinant halomonas expresses or overexpresses glutamate decarboxylase and / or glutamic acid / gamma-aminobutyric acid antiporter, and the recombinant halomonas also inactivates gamma-aminobutyric acid transaminase, so that the recombinant halomonas can be used for expressing or overexpressing glutamic acid decarboxylase and / or glutamic acid / gamma-aminobutyric acid antiporter. By culturing the recombinant halomonas disclosed by the invention, efficient production of gamma-aminobutyric acid can be realized, and other products (such as PHA) can be co-produced.

Description

Technical Field

[0001] The present invention relates to the technical field of engineering transformation of microorganisms, and in particular to a recombinant Halomonas bacterium for producing gamma-aminobutyric acid and an application thereof. Background Art

[0002] γ-Aminobutyric acid (GABA) is a four-carbon, non-protein amino acid found widely in vertebrates, plants, and microorganisms. It is an important inhibitory neurotransmitter in the mammalian central nervous system, reducing the activity of neurons to which it binds, inhibiting neurotransmission and balancing the effects of excitatory neurotransmitters. GABA can enhance immunity, reduce anxiety and depression, increase brain cell metabolism, and improve sleep. With the deepening of relevant research, the understanding of GABA's physiological functions has been gradually improved, making it safer and more reliable. Currently, GABA is gradually being widely used in industries such as medicine, food, healthcare, chemicals, and agriculture.

[0003] Currently, the production of GABA primarily relies on chemical synthesis, plant enrichment, and microbial fermentation. Chemical synthesis consumes non-renewable resources, is costly and energy-intensive, has poor safety, and has strict temperature requirements, polluting the environment and failing to meet the requirements of green production. Plant enrichment, on the other hand, only yields extremely low amounts of GABA, making it unsuitable for large-scale production. Furthermore, the process is complex and energy-intensive.

[0004] Microbial fermentation is highly specific, environmentally friendly, requires simple equipment, and is relatively low-cost, making it suitable for large-scale production. However, naturally bred strains have a low capacity to produce GABA, so metabolic engineering combined with synthetic biology and other disciplines is currently being used to increase production. Commonly used GABA-producing microorganisms, such as Lactobacillus, Escherichia coli, and Corynebacterium glutamicum, are subject to a long-term risk of contamination during growth. Large-scale industrial fermentation production requires significant labor and resources for high-temperature sterilization, thus reducing their competitive advantage in large-scale, low-cost industrial development. Summary of the Invention

[0005] The purpose of the present invention is to transform Halomonas, which does not produce γ-aminobutyric acid, into a microorganism that produces high γ-aminobutyric acid, and develop a low-cost production platform for γ-aminobutyric acid and its subsequent products or the co-production of γ-aminobutyric acid with other products, so as to achieve sterilization-free, open and continuous fermentation production of intracellular or extracellular products. In order to achieve the above-mentioned purpose, the present invention introduces a glutamate decarboxylase encoding gene and / or a glutamate / γ-aminobutyric acid reverse transporter (which can be an exogenous or endogenous gene) into Halomonas to achieve the production of γ-aminobutyric acid, and finally achieves high yield of γ-aminobutyric acid and efficient co-production with other products (such as PHA) by optimizing gene expression, inactivating proteins related to the γ-aminobutyric acid consumption pathway (such as γ-aminobutyric acid transaminase) and / or optimizing γ-aminobutyric acid culture and fermentation conditions. Specifically:

[0006] In a first aspect of the present invention, a recombinant Halomonas for producing γ-aminobutyric acid is provided, wherein the recombinant Halomonas expresses or overexpresses one or both of glutamate decarboxylase and glutamate / γ-aminobutyric acid antiporter; and / or the recombinant Halomonas does not express γ-aminobutyric acid transaminase or the expressed γ-aminobutyric acid transaminase is inactivated or has reduced function.

[0007] Preferably, one or both of the genes encoding glutamate decarboxylase and glutamate / γ-aminobutyric acid antiporter are introduced into Halomonas. The genes encoding glutamate decarboxylase and glutamate / γ-aminobutyric acid antiporter can be introduced simultaneously or separately.

[0008] Preferably, the glutamate decarboxylase or glutamate / γ-aminobutyric acid antiporter is endogenous or exogenous. Preferably, the recombinant Halomonas expresses glutamate decarboxylase.

[0009] Preferably, the glutamate decarboxylase is exogenous.

[0010] Further preferably, the glutamate decarboxylase is derived from Escherichia coli.

[0011] In a specific embodiment of the present invention, the glutamate decarboxylase is GadB.

[0012] Preferably, the amino acid sequence of glutamate decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 1, or comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or at least 99.9% identical to the amino acid sequence shown in SEQ ID NO: 1.

[0013] Preferably, the Halomonas expresses glutamate decarboxylase and glutamate / γ-aminobutyric acid antiporter.

[0014] Preferably, the glutamate decarboxylase and the glutamate / γ-aminobutyric acid antiporter are both exogenous.

[0015] Preferably, the sources of the glutamate decarboxylase or glutamate / γ-aminobutyric acid antiporter are the same or different.

[0016] Preferably, the glutamate / γ-aminobutyric acid antiporter is derived from Escherichia coli.

[0017] In a specific embodiment of the present invention, the glutamate decarboxylase is GadB; the glutamate / γ-aminobutyric acid antiporter is GadC.

[0018] Preferably, the amino acid sequence of the glutamate / gamma-aminobutyric acid antiporter comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or at least 99.9% identical to the amino acid sequence of SEQ ID NO:2.

[0019] Preferably, the expression of the glutamate decarboxylase and / or the glutamate / γ-aminobutyric acid antiporter is regulated by a promoter, and the promoter can be any conventional promoter in the prior art, as long as it can regulate the expression of the glutamate decarboxylase and / or the glutamate / γ-aminobutyric acid antiporter.

[0020] Preferably, the promoter includes a constitutive promoter and / or an inducible promoter.

[0021] Further preferably, the constitutive promoter includes P porinPromoter or its mutant (for example, as described in the literature: ShenR, Yin J, Ye JW, Xiang RJ, Ning ZY, Huang WZ, Chen GQ. Promoter Engineering for Enhanced P(3HB-co-4HB) Production by Halomonas blue phagenesis. ACS SynthBiol. 2018Aug17; 7(8): 1897-1906. doi: 10.1021 / acssynbio.8b00102. Epub 2018Jul31. PMID: 30024739.) or P Sp6 promoter or a variant thereof.

[0022] More preferably, P porin The promoter or its mutants include P porin 、P porin203 、P porin58 、P porin221 、P porin194 、P porin68 、P porin42 、P porin140 、P porin141 、P porin3 、P porin278 、P porin226 or P porin211 .

[0023] Preferably, the P porin Promoter mutants can be low-strength promoters, medium-strength promoters, or high-strength promoters depending on their expression strength.

[0024] Further preferably, the constitutive promoter includes P porin203 、P porin221 、P porin194 、P porin278 、P porin68 、P porin58 、P porin42 or P porin Any one or two or more.

[0025] Further preferably, the inducible promoter includes IPTG (isopropyl-β-D-thiogalactopyranoside) inducible promoter or AHL (homoserine lactone) inducible promoter. Preferably, the inducer includes but is not limited to IPTG or AHL. Preferably, the inducible promoter is selected from P frm promoter or its mutants, P luc Promoter or variant thereof, P lac Promoter or variant thereof, P trpPromoter or variant thereof, P tac Promoter or variant thereof, phage promoter or variant thereof, P araBAD Promoter or variant thereof, P phaP Promoter or variant thereof or P Mmp1 promoter or a variant thereof.

[0026] Preferably, the promoters regulating glutamate decarboxylase and / or glutamate / γ-aminobutyric acid antiporter are the same or different.

[0027] Preferably, the expression of the glutamate decarboxylase is regulated by a constitutive promoter or an inducible promoter. Preferably, the expression of the glutamate / γ-aminobutyric acid antiporter is regulated by a constitutive promoter or an inducible promoter.

[0028] Preferably, glutamate decarboxylase and / or glutamate / γ-aminobutyric acid antiporter can be independently expressed under the control of the same or different promoters, or the expression of both genes can be simultaneously regulated by the same promoter.

[0029] In a specific embodiment of the present invention, the promoter for regulating glutamate decarboxylase is a constitutive promoter or an inducible promoter, and the constitutive promoter is P porin promoter or its mutant; the inducible promoter is P Mmp1 promoter or a variant thereof.

[0030] Preferably, when the promoter is P Mmp1 When the promoter or its variant is used, an inducer needs to be added, and the inducer is preferably an IPTG inducer, and the concentration of the IPTG inducer is 0-400 mg / L, more preferably 0.1-200 mg / L, for example, 0, 0.1, 0.2, 0.5, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300 or 400 mg / L. In a specific embodiment of the present invention, the promoter for regulating the glutamate / γ-aminobutyric acid antiporter is a constitutive promoter or an inducible promoter, more preferably a constitutive promoter, and the constitutive promoter is preferably P porin Promoter or its mutant, more preferably P porin278 .

[0031] Preferably, the glutamate decarboxylase and / or glutamate / γ-aminobutyric acid antiporter is expressed on an expression vector and / or expressed on a chromosome.

[0032] Preferably, the glutamate decarboxylase or glutamate / γ-aminobutyric acid antiporter is expressed on an expression vector. Preferably, the two proteins can be expressed on one expression vector or on two expression vectors respectively.

[0033] Preferably, the expression on the chromosome comprises inserting one or both of glutamate decarboxylase or glutamate / gamma-aminobutyric acid antiporter into the chromosome (e.g., G3, G4, G7, or G51 site) of the recombinant Halomonas for expression. Preferably, glutamate decarboxylase and / or glutamate / gamma-aminobutyric acid antiporter are expressed at the same position or different positions on the chromosome.

[0034] Preferably, the two proteins are inserted into the same site of the Halomonas genome, or they can be inserted into different sites. Preferably, the two proteins are inserted into the G7 site of the Halomonas genome.

[0035] Preferably, the γ-aminobutyric acid transaminase comprises one, two, or three of GabT1, GabT2, or GabT3. Preferably, the non-expression of γ-aminobutyric acid transaminase or the inactivation or functional reduction of the expressed γ-aminobutyric acid transaminase comprises: 1) knocking out or knocking down the genes encoding one, two, or three of GabT1, GabT2, or GabT3; or, 2) mutating the genes encoding one, two, or three of GabT1, GabT2, or GabT3.

