An engineered halomonas sp. and a method for producing acetoin thereof

Abstract

The application provides an engineered halomonas and a method for producing acetoin, wherein the halomonas expresses acetyl-lactate synthase and acetyl-lactate decarboxylase, and does not express PHA hydratase and acetoacetyl-CoA or the expressed PHA hydratase and acetoacetyl-CoA are not functional or have reduced function. The engineered halomonas can realize economic, sustainable and efficient production of acetoin, and improve single batch fermentation yield.

Description

Technical Field

[0001] This invention relates to the fields of microbial metabolic engineering and fermentation engineering, specifically to an engineered Halomonas bacterium and a method for producing acetoin. Background Technology

[0002] Acetoin, also known as trihydroxybutanone, is naturally found in substances such as corn, wine, honey, cocoa, butter, coffee, and strawberries. Due to its distinctive creamy aroma, it is often used as a flavor enhancer in cream, cheese, coffee, and nuts. Acetoin also has a wide range of applications in the pharmaceutical, daily chemical, fragrance, and cosmetic fields.

[0003] Currently, the methods for producing acetoin include chemical synthesis and biosynthesis. Chemical synthesis of acetoin is energy-intensive, costly, and has a low product yield, making it difficult to industrialize and causing serious environmental pollution. Therefore, there is an urgent need for a sustainable acetoin production process.

[0004] Biosynthesis of acetoin is environmentally friendly, simple, and technologically mature, meeting the requirements of green chemistry. Currently, many high-yielding acetoin strains are pathogenic bacteria, such as Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter aerogenes, and Serratia marcescens. The safety principles of large-scale industrial production have greatly limited the application of these production strains. Therefore, there is an urgent need to develop more safe and reliable metabolic engineering strains. Summary of the Invention

[0005] The *Haloxylon* strain provided in this application expresses or overexpresses acetolactate synthase and acetolactate decarboxylase, and knocks out or knocks down PHA hydratase and acetyl-CoA, thus enabling the engineered *Haloxylon* strain to produce acetoin. Combined with various metabolic engineering strategies, including downregulating or blocking the acetoin degradation pathway in *Haloxylon*, downregulating or blocking the acetoin endocytosis transport system in *Haloxylon*, increasing the membrane permeability of *Haloxylon*, enhancing the precursor supply for acetoin synthesis in *Haloxylon*, and / or reducing the precursor consumption pathway for acetoin synthesis in *Haloxylon*, the ability of *Haloxylon* to produce acetoin is further improved. Compared with the whole-cell catalysis of pyruvate-to-acetoin synthesis, the *Haloxylon* strain modified in this application is stable and can produce acetoin de novo using glucose as a substrate under low-salt and low-oxygen conditions, which has profound significance for reducing acetoin production costs, improving production efficiency, and realizing large-scale production.

[0006] In a first aspect, the present invention provides an engineered Halomonas bacterium that expresses or overexpresses acetolactate synthase and acetolactate decarboxylase.

[0007] Preferably, engineered halomonas bacteria can produce acetoin.

[0008] The acetolactate synthase described above catalyzes the conversion of pyruvate to α-acetolactate.

[0009] The acetolactate synthase is derived from bacteria or fungi. Preferably, the acetolactate decarboxylase is derived from the orders Bacillales, Actinomycetales, or Endomycetales. For example, the acetolactate synthase is derived from the genera Bacillus, Bifidobacterium, or Saccharomyces. Another example is the acetolactate synthase derived from Bacillus subtilis, Bacillus rugosus, Bacillus vallismortis, Bacillus stercoris, Bacillus cabrialesii, Bacillus halotolerans, Bacillus tequilensis, or Bifidobacterium brevis.

[0010] The amino acid sequence of the acetolactate synthase comprises amino acid sequences 181-567 of SEQ ID NO: 25.

[0011] Preferably, the amino acid sequence of the acetolactate synthase includes SEQ ID NO: 25.

[0012] The acetolactate decarboxylase described above catalyzes the conversion of α-acetolactate to acetoin.

[0013] The acetyllactone decarboxylase is derived from bacteria or fungi. Preferably, the acetyllactone decarboxylase is derived from the orders Bacillales, Actinomycetales, or Endomycetales. For example, the acetyllactone decarboxylase is derived from the genera Bacillus, Bifidobacterium, or Saccharomyces. Another example is the acetyllactone decarboxylase derived from Bacillus subtilis, Bacillus rugosus, Bacillus vallismortis, Bacillus stercoris, Bacillus cabrialesii, Bacillus halotolerans, Bacillus tequilensis, or Bifidobacterium brevis.