[0036] In one embodiment of the present invention, the inactivated γ-aminobutyrate aminotransferase is any one of the following groups:

[0037] A) GabT1;

[0038] B) GabT2;

[0039] C) GabT3;

[0040] D) GabT1 and GabT2;

[0041] E) GabT1 and GabT3;

[0042] F) GabT2 and GabT3;

[0043] G) GabT1, GabT2 and GabT3.

[0044] Preferably, the inactivated γ-aminobutyrate aminotransferase is GabT1 and GabT2.

[0045] Preferably, the culture medium used to culture the recombinant Halomonas for producing metabolites can be any culture medium available in the art, preferably a culture medium suitable for the growth and metabolism of Halomonas. For example, it includes, but is not limited to, one or more of MM culture medium, MMG culture medium, LB culture medium, TB culture medium, beef extract peptone culture medium, or SB culture medium. Of course, it can also be a culture medium modified based on the above culture medium according to the desired purpose. In other words, those skilled in the art can routinely select a suitable culture medium as long as it can allow the growth and / or metabolism of the microorganism.

[0046] Preferably, the culture medium contains inorganic salts (preferably potassium salts, calcium salts, sodium salts, magnesium salts or zinc salts, more preferably sodium salts), including but not limited to one or a combination of two or more of sodium chloride, sodium sulfate, sodium phosphate, sodium citrate, sodium acetate, sodium gluconate, sodium nitrate and sodium carbonate.

[0047] In a specific embodiment of the present invention, the inorganic salt is sodium chloride.

[0048] Preferably, the concentration of inorganic salts in the culture medium is 2.5-100 g / L, more preferably 5-80 g / L, for example, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100 g / L.

[0049] Preferably, the culture medium contains a carbon source. Preferably, the carbon source includes but is not limited to one or more of glucose, gluconate, gluconate ester or amino acids or their salts.

[0050] Preferably, the gluconate includes but is not limited to one or more of potassium salt, sodium salt, calcium salt, magnesium salt or zinc salt, preferably sodium gluconate.

[0051] Preferably, the amino acid or its salt is added by directly adding one or more amino acids or their salts, or by adding a substance that produces an amino acid or its salt after fermentation, or its fermentation product or fermentation liquid.

[0052] Preferably, the substance that produces amino acids or their salts after fermentation includes one or both of monosodium glutamate and soybean meal.

[0053] Preferably, the amino acid comprises one or more of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine, preferably, the amino acid salt comprises one or more of potassium salt, sodium salt, calcium salt, magnesium salt or zinc salt of any one or more of the above amino acids. Preferably, the amino acid is glutamic acid (preferably L-glutamic acid). Preferably, the amino acid salt is sodium glutamate. In a specific embodiment of the present invention, the carbon source comprises one or more of glucose, sodium gluconate, glutamic acid, sodium glutamate or monosodium glutamate fermentation broth.

[0054] Preferably, the carbon source further comprises other carbon sources. Preferably, the other carbon sources include one or more of sucrose, butyrolactone, 4-hydroxybutyric acid, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol, 1,12-dodecanediol, fructose, xylose, cellulose, lactose, lactic acid, lauric acid, acetic acid, caproic acid, propionic acid, valeric acid, capric acid, butyric acid, palm oil or oleic acid.

[0055] Preferably, the concentration of the carbon source in the culture medium is greater than 1 g / L, preferably 10-300 g / L, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 35, 36, 37, 38, 39, 40, 45, 50, 51, 52, 53, 54, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300 g / L.

[0056] Preferably, the culture medium contains a nitrogen source, which includes an organic nitrogen source and an inorganic nitrogen source. The inorganic nitrogen source includes various ammonium salts, nitrates and / or ammonia water, and the organic nitrogen source includes one or a combination of two or more of urea, peanut cake powder, soybean cake powder, cottonseed cake powder, corn steep liquor, yeast powder, fish meal, silkworm pupa powder, peptone, bran or waste mycelium.

[0057] In one embodiment, the nitrogen source is urea.

[0058] Preferably, the concentration of the nitrogen source in the culture medium can be any value in the range of 0-10 g / L, preferably 0.5-5 g / L, for example, 0, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 g / L, etc.

[0059] In a specific embodiment, the culture medium further comprises yeast extract, magnesium sulfate, potassium dihydrogen phosphate, trace element solution I and trace element solution II, wherein trace element solution I comprises ammonium ferric citrate and CaCl2, prepared with HCl; trace element solution II comprises ZnSO4·7H2O, MnCl2·4H2O, H3BO3, CoCl2·6H2O, CuSO4·5H2O, NiCl2·6H2O and NaMoO4·2H2O, prepared with HCl.

[0060] Preferably, the culture is carried out under open conditions, preferably without sterilization.

[0061] Preferably, the equipment and / or culture medium used in the culture may be sterilized or not.

[0062] Preferably, the Halomonas includes Halomonas bluephagenesis or its derivatives, Halomonascampaniensis or its derivatives, Halomonas desiderata or its derivatives, Halomonas cupida or its derivatives, Halomonas smymensis or its derivatives, Halomonas levan or its derivatives, Halomonaslutescens or its derivatives, Halomonas elongata or its derivatives, Halomonas venusta or its derivatives or Halomonas alkaliantarctica or its derivatives.

[0063] In a specific embodiment of the present invention, the Halomonas includes but is not limited to Halomonas bluephagenesis TD1.0, Halomonas campaniensis LS21, Halomonasaydingkolgenesis M1, Halomonas bluephagenesis WZY254 or Halomonas bluephagenesis WZY278.

[0064] Preferably, the recombinant Halomonas also produces PHA. Preferably, the recombinant Halomonas can be a Halomonas that naturally produces PHA, or a Halomonas that has been modified to produce PHA.

[0065] In a second aspect of the present invention, a method for preparing the above-mentioned recombinant Halomonas is provided, the preparation method comprising introducing a nucleotide sequence encoding one or both of glutamate decarboxylase or glutamate / γ-aminobutyric acid reverse transporter into Halomonas; and / or, the preparation method knocks out or knocks down γ-aminobutyric acid transaminase in Halomonas.

[0066] Preferably, the amino acid sequence of glutamate decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 1, or comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or at least 99.9% identical to the amino acid sequence shown in SEQ ID NO: 1.

[0067] SEQ ID NO: 1:

[0068] MDKKQVTDLRSELLDSRFGAKSISTIAESKRFPLHEMRDDVAFQIINDELYLDGNARQNLATFCQTWDDENVHKLMDLSINKNWIDKEEYPQSAAIDLRCVNMVADLWHAPAPKNG QAVGTNTIGSSEACMLGGMAMKWRWRKRMEAAGKPTDKPNLVCGPVQICWHKFARYWDVELREIPMRPGQLFMDPKRMIEACDENTIGVVPTFGVTYTGNYEFPQPLHDALDKFQAD TGIDIDMHIDAASGGFLAPFVAPDIVWDFRLPRVKSISASGHKFGLAPLGCGWVIWRDEEALPQELVFNVDYLGGQIGTFAINFSRPAGQVIAQYYEFLRLGREGYTKVQNASYQVA AYLADEIAKLGPYEFICTGRPDEGIPAVCFKLKDGEDPGYTLYDLSERLRLRGWQVPAFTLGGEATDIVVMRIMCRRGFEMDFAELLLEDYKASLKYLSDHPKLQGIAQQNSFKHT*

[0069] Preferably, the amino acid sequence of the glutamate / gamma-aminobutyric acid antiporter comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or at least 99.9% identical to the amino acid sequence of SEQ ID NO:2.

[0070] SEQ ID NO: 2:

[0071] MATSVQTGKAKQLTLLGFFAITASMVMVYEYPTFATSGFSLVFFLLLGGILWFIPVGLCAAEMATVDGWEEGGVFAWVSNTLGPRWGFAAISFGYLQIAIGFIPMLYFVLGALSYILKWPALNEDPI TKTIAALIILWALALTQFGGTKYTARIAKVGFFAGILLPAFILIALAAIYLHSGAPVAIEMDSKTFFPDFSKVGTLVVFVAFILSYMGVEASATHVNEMSNPGRDYPLAMLLLMVAAICLSSVGGLSI AMVIPGNEINLSAGVMQTFTVLMSHVAPEIEWTVRVISALLLLGVLAEIASWIVGPSRGMYVTAQKNLLPAAFAKMNKNGVPVTLVISQLVITSIALIILTNTGGGNNMSFLIALALTVVIYLCAYFM LFIGYIVLVLKHPDLKRTFNIPGGKGVKLVVAIVGLLTSIMAFIVSFLPPDNIQGDSTDMYVELLVVSFLVVLALPFILYAVHDRKGKANTGVTLEPINSQNAPKGHFFLHPRARSPHYIVMNDKKH*

[0072] In a specific embodiment of the present invention, the preparation method comprises introducing the expression vector into Halomonas.

[0073] In a specific embodiment of the present invention, the preparation method comprises introducing the expression vector into the recombinant Halomonas by conjugation transformation, for example, by conjugation transformation with Escherichia coli.

[0074] Preferably, the expression vector comprises a nucleotide sequence encoding one or both of glutamate decarboxylase and glutamate / γ-aminobutyric acid antiporter. Preferably, the nucleotide sequence encoding glutamate decarboxylase and glutamate / γ-aminobutyric acid antiporter can be in the same expression vector or in different expression vectors.

[0075] Preferably, the expression vector comprises a promoter, and the relevant limitations on the promoter are the same as those in the first aspect of the present invention. The expression vector can be expressed in isolation in Halomonas, or one or both of the nucleotide sequences encoding glutamate decarboxylase or glutamate / γ-aminobutyric acid antiporter can be inserted into the chromosome of Halomonas for expression.

[0076] In a specific embodiment of the present invention, the expression vector is expressed in Halomonas, and the expression vector comprises, from the 5' end to the 3' end, a promoter and a nucleotide sequence encoding glutamate decarboxylase; preferably, the promoter is an inducible promoter, and more preferably P Mmp1 promoter.

[0077] In a specific embodiment of the present invention, the expression vector is expressed in Halomonas, and the expression vector comprises, from the 5' end to the 3' end, a promoter for regulating the expression of glutamate decarboxylase, a nucleotide sequence encoding glutamate decarboxylase, a promoter for regulating the expression of glutamate / γ-aminobutyric acid antiporter, and a nucleotide sequence encoding the glutamate / γ-aminobutyric acid antiporter; preferably, the promoter for regulating the expression of glutamate decarboxylase is an inducible promoter, and more preferably P Mmp1 Promoter; Preferably, the promoter regulating the expression of glutamate / γ-aminobutyric acid antiporter is a constitutive promoter, more preferably P porin promoter or its mutant, the P porin Promoter mutants include P porin58 、P porin221 、P porin194 、P porin68 、P porin42 、P porin140 、P porin141 、P porin3 、P porin278 、P porin226 or P porin211 More preferably, P porin203 、P porin221 、P porin194 、P porin278 、P porin68 、P porin58 、P porin42 or P porin140 Any one, more preferably Pporin203 、P porin221 、P porin194 、P porin278 、P porin68 、P porin58 or P porin42 In one embodiment of the present invention, the promoter for regulating the expression of glutamate / γ-aminobutyric acid antiporter is P porin278 .

[0078] In a specific embodiment of the present invention, the expression vector inserts one or both of the nucleotide sequences encoding glutamate decarboxylase or glutamate / γ-aminobutyric acid antiporter into the chromosome of Halomonas for expression, and the expression vector comprises, from the 5' end to the 3' end, an upstream homology arm, a promoter regulating the expression of glutamate decarboxylase, a nucleotide sequence encoding glutamate decarboxylase, a promoter regulating the expression of glutamate / γ-aminobutyric acid antiporter, a nucleotide sequence encoding glutamate / γ-aminobutyric acid antiporter, and a downstream homology arm; preferably, the promoters regulating the expression of glutamate decarboxylase and glutamate / γ-aminobutyric acid antiporter are both constitutive promoters, and more preferably P porin promoter or its mutant, the P porin Promoter mutants include P porin58 、P porin221 、P porin194 、P porin68 、P porin42 、P porin140 、P porin141 、P porin3 、P porin278 、P porin226 or P porin211 More preferably, P porin203 、P porin221 、P porin194 、P porin278 、P porin68 、P porin58 、P porin42 or P porin140 In one embodiment of the present invention, the promoter for regulating the expression of glutamate decarboxylase is P porin140 The promoter that regulates the expression of glutamate / γ-aminobutyric acid antiporter is P porin278 .

[0079] Preferably, inserting into the chromosome of Halomonas for expression comprises inserting into the genome of Halomonas by gene editing (e.g., CRISPR / Cas9), preferably inserting into one or more of the G3 site (SEQ ID NO: 51), G4 site (SEQ ID NO: 52), G7 site (SEQ ID NO: 53) or G51 site (SEQ ID NO: 54) of Halomonas.

[0080] SEQ ID NO: 51

[0081] TAACCATCTAATGATCAGCGAGTCATGCTGGGATGGTTAAACAAGCTGTTAATTAGTCTATTTCTCCAATAGTGGCGCGGCTAAACTTGGACTAAGGCCGCAGTTTTGACGCTAAAACTAACCGAGACGCTACTCATGCAGCCAACTGCACGCTTTTCATTGTCGCCGGCTCTTCCTGAGACCCACACTCAACACAGCCATGGAGCTTGCTGTTGTGGTTCACCA

[0082] SEQ ID NO: 52

[0083] GATGGCAACGCCCTCTGTAATACCTACGCATGAATTACTCCTTTAGAGCGTGTAAATCGCCAGCACAACGCCCTTATGAAAAGACGCTTAAAATCGAGCGTCTGATTAAAGGCCGATAGCCCAAAGCAGGCTCGAGCAATCAGCCTTCCACCCAAGCTATTTTCGTTTAATTCTTTACCCCAGAAAAACTCCACAAATACAAA ATATAGTTAACTTTTGAGGCGATGCAAATCCACCTTCCGAGCACATCGATCATTCACCTAGCTAGATGAGACAGGCAGCTAATCATGTGCATCATCATTAAAAATTAACCGAGTAGCTTGCGGCACTTCATCTCACCTTCATTTCCTTATGCTGAACACGGTAAGCTTGGCGATAACACCGCTACTGATGGCTAAAAATCAACG

[0084] SEQ ID NO: 53

[0085] AGGTAGTCGCATTATCTTCTTTTGTGCTTTCGCCTTATGGCTCTAGGCGCTTGGATGCTCGTGATATTTTCCTACTCATTGGTCGGAAGATTCGAGACTATCTTCGATGTACACCATTACGGGGGTTGTCACGGGGTGAT ATAGAGTGTTCGCAAAGTTCACAAATGTGTCACGGGGACTCTAACGATAGGGCGATGTCTGCATTAACGCCTCCCCTCATTTCAATAAGTGTGTTTCAGCAAGGAGAGCGCCCATGGCGACTACACGCCGTAAACTC

[0086] SEQ ID NO: 54

[0087] GTACGCAGTAATGCGCGCACTCAGTGCACGACTCTTGTTTAATTAATCAAAAGTAAGAAACTAGTTCTTAAAAACTAACCACATTGTTAAATTCTGTCATAGCGACTCAGTGTAAAAACTCATTCAACTAATCGCCTACAACAAAGACAAGCTTTTATATACCAGCTTTAAAACCGGGCAACTAAAT ATCACCCAAGGCTTTAAATTAAACAAATAAGAAACAATTATCGCATTAATAATTAACGCTAAAAATCATGAATTAATTAAACTGCTAAAAAGTGTTTCTTTAACACTTTGATTTTTAATTGACTAATAAAAACCATTAAGGAAGCACTAATGCTACTTAAAACGCCCGCTAGCGATTAGCGGTCATCGC

[0088] Preferably, insertion into the chromosome of Halomonas for expression comprises the use of sgRNA.

[0089] Preferably, the target site sequence of the sgRNA targeting the G3 site comprises the nucleotide sequence shown in SEQ ID NO: 55. Preferably, the target site sequence of the sgRNA targeting the G4 site comprises the nucleotide sequence shown in SEQ ID NO: 56. Preferably, the target site sequence of the sgRNA targeting the G7 site comprises the nucleotide sequence shown in SEQ ID NO: 57. Preferably, the target site sequence of the sgRNA targeting the G51 site comprises the nucleotide sequence shown in SEQ ID NO: 58.

[0090] SEQ ID NO: 55: AATAGTGGCGGCGGCTAAACT

[0091] SEQ ID NO: 56: TTCACCTAGCTAGATGAGAC

[0092] SEQ ID NO: 57: ACACCATTACGGGGGTGTCA

[0093] SEQ ID NO: 58: TTGGGTGATATTTAGTTGCC

[0094] In one embodiment of the present invention, inserting into the chromosome of Halomonas for expression comprises inserting into the G7 site of Halomonas.

[0095] Preferably, the expression vector can be a prokaryotic expression vector or a eukaryotic expression vector, preferably a prokaryotic expression vector. Preferably, the expression vector is a plasmid (such as a pSE series plasmid, preferably pSEVA341, pSEVA241, pSEVA321, etc.).

[0096] In a specific embodiment of the present invention, the preparation method comprises introducing the above-mentioned expression vector and the Cas9 protein expression plasmid (for example, pQ08 plasmid, see Qin Q, Ling C, Zhao Y, et al. CRISPR / Cas9 editing genome of extremophile Halomonas spp[J]. Metabolic engineering, 2018, 47: 219-229.) into Halomonas.

[0097] It should be understood that the expression vector is capable of replication, transcription and translation in the host cell. Therefore, the expression vector may also contain other conventional expression elements, such as RBS, terminator, restriction enzyme cleavage sites, etc., as long as the normal expression of the above-mentioned protein can be guaranteed.

[0098] Preferably, the γ-aminobutyrate aminotransferase comprises one, two or three of GabT1, GabT2 or GabT3. In a specific embodiment of the present invention, the knockout or knockdown comprises any one of the following groups:

[0099] A) Nucleotide sequence encoding GabT1;

[0100] B) Nucleotide sequence encoding GabT2;

[0101] C) nucleotide sequence encoding GabT3;

[0102] D) a nucleotide sequence encoding GabT1 and a nucleotide sequence encoding GabT2;

[0103] E) a nucleotide sequence encoding GabT1 and a nucleotide sequence encoding GabT3;

[0104] F) a nucleotide sequence encoding GabT2 and a nucleotide sequence encoding GabT3;

[0105] G) Nucleotide sequence encoding GabT1, nucleotide sequence encoding GabT2 and nucleotide sequence encoding GabT3 In one embodiment of the present invention, the nucleotide sequence encoding GabT1 and the nucleotide sequence encoding GabT2 are knocked out or knocked down.

[0106] Among them, the nucleotide sequences encoding GabT1, GabT2, and GabT3 respectively comprise the nucleotide sequences shown in SEQ ID NOs: 3-5, or comprise nucleotide sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or at least 99.9% identical to the nucleotide sequences shown in SEQ ID NOs: 3-5.

[0107] SEQ ID NO: 3

[0108]

[0109] SEQ ID NO:4

[0110]

[0111] SEQ ID NO:5

[0112]

[0113] Preferably, the knockout or knockdown comprises the use of gene editing technology, including but not limited to CRISPR-Cas9 technology or tissue-specific knockout technology (such as Loxp-cre system).

[0114] In a specific embodiment of the present invention, the knockout or knockdown is achieved by utilizing CRISPR-Cas9 technology, including designing and synthesizing sgRNA targeting the gabT gene.

[0115] In a specific embodiment of the present invention, the target site sequence of the sgRNA for knocking out or knocking down the nucleotide sequence encoding GabT1 includes the nucleotide sequence shown in SEQ ID NO: 16,

[0116] In a specific embodiment of the present invention, the target site sequence of the sgRNA for knocking out or knocking down the nucleotide sequence encoding GabT2 includes the nucleotide sequence shown in SEQ ID NO: 17. In a specific embodiment of the present invention, the target site sequence of the sgRNA for knocking out or knocking down the nucleotide sequence encoding GabT3 includes the nucleotide sequence shown in SEQ ID NO: 18.

[0117] The third aspect of the present invention provides a recombinant Halomonas obtained by the preparation method described in the second aspect.

[0118] A fourth aspect of the present invention provides a method for culturing a microorganism or a method for producing γ-aminobutyric acid, wherein the method comprises culturing any of the above-mentioned recombinant Halomonas.

[0119] Preferably, the Halomonas bacteria can be selected to be those that can rapidly self-flocculate, and the Halomonas bacteria and the supernatant containing γ-aminobutyric acid are separated by self-flocculation, and then glutamate (preferably L-glutamate) is added to the Halomonas bacteria for whole-cell catalysis. Preferably, the method further comprises a step of purifying the γ-aminobutyric acid, preferably without centrifugation, utilizing the self-precipitation property of the Halomonas bacteria.

[0120] Preferably, the method comprises using a culture medium.