[0014] The amino acid sequence of the acetolactate decarboxylase comprises amino acid sequences 40-251 of SEQ ID NO: 26.

[0015] Preferably, the amino acid sequence of the acetolactate decarboxylase includes SEQ ID NO: 26.

[0016] Preferably, the *Halomonas* contains exogenous genes, including alsS and alsD.

[0017] The exogenous gene can be one or more copies, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies.

[0018] The gene clusters formed by alsS and alsD can have one or more copy numbers. Preferably, overexpression of the gene clusters formed by alsS and alsD can increase acetoin production. The gene clusters formed by alsS and alsD can be expressed on plasmids or on chromosomes. Preferably, multiple copy numbers of the gene clusters formed by alsS and alsD are expressed at one or more loci (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more copy numbers at one or more loci on the genome).

[0019] Preferably, the promoters in the gene clusters formed by alsS and alsD can be P respectively. mmp1 and P lux Regulation.

[0020] Preferably, a portion of the sequence in the spacer region between alsS and alsD in the gene cluster formed by alsS and alsD is replaced with a constitutive promoter (preferably a strong promoter, such as P). porin (or its mutants) and an artificially designed ribosome strong binding site (RBS) sequence (preferably nucleotides 677-714 of SEQ ID NO: 3).

[0021] The acetolactate synthase encoding gene is alsS, and the nucleotide sequence of alsS includes any one or more of the following: nucleotide sequence 1003-2715 in SEQ ID NO: 1, nucleotide sequence 193-1904 in SEQ ID NO: 2, or nucleotide sequence 2056-3810 in SEQ ID NO: 3.

[0022] The acetolactate decarboxylase encoding gene is alsD, and the nucleotide sequence of alsD includes nucleotides 193-959 in SEQ ID NO: 1 and / or nucleotides 1948-2715 in SEQ ID NO: 2.

[0023] Preferably, this is achieved by introducing a plasmid containing the alsD and / or alsS genes into Halomonas.

[0024] The plasmid containing the alsD and alsS genes, from 5' to 3', sequentially includes a promoter, RBS, alsD gene sequence, RBS, and alsS gene sequences; or,

[0025] The plasmid containing the alsD and alsS genes includes, from 5' to 3', a promoter, RBS, alsS gene sequence, RBS, and alsD gene sequence; or,

[0026] The plasmid containing the alsD and alsS genes, from 5' to 3', sequentially includes a promoter, RBS, alsD gene sequence, terminator, luxR gene sequence, promoter, RBS, and alsS gene sequence; or,

[0027] The plasmid containing the alsD and alsS genes, from 5' to 3', sequentially includes an sgRNA expression module, an upstream homologous arm, a promoter, an alsSD gene cluster, and a downstream homologous arm; or,

[0028] The plasmid containing the alsD and alsS genes, from 5' to 3', sequentially includes an sgRNA expression module, an upstream homologous arm, a promoter, an RBS, the alsD gene sequence, the promoter, the RBS, the alsS gene sequence, and a downstream homologous arm; or,

[0029] The plasmid containing the alsD and alsS genes includes, from 5' to 3', an sgRNA expression module, an upstream homologous arm, a promoter, an alsDS gene cluster, and a downstream homologous arm; or

[0030] The plasmid containing the alsD and alsS genes includes, from 5' to 3', one or more of the following: sgRNA expression module, upstream homologous arm, promoter, RBS, alsDS gene cluster, and downstream homologous arm.

[0031] The sequence of the plasmid containing the alsD and / or alsS genes includes one or more of SEQ ID NO: 1, 2, 3, 10, 11, 12, 13, 17 or 18.

[0032] The *Halomonas* strain can be further modified by the following methods or any combination thereof:

[0033] i) Downregulate or block the acetoin competitive pathway in the aforementioned Halomonas;

[0034] ii) Downregulate or block the acetoin degradation pathway in the aforementioned Halomonas;

[0035] iii) Downregulate or block the acetoin endocytotransport system in the aforementioned Halomonas;

[0036] iv) Increase the membrane permeability of the aforementioned Halomonas bacteria;

[0037] v) Enhance the supply of precursors for acetoin synthesis in the aforementioned Halomonas;

[0038] vi) Reduce the consumption of precursors for acetoin synthesis in the aforementioned *Halomonas*; and / or,

[0039] vii) Adjust the expression levels of acetolactate synthase and / or acetolactate decarboxylase.