[0121] Preferably, the culture medium contains a nitrogen source. Further preferably, the nitrogen source includes an organic nitrogen source and an inorganic nitrogen source. The inorganic nitrogen source includes various ammonium salts, nitrates and / or ammonia water, and the organic nitrogen source includes one or a combination of two or more of urea, peanut cake powder, soybean cake powder, cottonseed cake powder, corn steep liquor, yeast powder, fish meal, silkworm pupa powder, peptone, bran or waste mycelium.

[0122] In one embodiment, the nitrogen source is urea.

[0123] Preferably, the concentration of the nitrogen source in the culture medium can be any value in the range of 0-10 g / L, preferably 0.5-5 g / L, for example, 0, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 g / L, etc.

[0124] Preferably, the culture medium contains an inorganic salt (preferably a potassium salt, a calcium salt, a sodium salt, a magnesium salt, or a zinc salt, more preferably a sodium salt), including but not limited to one or a combination of two or more of sodium chloride, sodium sulfate, sodium phosphate, sodium citrate, sodium acetate, sodium gluconate, sodium nitrate, and sodium carbonate. In a specific embodiment of the present invention, the inorganic salt is sodium chloride.

[0125] Preferably, the concentration of inorganic salts in the culture medium is 2.5-100 g / L, more preferably 5-80 g / L, for example, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100 g / L.

[0126] Preferably, the culture medium contains a carbon source.

[0127] Preferably, the carbon source includes but is not limited to one or more of glucose, gluconate, gluconate ester or amino acid or its salt.

[0128] Preferably, the gluconate includes but is not limited to one or more of potassium salt, sodium salt, calcium salt, magnesium salt or zinc salt, preferably sodium gluconate.

[0129] Preferably, the amino acid or its salt is added by directly adding one or more amino acids or their salts, or by adding a substance that produces an amino acid or its salt after fermentation, or its fermentation product or fermentation liquid.

[0130] Preferably, the amino acid or its salt substance produced after the fermentation includes one or two of monosodium glutamate fermentation broth or soybean meal. Preferably, the amino acid includes one or more of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine, and preferably, the amino acid salt includes one or more of potassium salt, sodium salt, calcium salt, magnesium salt or zinc salt of any one or more of the above amino acids. Preferably, the amino acid is glutamic acid (preferably L-glutamic acid). Preferably, the amino acid salt is sodium glutamate. In a specific embodiment of the present invention, the carbon source includes one or more of glucose, sodium gluconate, glutamic acid, sodium glutamate or monosodium glutamate fermentation broth.

[0131] Preferably, the carbon source further comprises other carbon sources. Preferably, the other carbon sources include one or more of sucrose, butyrolactone, 4-hydroxybutyric acid, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol, 1,12-dodecanediol, fructose, xylose, cellulose, lactose, lactic acid, lauric acid, acetic acid, caproic acid, propionic acid, valeric acid, capric acid, butyric acid, palm oil or oleic acid.

[0132] Preferably, the concentration of the carbon source in the culture medium is greater than 1 g / L, preferably 10-300 g / L, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 35, 36, 37, 38, 39, 40, 45, 50, 51, 52, 53, 54, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300 g / L. Preferably, the culture medium is defined as in the first aspect.

[0133] Preferably, the culturing is carried out under open conditions.

[0134] Preferably, the equipment and culture medium used in the culture do not need to be sterilized.

[0135] A fifth aspect of the present invention provides a use of any of the above-mentioned recombinant Halomonas for producing a metabolite, wherein the metabolite is a natural or non-natural product.

[0136] Preferably, the metabolite comprises gamma-aminobutyric acid.

[0137] Further preferably, the metabolites also include intracellular products and / or extracellular products other than γ-aminobutyric acid.

[0138] More preferably, the metabolites also include PHA.

[0139] In a sixth aspect, the present invention provides a method for co-producing γ-aminobutyric acid and other products, wherein the method comprises culturing the above-mentioned recombinant Halomonas.

[0140] Preferably, the method further comprises adding a carbon source during the culture process. The relevant limitations on the carbon source are preferably the same as those in the first aspect of the present invention.

[0141] Preferably, the other products include intracellular products and / or extracellular products;

[0142] Preferably, the other products include PHA.

[0143] Preferably, the culturing is carried out under open conditions.

[0144] Preferably, the equipment and culture medium used in the culture do not need to be sterilized.

[0145] The seventh aspect of the present invention provides an expression vector as described in any one of the above aspects.

[0146] In an eighth aspect, the present invention provides a cell comprising the above-mentioned expression vector.

[0147] The ninth aspect of the present invention provides a use of the above-mentioned expression vector or the above-mentioned cell in preparing a recombinant bacterium for producing γ-aminobutyric acid, or producing γ-aminobutyric acid, or co-producing γ-aminobutyric acid and other products.

[0148] Beneficial effects of the present invention:

[0149] The present invention integrates and coordinates the optimization of the γ-aminobutyric acid production pathway and the degradation pathway, optimizes the culture medium, and utilizes a low-cost carbon source to produce γ-aminobutyric acid and co-produce other products (such as PHA). It has the characteristics of no need for sterilization, simple equipment, and continuous production. It can save production costs, is easy to expand the scale, and has a very broad application prospect.

[0150] The "PHA" described in the present invention is a homopolymer PHA and / or a copolymer PHA. Preferably, the PHA is selected from 3-hydroxybutyric acid (3HB) homopolymer PHB, 3-hydroxybutyric acid (3HB) and 4-hydroxybutyric acid (4HB) binary copolymer P3HB4HB, 3-hydroxybutyric acid (3HB), 4-hydroxybutyric acid (4HB) and 3-hydroxyvaleric acid terpolymer P(3HB-co-4HB-co-3HV), 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid binary copolymer PHBHHx, 3-hydroxypropionic acid (3HP) homopolymer or copolymer, further preferably, the 3-hydroxypropionic acid (3HP) homopolymer is P3HP (poly-3-hydroxypropionate), and the 3-hydroxypropionic acid (3HP) copolymer is P(3HB-co-3HP) (poly(3-hydroxybutyrate-co-3-hydroxypropionate)). In a specific embodiment of the present invention, the PHA is selected from 3-hydroxybutyric acid homopolymer PHB, 3-hydroxybutyric acid and 4-hydroxybutyric acid copolymer P3HB4HB, 3-hydroxybutyric acid, 4-hydroxybutyric acid and 3-hydroxyvaleric acid terpolymer PHBV4HB, 3-hydroxybutyric acid and 3-hydroxyhexanoic acid copolymer PHBHHx, a homopolymer or copolymer of 3-hydroxypropionic acid, the homopolymer of 3-hydroxypropionic acid is P3HP, and the copolymer of 3-hydroxypropionic acid is P(3HB-co-3HP) or PHBHP.

[0151] The terms “include” or “comprising” described in the present invention are open-ended descriptions, which include the specified components or steps described and other specified components or steps that will not be substantially affected.

[0152] The term "and / or" as used herein includes all combinations of the items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A," "A and B," and "B." For another example, "A, B and / or C" includes "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C." A comparison of some of the English abbreviations used in this application with their full Chinese and English names is shown in Table 1 below.

[0153] Table 1

[0154]

[0155] BRIEF DESCRIPTION OF THE DRAWINGS

[0156] Figure 1 : GABA de novo synthesis pathway and glutamate to GABA synthesis pathway;

[0157] Figure 2 :The intracellular and extracellular transport system of γ-aminobutyric acid and glutamate;

[0158] Figure 3 : Schematic diagram of module A and module B; P1, P2, and P3 represent different Pporin promoter mutants;

[0159] Figure 4 : Results of GABA production and conversion rate induced by different concentrations of IPTG, where the control group was Halomonas bluephagenesis TD1.0;

[0160] Figure 5 :GABA production results under the regulation of different Pporin promoter mutants;

[0161] Figure 6 : Characterization of the ability of TD1.0△gabT1 / 2 to decompose γ-aminobutyric acid. The experiment was conducted with a carbon source of 30 g / L glucose plus 10 g / L γ-aminobutyric acid. The left figure shows the residual γ-aminobutyric acid after 48 hours of fermentation, and the right figure shows the OD600 value at 48 hours. TD1.0 is the wild-type control group, and TD1.0△gabT1 / 2 is the experimental group with gabT1 and gabT2 genes knocked out.

[0162] Figure 7 :The effects of different culture media on the γ-aminobutyric acid production and conversion rate of recombinant Halomonas. TD1.0 is the wild-type control group, and gadBO is the Halomonas bluephagenesis TGE04 conjugated with pE01A.

[0163] Figure 8 :The effects of different culture media on the PHB production and dry cell weight of recombinant Halomonas. TD1.0 is the wild-type control group, and gadB is the Halomonas bluephagenesis TGE04 conjugated with pE01A.

[0164] Figure 9 :Effects of nitrogen source concentration on the production and conversion rate of γ-aminobutyric acid by recombinant Halomonas sp.;

[0165] Figure 10 :Effect of nitrogen source concentration on PHB production and cell dry weight of recombinant Halomonas;

[0166] Figure 11 Effects of different carbon sources on the production and conversion of γ-aminobutyric acid in recombinant Halomonas (Halomonas bluephagenesis TGE04 conjugated with pE01A). Glu represents the pure glutamate group, crude Glu represents the crude glutamate content group, MSG represents the pure sodium glutamate group, and NC represents the no-substrate group.

[0167] Figure 12 :Effects of different carbon sources on the PHB production and dry cell weight of recombinant Halomonas bluephagenesis TGE04;

[0168] Figure 13 : Dry cell weight and production of γ-aminobutyric acid and PHB by open fermentation of recombinant Halomonas (Halomonas bluephagenesis TGE04 conjugated with pE01A) in a 7 L fermentor;

[0169] Figure 14 :Shake flask fermentation experiment of γ-aminobutyric acid produced by recombinant Halomonas bluephagenesis TGE08;

[0170] Figure 15 :Dry cell weight and production of γ-aminobutyric acid and PHB by open fermentation of recombinant Halomonas bluephagenesis TGE08 in 7L fermentor; DETAILED DESCRIPTION

[0171] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0172] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0173] 1. Halomonas bluephagenesis TD1.0 (Accession No. CGMCC No. 4353):

[0174] The applicant has screened a salt- and alkali-tolerant, Gram-negative halophilic bacterium that naturally produces PHB. This bacterium can naturally accumulate high levels of polyhydroxyalkanoates (PHB), demonstrating promising prospects for industrial production. This information is described in the paper "Tan Dan, Wu Qiongg, Chen, Jin-Chun and Chen GQ. Engineering Halomonas TD01 for Low-Cost Production of Polyhydroxyalkanoates. Metabolic Engineering 26 (2014) 34–47," and patent application publication number CN102120973A. The strain is available to the public from the applicant for use solely in replicating the experiments described herein.