[0040] Preferably, i) the Halomonas strain:

[0041] A) Does not express PHA hydratase and acetyl-CoA; or,

[0042] B) The expressed PHA hydratase and acetyl-CoA are nonfunctional or have reduced function; or,

[0043] C) Does not express PHA hydratase; or,

[0044] D) The expressed PHA hydratase is either nonfunctional or has reduced function.

[0045] The PHA hydratase catalyzes the synthesis of poly-3-hydroxybutyryl coenzyme A (P3HB).

[0046] The acetyl-CoA catalyzes the conversion of acetyl-CoA to 3-hydroxybutyryl-CoA.

[0047] Preferably, downregulating or blocking the acetoin competitive pathway in the halomonas includes knocking out or knocking down phaC.

[0048] Preferably, downregulating or blocking the acetoin competitive pathway in the halomonas includes knocking out or reducing phaB and phaC.

[0049] The nucleotide sequence of the phaB contains SEQ ID NO: 30.

[0050] This application downregulates or blocks the acetoin competitive pathway in *Halomonas*, preferably downregulating enzymes related to the acetoin competitive pathway, such as PHA hydratase and acetyl-CoA. This achieves an increase in acetoin production, and unexpectedly reveals that downregulation or blocking of the acetoin competitive pathway alters the intracellular environment of *Halomonas*, adapting it to a low-salt environment. Experiments have confirmed that acetoin production is further increased under low-salt conditions.

[0051] ii) Downregulating or blocking the acetoin degradation pathway in *Halomonas* involves downregulating or blocking the degradation of acetoin to 2,3-butanediol. Preferably, this is done by preventing *Halomonas* from expressing enzymes involved in the acetoin degradation pathway, or by rendering the expressed enzymes nonfunctional or functionally weakened; more preferably, the enzymes involved in the acetoin degradation pathway include one or both of 2,3-butanediol dehydrogenase and acetoin-utilizing protease.

[0052] The 2,3-butanediol dehydrogenase catalyzes the conversion of acetoin to 2,3-butanediol.

[0053] This application further improves the yield of acetoin by downregulating or blocking the acetoin degradation pathway in the aforementioned Halomonas bacteria. More importantly, the experiment showed that downregulating or blocking the acetoin competitive pathway and downregulating or blocking the acetoin degradation pathway simultaneously, which is far superior to the effect of downregulating or blocking the acetoin competitive pathway or downregulating or blocking the acetoin degradation pathway alone. The simultaneous downregulation or blocking of these two pathways has a synergistic effect on the production of acetoin.

[0054] Preferably, downregulating or blocking the acetoin degradation pathway in the *Haloxymonas* includes knocking out or reducing one or more of butA, bdhA, or acuC.

[0055] iii) Downregulating or blocking the acetoin endocytotransport system in the *Halomonas* strain involves downregulating or blocking the secretion of acetoin across the cell membrane to the extracellular space. Preferably, this involves preventing the *Halomonas* strain from expressing enzymes of the acetoin endocytotransport system, or rendering the expressed enzymes nonfunctional or functionally weakened. More preferably, the enzymes of the acetoin endocytotransport system include one or more of the following: a four-carbon dicarboxylic acid (4CDI) binding protein, a 4CDI TRAP transporter small permease protein, or a 4CDI TRAP transporter large permease protein.

[0056] More preferably, downregulating or blocking the acetoin endocytosis transport system in the halomonas includes knocking out or reducing one or more of DctP, DctQ, or DctM.

[0057] iv) Increasing the membrane permeability of the halomonas is preferably achieved by reducing the membrane thickness, for example by preventing the halomonas from expressing outer membrane proteins or by making the expressed outer membrane proteins non-functional or functionally weakened; more preferably, knocking out or knocking down one or both of lpxM or lpxL.

[0058] v) Enhancing the precursor supply for acetoin synthesis in *Halomonas* by increasing the supply of the direct precursor (pyruvate) for acetoin synthesis. This includes expressing or overexpressing glucose-converting enzyme, and / or enhancing the precursor supply for acetoin synthesis in *Halomonas* by not expressing phosphoenolpyruvate carboxylase or by expressing phosphoenolpyruvate carboxylase that is nonfunctional or functionally weakened;

[0059] The glucose-activated sugar catalyzed by the glucose invertase is linked to different receptor molecules.