[0175] 2. Culture medium formula:

[0176] LB medium contains: 5 g / L yeast extract (OXID, catalog number LP0021), 10 g / L peptone (OXID, catalog number LP0042), 10 g / L NaCl, and the remainder is water. Adjust the pH to 7.0-7.2 and sterilize by autoclaving. 60LB medium is LB medium supplemented with 60 g / L NaCl. The remaining ingredients and preparation conditions are the same as those for LB medium.

[0177] 60MM culture medium contains: 60g / L NaCl, 1g / L yeast extract (OXID, catalog number LP0021), 35g-55g glucose (depending on the microorganism's sugar intake), and the remainder water. The optimal pH for the halomonas used in the experiment is around 8-9, and the pH was adjusted with NaOH.

[0178] MM medium: The medium contains: varying concentrations of NaCl, 1 g / L yeast extract (OXID, catalog number LP0021), 35 g–55 g glucose (depending on the microorganism's sugar uptake), and the remainder water. The optimal pH for the halomonas used in the experiment is around 8–9, and the pH was adjusted with NaOH.

[0179] 60MMG medium (mineral modified medium) contains: 1g / L yeast extract (OXID, catalog number LP0021), 60g / L sodium chloride, 30g / L glucose, 2% component I, 2% component II, 2% components III and IV, and the remainder is water. The optimal pH for the halomonas used in the experiment is around 8-9, and the pH was adjusted with NaOH.

[0180] Component I contains: 25g / L urea, 10g / L magnesium sulfate, and the rest is water.

[0181] Component II contains: 191.25 g / L disodium hydrogen phosphate, 75 g / L potassium dihydrogen phosphate, and the remainder is water.

[0182] Component III contains: 5 g / L ammonium ferric citrate, 2 g / L calcium chloride dihydrate, and the remainder is water.

[0183] Component IV contains: 0.03 g / L sodium molybdate dihydrate, 0.03 g / L manganese chloride tetrahydrate, 0.01 g / L copper sulfate pentahydrate, 0.02 g / L nickel chloride hexahydrate, 0.2 g / L cobalt chloride hexahydrate, 0.1 g / L zinc sulfate heptahydrate, 0.3 g / L boric acid, and the remainder is water.

[0184] 3. pSEVA321 plasmid and pQ08, pQ41 plasmid:

[0185] The present invention is described in the following document: “Qin Qin, Ling Chen, Zhao Yiqing, Yang Tian, ​​Yin Jin, Guo Yingying, Chen GQ. CRISPR / Cas9 editing genome of extremophile Halomonas spp. Metabolic Engineering 47 (2018) 219-229”. The public can obtain the data from the applicant and may only be used to repeat the experiments of the present invention.

[0186] 4. Unless otherwise specified, the gene editing technology used in the present invention is CRISPR-Cas9 gene editing technology. The specific operating methods are described in the following document: "Qin Qin, Ling Chen, Zhao Yiqing, Yang Tian, ​​Yin Jin, Guo Yingying, Chen GQ. CRISPR / Cas9 editing genome of extremophile Halomonas spp. Metabolic Engineering 47 (2018) 219-229". The public can obtain it from the applicant and may only be used to replicate the experiments of the present invention.

[0187] 5. Genes introduced into recombinant halophilic bacteria in the examples:

[0188] gadB is derived from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO: 1;

[0189] gadC is derived from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO:2.

[0190] Example 1 Construction of a recombinant halophilic bacterium Halomonas bluephagenesis TD1.0 for producing γ-aminobutyric acid by modular design

[0191] Using a heterologous expression strategy, a heterologous synthesis pathway for γ-aminobutyric acid was designed in Halomonas bluephagenesis TD1.0. This pathway can start from a single carbon source, glucose, and require more steps to synthesize γ-aminobutyric acid from scratch, or it can start from glutamate and synthesize γ-aminobutyric acid in a single step, such as Figure 1 As shown. The present invention utilizes two synthetic pathways simultaneously. At the same time, the intracellular and extracellular transport pathways of the substrate glutamate product γ-aminobutyric acid are as follows Figure 2 As shown, the introduction of this gene can promote the intracellular conversion of substrates to products and the extracellular transport of products, thereby increasing the yield and facilitating the subsequent separation and purification of products. In order to facilitate the expression regulation of the two genes, the concept of modular design was introduced, and the two genes were placed in two complete expression modules (modules A and B), which include the corresponding promoters, RBS, target genes and terminators (such as Figure 3 Inducible MmP1 expression system and constitutive P porin Promoter libraries provide a technical basis for gene expression regulation (reference Shen et al. (2018) Promoter Engineering for Enhanced P(3HB-co-4HB) Production by Halomonas bluephagenesis, ACS Synthetic Biology).

[0192] 1. Module A: Regulated expression of the gadB gene

[0193] Using IPTG-induced P MmP1 The promoter induces the expression of the gadB gene.

[0194] 1) gadB inducible expression plasmid pE01:pSEVA321-P MmP1 -gadB was constructed with P MmP1 -LacO-p321 plasmid (reference Zhao et al. (2017) Novel T7-like expression systems used for Halomonas, Metabolic Engineering) was used as a template, and the vector backbone fragment containing the Mmp1 promoter was amplified by primers F1 and R1. The synthetic gene fragment gadB (SEQ ID NO: 1) was used as a template, and the gadB gene fragment was amplified by primers F2 and R2.

[0195] Among them, primer: F1: 5'-agcctgataagccaggcatca-3' (SEQ ID NO: 6)

[0196] R1:5'-ctagtatttctcctctttctctagtattaaacaaaattatttgtagag-3' (SEQ ID NO: 7)

[0197] F2:5'-ctagagaaagaggagaaatactagatggacaaaaaacaggtgaccg-3' (SEQ ID NO: 8)

[0198] R2:5'-gatgcctggcttatcaggctttaggtgtgtttgaagctgttctg-3' (SEQ ID NO: 9)

[0199] The two PCR products were connected by Gibson assembly homologous recombination method to obtain a ligation product, which was transformed into E. coli S17-1 to obtain transformants and sequenced. The correctly sequenced transformants were selected and preserved and cultured overnight in LB medium to extract the plasmid, which was named pE01.

[0200] 2) Determine the appropriate gadB expression intensity by inducing with different IPTG concentrations

[0201] The chloramphenicol-resistant plasmid pE01 was transformed into Halomonas blue phagenesis TD1.0 using the conjugation transformation method and cultured on a plate containing chloramphenicol for one day until a single colony grew.

[0202] The TD1.0 strain conjugated with plasmid pE01 was cultured in 60LB medium and cultured at 37°C for 12 hours as a primary seed solution. The solution was then transferred to a new 60LB medium at a transfer rate of 1% and cultured for 12 hours as a secondary seed solution. The solution was then transferred to a shake flask containing 60MMG medium at a transfer rate of 5%. The final IPTG induction concentrations of 0, 5, 10, 50, and 200 mg / L were selected for initial induction. 30 g / L sodium glutamate was added at 24 hours of fermentation in a constant temperature incubator. The cells were cultured at 37°C for 36 hours. 5 ml of the fermentation broth was diluted 10-fold and the cells were lysed using a high-pressure disruptor. The γ-aminobutyric acid content in the lysed fermentation broth was detected by HPLC. The experimental results showed that in the plasmid-expressing strain, the highest yield of γ-aminobutyric acid was achieved at an IPTG concentration of 10 mg / L. The total (intracellular + extracellular) γ-aminobutyric acid yield in the whole cell was 12.8 g / L, and the conversion rate was 89.4%. Figure 4 .

[0203] The conversion rate calculation formula is:

[0204]

[0205]

[0206] 2. Module B: Regulated expression of the gadC gene

[0207] Based on module A, pE01:pSEVA321-P MmP1 -gadB is the skeleton, building the combination of modules A+B. Module B is achieved by P with different strengths. porin The promoter regulates the expression of the gadC gene.

[0208] 1) gadB and gadC co-expression plasmid pE01A-pE01F:pSEVA321-P MmP1 -gadB-P porin -gadC was constructed using plasmid pE01 (pSEVA321-P MmP1 -gadB) as a template, primers F3, R3 were used to amplify the vector backbone fragment containing the Mmp1 promoter and gadB gene, and the E. coli MG1655 genome was used as a template, primers F4, R4 were used to amplify the gadC gene fragment, and P porin The promoter library was used as a template and primers F5 and R5 were used to amplify the P porin The promoter fragment includes 6 P porin promoter, respectively P porin278 , P porin68 , P porin42 , P porin58 , P porin226 , P porin140 The three fragments were connected by Gibson assembly homologous recombination method to obtain the connection product, which was transformed into E. coli S17-1 to obtain transformants and sequenced. The correct sequence was selected and preserved and cultured overnight in LB medium to extract the plasmid. The plasmid was obtained according to P porin278 , P porin68 , P porin42 , P porin58 , P porin226 , P porin140 The promoter sequences were named pE01A-pE01F.

[0209] Among them, primer: F3: 5'-agcggccgctgcaggc-3' (SEQ ID NO: 10)

[0210] R3:5'-actagtaatcttgcaag-3' (SEQ ID NO: 11)

[0211] F4:5'-agaaagaggagaaatactagatggctacatcagtacagac-3' (SEQ ID NO: 12)

[0212] R4:5'-gcctgcagcggccgctttagtgtttcttgtcattca-3' (SEQ ID NO: 13)

[0213] F5:5'-ttcttgcaagattactagtatgcctccacaccgctcg-3' (SEQ ID NO: 14)

[0214] R5:5'-ctagtatttctcctctttctctagtaactctt-3' (SEQ ID NO: 15)

[0215] 2) Combined Regulation of gadB (Module A) and gadC (Module B) to Enhance GABA Production Chloramphenicol-resistant plasmids pE01A-pE01F were individually transformed into TD1.0 using conjugation transformation and cultured on plates containing chloramphenicol for one day until single colonies grew.

[0216] The six TD1.0 strains conjugated with plasmids pE01A-pE01F were cultured separately in 60LB medium and cultured at 37°C for 12 hours as the first-level seed liquid. The liquid was transferred to a new 60LB medium at a transfer rate of 1% and cultured for 12 hours as the second-level seed liquid. The liquid was transferred to a shake flask with 60MMG medium at a ratio of 5%. The IPTG induction concentration was 10 mg / L, the pH value was about 9, and the culture was carried out at 37°C for 48 hours. The content of γ-aminobutyric acid in the fermentation broth was detected by HPLC. The combined expression results of modules A and B showed that only the expression of the gadB gene in the γ-aminobutyric acid synthesis pathway could only produce 5.9 g / L of γ-aminobutyric acid extracellularly. However, when the gadC gene was expressed in combination, the extracellular γ-aminobutyric acid content could be increased to 12.8 g / L, and with the increase of P porin As the intensity of gadC increases, its expression intensity increases, its toxicity as a membrane protein increases, and the extracellular γ-aminobutyric acid production gradually decreases (see Figure 5 ). For example, the following embodiments are all based on P porin278 driven by gadC.

[0217] Example 2: Construction of a γ-aminobutyric acid auxotrophic recombinant halophilic bacterium Halomonas bluephagenesis TDΔgabT by gene knockout

[0218] Any one, two or three of the gabT1, gabT2 or gabT3 genes were knocked out (see Table 2), and the nucleotide sequences thereof were shown in SEQ ID NOs: 3-5.

[0219] Table 2 Combination knockout strategies for gabT1 / gabT2 / gabT3 genes

[0220] Gene knockout combination △gabT1 △gabT2 △gabT3 △gabT1 / 2 △gabT1 / 3 △gabT2 / 3 △gabT1 / 2 / 3

[0221] 1) Construction of gabT1 / 2 / 3 knockout plasmids

[0222] ① Use CRISPR-Cas9 to knock out the gene and find a suitable 20bp guide RNA near the site. The target site sequence of the guide RNA is:

[0223] gabT1sgRNA:gtgctgagaacgcgattatc (SEQ ID NO: 16)

[0224] gabT2sgRNA: gcgctgaaaacgctgagatc (SEQ ID NO: 17)

[0225] gabT3sgRNA: caaccgctggatcgattttg (SEQ ID NO: 18);

[0226] Using the pQ41 plasmid as a template, the guide RNA sequence was designed into the primers, circular PCR was performed using the primers, and the PCR products were blunt-ended with T4 ligase to obtain a plasmid containing the guide RNA.

[0227] Among them, the primers are:

[0228] gabT1 / 2 / 3sgRNA-F:5'-gttttagagctagaaatagcaagt-3' (SEQ ID NO: 19)

[0229] gabT1sgRNA-R:5'-gataatcgcgttctcagcacactagtattatacctaggactgagc-3'(SEQID NO: 20)

[0230] gabT2sgRNA-R:5'-gatctcagcgttttcagcgcactagtattatacctaggactgagc-3' (SEQID NO: 21)

[0231] gabT3sgRNA-R:5'-caaaatcgatccagcggttgactagtattatacctaggactgagc-3' (SEQ ID NO: 22) PCR reaction conditions:

[0232] Pre-denaturation at 95°C for 5 minutes; 30 cycles of denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds; post-extension at 72°C for 10 minutes.

[0233] ② Select two 500-base stretches near the guide RNA as homology arms. Using the Halomonas blue phagenesis TD1.0 genome as a template, amplify the upstream homology arm using primers F6 / R6, F7 / R7, and F8 / R8, respectively. Amplify the downstream homology arm using primers F9 / R9, F10 / R10, and F11 / R11.

[0234] Among them, primer: F6: 5'-gtcggtgctttttttgaacccggatgggctggatgaagg-3' (SEQ ID NO: 23)

[0235] R6:5'-ttactgctttccttattaaattagtgaaa-3' (SEQ ID NO: 24)

[0236] F7:5'-gtcggtgctttttttgaacccggcaacatcatcgaagcctac-3' (SEQ ID NO: 25)

[0237] R7:5'-gctaataactgggctcaactcctttggat-3' (SEQ ID NO: 26)

[0238] F8:5'-gtcggtgctttttttgaacccggggtcggcgatggtttaaca-3' (SEQ ID NO: 27)

[0239] R8:5'-gaaggacggtcgttttccttatagaccgccaac-3' (SEQ ID NO: 28)

[0240] F9:5'-taatttaataaggaaagcagtaacgtctgatgcttgtgg-3' (SEQ ID NO: 29)

[0241] R9:5'-tcccagtcacgacgcttcgccacgctctatcg-3' (SEQ ID NO: 30)

[0242] F10:5'-ggagttgagcccagttattagcgccaaca-3' (SEQ ID NO: 31)

[0243] R10:5'-tcccagtcacgacgcttcacgtcgagttggtgtg-3' (SEQ ID NO: 32)

[0244] F11:5'-ggcggtctataaggaaaacgaccgtccttctaggtatcaccc-3' (SEQ ID NO: 33)

[0245] R11:5'-ttttcccagtcacgacgcttcgccatgttgctctcccac-3' (SEQ ID NO: 34)

[0246] ③ The guide RNA plasmid from ① was amplified into a linear fragment using primers F12 and R12. This fragment was assembled with the three sets of upstream and downstream homology arms from ② using Gibson ligation to construct plasmids pE02:pSEVA341-gabT1-ko, pE03:pSEVA341-gabT2-ko, and pE04:pSEVA341-gabT3-ko. These plasmids were transformed into E. coli S17-1, obtained transformants, and sequenced. Transformants that correctly sequenced were selected and stored, cultured overnight in LB medium, and then plasmids were extracted.

[0247] Among them, primer: F12: 5'-aagcgtcgtgactggga-3' (SEQ ID NO: 35)

[0248] R12:5'-ccgggttcaaaaaaagcaccgac-3' (SEQ ID NO: 36)

[0249] 2) Knockout of gabT1 / 2 / 3 genes in the Halomonas bluephagenesis TD1.0 genome

[0250] The chloramphenicol-resistant plasmid pQ08 containing Cas9 was transformed into Halomonas blue phagenesis TD1.0 using conjugation. Plasmids pE02, pE03, and pE04 were then transferred into the strain according to the combination in Table 2. The strain was cultured for two days on plates containing chloramphenicol and spectinomycin until a single colony emerged.

[0251] After confirmation by colony PCR and gene sequencing, three pairs of primers, gabT1-F / R, gabT2-F / R, and gabT3-F / R, were designed upstream and downstream of the insertion site for verification by colony PCR and further verification by sequencing.

[0252] Among them, primers:

[0253] gabT1-F:5'-ctaagcgcgtggcgca-3' (SEQ ID NO: 37)

[0254] gabT1-R:5'-agctcatttctgtcagctttgctaat-3' (SEQ ID NO: 38)

[0255] gabT2-F:5'-ccgaacgcgtacgtgac-3' (SEQ ID NO: 39)

[0256] gabT2-R:5'-atatttcgccatctgccc-3' (SEQ ID NO: 40)

[0257] gabT3-F:5'-caaactgaccgagcgt-3' (SEQ ID NO: 41)

[0258] gabT3-R:5'-gcttaactttcgcatgcg-3' (SEQ ID NO: 42)

[0259] The strain with correct sequencing was selected and cultured in an antibiotic-free seed culture medium until the strain completely lost the plasmid, thereby obtaining the gabT combination knockout strain Halomonas bluephagenesis TD1.0△gabT combination.

[0260] 3) Characterization of the gabT knockout strain TD1.0ΔgabT

[0261] To verify the ability of TD1.0△gabT to decompose γ-aminobutyric acid, Halomonas bluephagenesis TD1.0 and each TD1.0△gabT knockout strain were cultured in mineral medium MM medium with 30 g / L glucose and 5 g / L γ-aminobutyric acid as carbon sources. After 12 hours of culture, the OD 600 and the remaining amount of γ-aminobutyric acid, OD 600represents the effect of knocking out genes on growth. The test results showed that the double knockout TD1.0△gabT1 / 2 (named Halomonasbluephagenesis TGE04) had the best results. Figure 6 As shown, the growth of the knockout strain TD1.0△gabT1 / 2 was consistent with that of the wild type, indicating that knockout of this gene did not affect growth. The amount of residual GABA in the knockout strain TD1.0△gabT1 / 2 after culture was approximately 4.5 g / L, while the control group completely utilized GABA. Furthermore, knockout of gabT3 significantly affected growth, resulting in a higher amount of GABA remaining. Therefore, knockout of the gabT gene can block the catabolism of GABA.

[0262] Example 3: Effects of different culture media on the γ-aminobutyric acid conversion rate and PHB production of recombinant Halomonas;

[0263] like Figure 1 As shown, the conversion of glutamate to γ-aminobutyric acid is shorter and faster than that of glucose, relying on only one enzymatic reaction. This requires sufficient accumulation of the enzyme. To select a more suitable culture medium, shake flask experiments were conducted in 60 MMG and 60 LB + 30 g / L glucose culture media. The specific experimental procedures are as follows.

[0264] The chloramphenicol-resistant plasmid pE01A was transformed into Halomonas blue phagenesis TGE04 using the conjugation transformation method and cultured on a plate containing chloramphenicol for one day until a single colony grew.

[0265] Halomonas bluephagenesis TGE04 conjugated with pE01A was cultured in 60LB medium and cultured at 37°C for 12 hours as the primary seed solution. The solution was then transferred to a new 60LB medium at a transfer rate of 1% and cultured for 12 hours as the secondary seed solution. The solution was then transferred to shake flasks with different culture media at a transfer rate of 5%. The IPTG induction concentration was 10 mg / L, the pH was about 9, and 30 g / L glutamate was added at 24 hours of fermentation. The culture was cultured at 37°C for 36 hours. The γ-aminobutyric acid content in the fermentation broth was detected by HPLC. The experimental results are shown in the figure. Figure 7 and Figure 8 The highest γ-aminobutyric acid yield was 13.9 g / L in 60 MMG medium, with a conversion rate of 100%. GABA and PHB were also co-produced, with a PHB content of 62.8%.

[0266] Example 4: Effect of nitrogen source concentration on γ-aminobutyric acid conversion rate and PHB production of recombinant Halomonas sp.

[0267] The optimal nitrogen source concentration for the culture medium of wild Halomonas bluephagenesis TD1.0 is approximately 0.5 g / L. Since the synthesis of the catalytic enzyme GadB requires a nitrogen source during the culture process, and the synthesis of PHB is a nitrogen-limited process, it is necessary to re-evaluate the optimal nitrogen source concentration for the fermentation of Halomonas bluephagenesis TGE04. The specific experimental procedures are as follows:

[0268] The chloramphenicol-resistant plasmid pE01A was transformed into Halomonas blue phagenesis TGE04 using the conjugation transformation method and cultured on a plate containing chloramphenicol for one day until a single colony grew.