[0060] Preferably, the glucose invertase is derived from the order Bacillales; for example, the glucose invertase is derived from the genus Bacillus, or even from Bacillus subtilis.

[0061] Preferably, the amino acid sequence of the glucose invertase includes SEQ ID NO: 27.

[0062] The expression or overexpression of glucose invertase involves introducing a foreign gene into *Halomonas*, wherein the foreign gene includes gluC.

[0063] The plasmid containing the gluC gene includes, from 5' to 3', an sgRNA expression module, an upstream homologous arm, a promoter, the gluC gene sequence, and a downstream homologous arm.

[0064] The gluC mentioned refers to one or more copies.

[0065] Preferably, the expression level of gluC can be upregulated by increasing the strength of the promoter that regulates gluC.

[0066] The glucose invertase encoding gene is gluC, and the nucleotide sequence of gluC includes nucleotides 926-1795 of SEQ ID NO: 7.

[0067] The phosphoenolpyruvate carboxylase catalyzes the reaction of phosphoenolpyruvate with carbon dioxide to produce oxaloacetic acid.

[0068] Preferably, the expression of phosphoenolpyruvate carboxylase or the expression of phosphoenolpyruvate carboxylase is non-functional or functionally weakened, including knockout or knockdown of ppc.

[0069] vi) Reducing the consumption of precursors for acetoin synthesis in the aforementioned *Halomonas* involves reducing the consumption of pyruvate in metabolic pathways other than acetoin synthesis. This includes not expressing one or more of the following: phosphorylacetyltransferase, pyruvate carboxylase, lactate dehydrogenase, pyruvate dehydrogenase, or ketoate reductase; or expressing one or more of the following: phosphorylacetyltransferase, pyruvate carboxylase, lactate dehydrogenase, pyruvate dehydrogenase, or ketoate reductase, which are either nonfunctional or functionally weakened.

[0070] The phosphoacetyltransferase catalyzes the conversion of acetyl-CoA to acetyl phosphate.

[0071] The pyruvate carboxylase catalyzes the fixation of carbon dioxide onto pyruvate to generate oxaloacetic acid.

[0072] The lactate dehydrogenase described above catalyzes the conversion of lactate to pyruvate.

[0073] The pyruvate dehydrogenase catalyzes the conversion of pyruvate to acetyl-CoA.

[0074] The keto acid reductase catalyzes the conversion of 2-acetolactate to 2,3-dihydroxy-3-methylbutyric acid.

[0075] More preferably, reducing the consumption of precursors for acetoin synthesis in the *Haloxymonas* includes knocking out or reducing one or more of pta, pyc, ldh, nagD, or ilvC.

[0076] vii) Adjusting the expression levels of acetolactate synthase and / or acetolactate decarboxylase includes increasing the expression levels or activities of acetolactate synthase and / or acetolactate decarboxylase, or making the expression levels of acetolactate synthase and / or acetolactate decarboxylase different. For example, making the expression level of acetolactate decarboxylase greater than that of acetolactate synthase can be achieved by adjusting the promoter strength of the two enzymes.

[0077] Preferably, the promoters that regulate acetolactate synthase and acetolactate decarboxylase are such that the promoter strength regulating acetolactate synthase is weaker than the promoter strength regulating acetolactate decarboxylase.

[0078] Preferably, the exogenous gene is expressed on a plasmid and / or on a chromosome.

[0079] The exogenous genes mentioned can be integrated into the same or different sites in the genome. The same gene can be integrated into one site or multiple identical or different sites.

[0080] The sites include one or more of G3, G4, G43, G51 or GY2.

[0081] The G4 site sgRNA or its target sequence comprises nucleotides 36-55 of SEQ ID NO: 12;

[0082] The G43 site sgRNA or its target sequence comprises nucleotides 36-55 of SEQ ID NO: 13;

[0083] The G51 site sgRNA or its target sequence contains the nucleotide sequence of positions 36-55 in SEQ ID NO: 7 or SEQ ID NO: 17.

[0084] The GY2 site sgRNA or its target sequence contains nucleotides 36-55 of SEQ ID NO: 18.

[0085] The exogenous gene is one or more copies. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more copies.

[0086] The exogenous gene is regulated by the promoter.

[0087] The exogenous genes mentioned can be regulated by the same promoter or by different promoters.