[0269] Halomonas bluephagenesis TGE04 conjugated with pE01A was cultured in 60LB medium and cultured at 37°C for 12 hours as the primary seed solution. The solution was transferred to a new 20mL 60LB medium at a transfer rate of 1% and cultured for 12 hours as the secondary seed solution. The solution was transferred to a shake flask of 60MMG medium at a ratio of 5%. Six gradients of urea concentration were set, namely 0.5, 1, 2, 3, 4 and 5 g / L, of which the 0.5 g / L group was used as the control group. The IPTG induction concentration was 10 mg / L, the pH value was about 9, and the substrate 90 g / L glutamate was added at 24 hours of fermentation. The solution was cultured at 37°C for 36 hours. The content of γ-aminobutyric acid in the fermentation broth and the percentage of PHB accumulated in the recombinant bacteria were detected. The results are shown in Figure 2. Figure 9 and Figure 10 As shown, in the culture of recombinant Halomonas, cell dry weight, PHB percentage, γ-aminobutyric acid (GABA) content, and conversion rate varied with urea concentration. With increasing urea concentration, cell dry weight initially increased and then decreased, reaching its highest level at 1 g / L urea. PHB percentage reached over 57.6% at both 0.5 g / L and 1 g / L urea concentrations, and GABA content also reached its highest level at 1 g / L urea, reaching 59.8 g / L. Furthermore, the conversion rate of recombinant Halomonas was high under all urea conditions, exceeding 95%. This indicates that urea primarily affects cell growth and enzyme accumulation. Recombinant Halomonas can achieve high yields of GABA and PHB co-production at 1 g / L urea.

[0270] Example 5: Utilization of different cheap substrates by recombinant Halomonas;

[0271] In line with the concept of green production, the glutamate and sodium glutamate substrates we use can be replaced with MSG fermentation wastewater. To reduce costs, MSG fermentation wastewater is also used as a carbon source. The specific experimental procedures are as follows.

[0272] The chloramphenicol-resistant plasmid pE01A was transformed into Halomonas blue phagenesis TGE04 using the conjugation transformation method and cultured on a plate containing chloramphenicol for one day until a single colony grew.

[0273] Halomonas bluephagenesis TGE04 conjugated with pE01A was cultured in 60LB medium and cultured at 37°C for 12 hours as a primary seed solution. 1% of the solution was transferred to a new 20mL 60LB medium and cultured for 12 hours as a secondary seed solution. 5% of the solution was transferred to a shake flask containing 60MMG medium. The IPTG induction concentration was 10mg / L and the pH was approximately 9. At 24 hours of fermentation, 90g / L glutamate was added as a substrate. The culture was cultured at 37°C for 36 hours. The γ-aminobutyric acid content in the fermentation broth and the percentage of PHB accumulated in the recombinant bacteria were detected. The results are shown in Figure 2. Figure 11-12 As shown, in the culture of recombinant Halomonas, the highest γ-aminobutyric acid yield can be obtained by using pure glutamic acid, but γ-aminobutyric acid can also be produced by using MSG fermentation waste liquid. The waste liquid containing a crude amount of 30 g / L glutamic acid can produce 10.4 g / L γ-aminobutyric acid.

[0274] Example 6. Verification of the production of γ-aminobutyric acid and PHB by open fermentation of recombinant Halomonas in a 7-L fermentor. Halomonas bluephagenesis TGE04 harboring pE01A was inoculated into 20 mL of 60LB medium. After culturing for 12-16 h, the culture was transferred to new 60LB medium at a volume ratio of 1%. The culture was continued for 8-12 h, and 300 mL of seed solution was prepared as the inoculum for a 7-L bioreactor (NBS Bioflo3000).

[0275] Prepare 2.7 L of base medium containing sodium chloride (90 g), glucose (45 g), sodium gluconate (45 g), urea (9 g), yeast extract (15 g), magnesium sulfate (0.6 g), potassium dihydrogen phosphate (10.5 g), 30 ml / L of trace element solution I, and 3 ml / L of trace element solution II. Trace element solution I consists of 5 g / L ammonium ferric citrate and 2 g / L CaCl2, prepared in 1 M HCl. Trace element solution II consists of 100 mg / L ZnSO4·7H2O, 30 mg / L MnCl2·4H2O, 300 mg / L H3BO3, 200 mg / L CoCl2·6H2O, 10 mg / L CuSO4·5H2O, 20 mg / L NiCl2·6H2O, and 30 mg / L NaMoO4·2H2O, prepared in 1 M HCl. The final pH of the culture medium was adjusted to 8-9 using 5 M NaOH solution.

[0276] During the fermentation process, the dissolved oxygen content was adjusted by stirring and aeration, and the glucose content in the fermentation medium was monitored by measuring the residual sugar with a blood glucose meter every hour, thereby regulating the feed amount.

[0277] After 22 hours of fermentation, glutamic acid was added to enter the production conversion stage. The glutamic acid content in the fermentation medium was monitored by measuring the residual glutamic acid using HPLC every hour, thereby regulating the feed amount.

[0278] Fermentation results such as Figure 13 As shown in Figure 2, in a 7L fermentor tank, after 40 hours of fermentation, the gamma-aminobutyric acid accumulation amount was increased to 250g / L, and the productive rate was 13.2g / L / h. Before entering the conversion stage (22h), 35mL culture fluid was taken to collect a sample, and the percentage of PHB was detected by GC. It was found that PHB had 35% accumulation. PHB is a product accumulated in the cell and gamma-aminobutyric acid is a product accumulated outside the cell. Both do not interfere with each other in the extraction of later products. Therefore, Halomonas bluephagenesis TGE04 also has the advantage of producing gamma-aminobutyric acid and PHB together. Moreover, the Halomonas bluephagenesis TGE04 bacterial strain did not have any pollution in the fermentation under the situation of sterilization operation, which has proved that the production of gamma-aminobutyric acid with Halomonas as the base bacteria has a low cost advantage.

[0279] Example 7: Construction of Halomonas bluephagenesis TGE08 for producing γ-aminobutyric acid

[0280] 1. Inserting the γ-aminobutyric acid synthesis metabolic pathway into the halophilic bacteria genome

[0281] Using the pQ41 plasmid as a template, the guide RNA sequence was designed into the primers, circular PCR was performed using the primers, and the PCR products were blunt-ended with T4 ligase to obtain a plasmid containing the guide RNA.

[0282] 1) Construction of gadB and gadC insertion plasmid pE11:pSEVA241-G7-P porin -gadB-P porin -gadC

[0283] ①Use CRISPR-Cas9 to insert the target DNA sequence into the genome, use pQ41-G7 plasmid as a template, and amplify the plasmid backbone fragment containing the homology arm of the G7 site using primers F13 and R13.

[0284] Primer: F13:5'-actagtagcggccgctgcagggtgatatagagtgtatcgcgcaaag-3' (SEQ IDNO: 43)

[0285] R13:5'-ggtgatatagagtgtatcgcgcaaag-3' (SEQ ID NO: 44)

[0286] ②Use P porin Promoter library, based on promoter P porin140 The promoter fragment was obtained by amplification using primers F14 and R14. Primer F14: 5'-atgcctccacaccgctcgt-3' (SEQ ID NO: 45)

[0287] R14:5'-ctagtatttctcctctttctctagtaactctt-3' (SEQ ID NO: 46)

[0288] ③ Using plasmid pE01A as a template, primers F15 and R15 were used to amplify the gadB-Pporin278-gadC gene fragment. Primer F15: 5'-tactagagaaagaggagaaatactagatggacaaa-3' (SEQ ID NO: 47)

[0289] R15:5'-gcgatacactctatatcaccttagtgtttcttgtcattca-3' (SEQ ID NO: 48)

[0290] ④ The three fragments were connected by Gibson assembly homologous recombination method to obtain a connection product, which was transformed into E. coli S17-1 to obtain transformants and sequenced. The correctly sequenced ones were selected and preserved and cultured overnight in LB medium to extract the plasmid, which was named pE11.

[0291] 2) Insert the gadB and gadC genes into the G7 site of the Halomonas bluephagenesis TGE04 genome

[0292] ① Using conjugation, the chloramphenicol-resistant plasmid pQ08 containing Cas9 was transformed into Halomonas bluephagenesis TGE04 prepared in Example 2. Plasmid pE11 was then transferred into the strain and cultured on plates containing chloramphenicol and spectinomycin for two days until a single colony emerged.

[0293] ② Verification: Design a pair of primers G7-F and G7-R upstream and downstream of the insertion site, perform colony PCR verification, and further verify by sequencing.

[0294] Among them, primer: G7-F: 5'-gcgctcgcaaagaatagg-3' (SEQ ID NO: 49)

[0295] G7-R:5'-gcacttgctcaggaggtat-3' (SEQ ID NO: 50)

[0296] ③ Select the strain with the correct sequencing and culture it in the antibiotic-free seed culture medium until the strain completely loses the plasmid, and obtain the strain TD1.0△gabT1 / 2G7::P with gadB and gadC inserted in the genome porin -gadB-P porin -gadC, named Halomonas bluephagenesis TGE08.

[0297] 2. Shake flask fermentation experiment of γ-aminobutyric acid production by Halomonas bluephagenesis TGE08: Halomonas bluephagenesis TGE08 was cultured in 60LB medium and incubated at 37°C for 12 hours. The first seed liquid was transferred at a transfer rate of 1% to a new 20mL 60LB medium and incubated for 12 hours. The second seed liquid was transferred at a transfer rate of 5% to a shake flask containing 20mL 60MM medium. It is worth noting that the pH value was about 9. 90g / L glutamic acid was added at 24 hours of fermentation. The culture was incubated at 37°C for 48 hours. The γ-aminobutyric acid content in the fermentation broth and the percentage of intracellular PHB accumulation in the strain were detected. The experimental results are shown in Figure 2. Figure 14 The results showed that in a 50 ml shake flask, the yield of GABA was 59 g / L and the PHB percentage was 57%, which were similar to the results of expression on the plasmid.

[0298] 3. Verification of the production of γ-aminobutyric acid and PHB by Halomonas bluephagenesis TGE08 in a 7L fermenter

[0299] Halomonas bluephagenesis TGE08 was inoculated into 20 mL of 60 LB medium and cultured for 12-16 h. The culture was then transferred to new 60 LB medium at a volume ratio of 1% and cultured for another 8-12 h. 300 mL of seed solution was prepared as inoculum for a 7 L bioreactor (NBS Bioflo3000).

[0300] Prepare 2.7 L of base medium containing sodium chloride (90 g), glucose (45 g), sodium gluconate (45 g), urea (9 g), yeast extract (12 g), magnesium sulfate (0.6 g), potassium dihydrogen phosphate (10.5 g), 30 ml / L of trace element solution I, and 3 ml / L of trace element solution II. Trace element solution I consists of 5 g / L ammonium ferric citrate and 2 g / L CaCl2, prepared in 1 M HCl. Trace element solution II consists of 100 mg / L ZnSO4·7H2O, 30 mg / L MnCl2·4H2O, 300 mg / L H3BO3, 200 mg / L CoCl2·6H2O, 10 mg / L CuSO4·5H2O, 20 mg / L NiCl2·6H2O, and 30 mg / L NaMoO4·2H2O, prepared in 1 M HCl. The final pH of the culture medium was adjusted to 8-9 using 5 M NaOH solution.

[0301] During the fermentation process, the dissolved oxygen content was adjusted by stirring and aeration, and the glucose content in the fermentation medium was monitored by measuring the residual sugar with a blood glucose meter every hour, thereby regulating the feed amount.

[0302] After 22 hours of fermentation, glutamic acid was added to enter the production conversion stage. The glutamic acid content in the fermentation medium was monitored by measuring the residual glutamic acid using HPLC every hour, thereby regulating the feed amount.

[0303] Fermentation results such as Figure 15 As shown, after 40 hours of fermentation in a 7-L fermentor, GABA accumulation reached 230 g / L, with a yield of 12.8 g / L / h. Every four hours, 35 mL of culture fluid was sampled and analyzed by GC for PHB percentage, revealing 49% PHB accumulation.

[0304] Compared to the Halomonas bluephagenesis TGE04 strain conjugated with pE01A, the Halomonas bluephagenesis TGE08 strain does not require the addition of antibiotics or the inducer IPTG during fermentation, which can reduce fermentation costs and achieve a higher yield of γ-aminobutyric acid. The Halomonas bluephagenesis TGE08 strain also ferments without any contamination without any sterilization operations, demonstrating the low-cost advantage of using Halomonas as the base strain for producing γ-aminobutyric acid.

[0305] Example 8: Construction of a recombinant halophilic bacterium Halomonas campaniensis LS21CGMCC No. 6593 producing γ-aminobutyric acid

[0306] First, gabT1 / 2 was knocked out in Halomonas campaniensis LS21 according to the method described in Example 2 to obtain strain LSΔgabT1 / 2. MmP1 -gadB-P porin278 The LSΔgabT1 / 2 plasmid was ligated to achieve the production of γ-aminobutyric acid and PHB.

[0307] The specific operations are as follows:

[0308] The chloramphenicol-resistant plasmid pE01A was transformed into LSΔgabT1 / 2 using the conjugative transformation method and cultured on a plate containing chloramphenicol for one day until a single colony grew.

[0309] The LSΔgabT1 / 2 strain, harboring plasmid pE01A, was cultured in 60LB medium at 37°C for 12 hours. This was used as a primary seed culture, which was then transferred at a 1% transfer rate to fresh 60LB medium and incubated for another 12 hours. This culture then served as a secondary seed culture, which was then transferred at a 5% transfer rate to 60MMG shake flasks. IPTG was used for induction at a concentration of 10 mg / L, and the pH was approximately 9. 90 g / L of glutamate was added 24 hours into the fermentation, and the culture was incubated at 37°C for 36 hours. After freeze-drying, weighing, and liquid chromatography analysis, the recombinant strain produced 50 g / L of γ-aminobutyric acid (GABA) after 36 hours of shake flask fermentation, with a dry cell weight of 6 g / L and a PHB content of 60%.

[0310] The results showed that the introduced gadB and gadC genes could be expressed in Halomonas campaniensis LS21 and produce high levels of γ-aminobutyric acid.

[0311] Example 9: Construction of a recombinant bacterium Halomonas aydingkolgenesis M1CGMCCNO.19880 for producing γ-aminobutyric acid

[0312] First, according to the method described in Example 2, gabT1 / 2 in Halomonas aydingkolgenesis M1 was knocked out to obtain strain M1ΔgabT1 / 2. MmP1 -gadB-P porin278 The -gadC) plasmid was ligated into M1ΔgabT1 / 2 to achieve the production of γ-aminobutyric acid and PHB.

[0313] The specific operations are as follows:

[0314] The chloramphenicol-resistant plasmid pE01A was transformed into M1ΔgabT1 / 2 using the conjugative transformation method and cultured on a plate containing chloramphenicol for one day until a single colony grew.

[0315] The M1ΔgabT1 / 2 strain, conjugated with plasmid pE01A, was cultured in 60LB medium at 37°C for 12 hours. This was used as a primary seed solution, which was then transferred at a 1% transfer rate to fresh 60LB medium and incubated for another 12 hours. This served as a secondary seed solution, which was then transferred at a 5% transfer rate to 60MMG shake flasks. IPTG was used for induction at a concentration of 10 mg / L, and the pH was approximately 9. Glutamate was added at 90 g / L after 24 hours of fermentation, and the cells were incubated at 37°C for 36 hours. After freeze-drying, weighing, and liquid chromatography analysis, the recombinant strain produced 45 g / L of γ-aminobutyric acid (GABA) after 36 hours of shake flask fermentation, with a dry cell weight of 5.6 g / L.

[0316] The results showed that the introduced gadB and gadC genes could be expressed in Halomonas aydingkolgenesis M1 and successfully synthesize γ-aminobutyric acid.

[0317] From the above description, it should be noted and understood that various modifications and improvements can be made to the present invention described in detail above without departing from the spirit and scope of the present invention required by the appended claims. Therefore, the scope of the technical solution claimed is not limited by any specific exemplary teaching given.

[0318] The applicant declares that the above description is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention should not be construed as being limited to these descriptions. A person skilled in the art of the present invention may make several simple deductions or substitutions without departing from the scope of the present invention, and all of these should be considered to fall within the scope of protection of the present invention.

Claims

1. A recombinant Halomonas bacteria for producing γ-aminobutyric acid, characterized in that: The recombinant Halomonas expresses or overexpresses one or both of glutamate decarboxylase and glutamate / γ-aminobutyric acid antiporter; and / or the recombinant Halomonas does not express γ-aminobutyric acid transaminase or the expressed γ-aminobutyric acid transaminase is inactivated or functionally reduced; Preferably, the glutamate decarboxylase or glutamate / γ-aminobutyric acid antiporter is endogenous or exogenous.

2. The recombinant Halomonas according to claim 1, characterized in that The glutamate decarboxylase is derived from Escherichia coli; Preferably, the amino acid sequence of the glutamate decarboxylase comprises the amino acid sequence shown in SEQ ID NO:

1.

3. The recombinant Halomonas according to claim 1 or 2, characterized in that The glutamate / γ-aminobutyric acid antiporter is derived from Escherichia coli; Preferably, the amino acid sequence of the glutamate / γ-aminobutyric acid antiporter comprises the amino acid sequence shown in SEQ ID NO:

2.

4. The recombinant Halomonas according to any one of claims 1 to 3, characterized in that The gamma-aminobutyric acid transaminase includes one, two or three of GabT1, GabT2 or GabT3.

5. The recombinant Halomonas according to any one of claims 1 to 4, characterized in that The expression of the glutamate decarboxylase and / or glutamate / γ-aminobutyric acid antiporter is regulated by a promoter, Preferably, the promoter includes a constitutive promoter and / or an inducible promoter; Further preferably, the constitutive promoter includes P porin promoter or its mutant or P Sp6 promoter or a variant thereof; more preferably, P porin The promoter or its mutants include P porin 、P porin203 、P porin58 、P porin221 、P porin194 、P porin68 、P porin42 、P porin140 、P porin141 、P porin3 、P porin278 、P porin226 or P porin211 ; Further preferably, the inducible promoter includes an IPTG-inducible promoter or an AHL-inducible promoter.

6. The recombinant Halomonas according to any one of claims 1 to 5, characterized in that The glutamate decarboxylase and / or glutamate / γ-aminobutyric acid antiporter are expressed on an expression vector and / or on a chromosome.

7. The recombinant Halomonas according to any one of claims 1 to 6, characterized in that The recombinant Halomonas bacteria include Halomonas bluephagenesis, Halomonas campaniensis, Halomonas desiderata, Halomonas cupida, Halomonas smymensis, Halomonas levan, Halomonas lutescens, Halomonas elongata, Halomonas venusta or Halomonas alkaliantarctica.

8. A method for preparing the recombinant Halomonas according to any one of claims 1 to 7, characterized in that: The preparation method comprises introducing a nucleotide sequence encoding one or both of glutamate decarboxylase and glutamate / γ-aminobutyric acid reverse transporter into Halomonas; and / or, the preparation method knocks out or knocks down γ-aminobutyric acid transaminase in Halomonas.

9. The preparation method according to claim 8, characterized in that sgRNA is used to knock out or knock down γ-aminobutyrate aminotransferase in Halomonas, wherein the target site sequence of the sgRNA comprises any one or a combination of two or more of SEQ ID NOs: 16-18.

10. A method for culturing microorganisms or producing γ-aminobutyric acid, characterized in that: The method comprises culturing the recombinant Halomonas according to any one of claims 1 to 7; Preferably, the method comprises using a culture medium; Preferably, the culture medium contains a nitrogen source; Preferably, the concentration of the nitrogen source in the culture medium is 0-10 g / L.

11. The method according to claim 10, characterized in that The culture medium contains a carbon source, and the carbon source contains one or more of glucose, gluconate, gluconate ester or amino acid or its salt; Preferably, the amino acid or its salt is added by directly adding one or more amino acids or their salts, or by adding a substance or fermentation liquid that produces amino acids or their salts after fermentation; Preferably, the concentration of the carbon source in the culture medium is 10-300 g / L.

12. The method according to claim 11, characterized in that The substance that produces amino acids after fermentation includes one or both of monosodium glutamate and soybean meal; Preferably, the amino acids include one or more of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine, Preferably, the amino acid salt comprises one or more of potassium salt, sodium salt, calcium salt, magnesium salt or zinc salt of an amino acid.

13. Use of the recombinant Halomonas according to any one of claims 1 to 7 in producing metabolites; Preferably, the metabolites include γ-aminobutyric acid; Further preferably, the metabolites further include intracellular products and / or extracellular products in addition to γ-aminobutyric acid; More preferably, the metabolites also include PHA.

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