[0088] Preferably, the promoter includes a constitutive promoter and / or an inducible promoter;

[0089] More preferably, the constitutive promoter includes wild-type P porin (Preferably nucleotides 655-925 of SEQ ID NO: 7) or a mutant thereof, more preferably, the P porin Mutants include P porin58 P porin42 P porin68 P porin140 P porin278 P porin194P porin211 P porin221 P porin203 P porin197 P porin259 P porin70 P porin60 P porin185 P porin138 P porin141 or P porin3 More preferably, the constitutive promoter includes P porin68 (Preferred nucleotides 2325-2327 in SEQ ID NO: 11 are nucleotides 2113-2390 when they are GAC), P porin203 (Preferred replacement of nucleotides 2325-2327 in SEQ ID NO: 11 with GTA, and nucleotides 2329-2332 with nucleotides 2113-2390 of CCCT), P porin197 (Preferred nucleotides 53-56 in SEQ ID NO: 14 are nucleotides 1-176 when they are CACC), P porin259 (Preferred nucleotides 53-56 in SEQ ID NO: 14 are nucleotides 1-176 of TAGC), P porin194 (Preferred nucleotides 2325-2327 in SEQ ID NO: 11 are replaced with GTA, and nucleotides 2329-2332 are replaced with CTAA, i.e., nucleotides 2113-2390), P porin70 (Preferred nucleotides 1-176 when nucleotides 49-51 of SEQ ID NO: 14 are CAC), P porin60 (Preferred nucleotides 1-176 when nucleotides 49-51 of SEQ ID NO: 14 are CAT, or nucleotides 653-828 of SEQ ID NO: 18), P porin58 (Preferred nucleotides 1-176 when nucleotides 49-51 in SEQ ID NO: 14 are ATA), P porin185 (Preferred nucleotides 1-176 when nucleotides 53-56 in SEQ ID NO: 14 are ACTC), P porin138 (Preferred nucleotides 1-176 when nucleotides 53-56 in SEQ ID NO: 14 are AGAC), P porin141 (Preferred nucleotides 53-56 in SEQ ID NO: 14 are ATAC), P porin3 (Preferably, nucleotides 49-51 in SEQ ID NO: 14 are nucleotides 1-176 when TGA is used) or P porin140 .

[0090] More preferably, the inducible promoter includes P lux The promoter (preferably nucleotides 1943-2040 of SEQ ID NO: 16) or its mutant, P mmp promoter or its mutant, P tac promoter or its mutant, P trp A promoter or a mutant thereof, more preferably, the P mmp Mutants include P mmp1 (Preferred nucleotides 1-165 of SEQ ID NO: 1), P mmp3 P mmp9 .

[0091] In one specific embodiment of the present invention, alsD and alsS are generated by P mmp1 Promoter regulation of expression.

[0092] In one specific embodiment of the present invention, alsD and alsS are generated by P porin Promoter regulation of expression

[0093] In one specific embodiment of the present invention, the alsD is composed of P mmp1 Promoter-regulated expression, alsS is regulated by P lux Promoter regulation of expression.

[0094] In one specific embodiment of the present invention, the alsD is composed of P porin185 Promoter-regulated expression, alsS is regulated by P porin60 Promoter regulation of expression.

[0095] In one specific embodiment of the present invention, the gluC gene is derived from P porin58 Promoter regulation of expression.

[0096] The exogenous gene was introduced into Halomonas bacteria via plasmid.

[0097] The plasmid contains a promoter.

[0098] The plasmid can integrate foreign genes into the chromosome genome for expression or allow foreign genes to be expressed on the plasmid.

[0099] When expressed on a plasmid, the plasmid can be a high-copy plasmid or a low-copy plasmid.

[0100] The high-copy plasmids mentioned include ColE1, pMB1, pUC, pBluescript, pTZ57R, pSEVA341, etc.

[0101] The low-copy plasmids mentioned include, for example, pSC101, pACYC184, pWE15, and pSEVA321.

[0102] The knockout can be achieved using CRISPR / Cas9 genome editing, chemical mutagenesis, T-DNA insertion mutation, virus-induced gene silencing, suicide plasmids, transcription activation-like effector nuclease (TALEN) technology, or zinc finger nuclease technology.

[0103] Preferably, the suicide plasmid includes one or more of pRE112, pK18mobsacB, or pDM4. In one specific embodiment of the present invention, the suicide plasmid is pRE112.

[0104] The knockdown can be achieved using RNA interference technology, which includes using siRNA or shRNA to target and degrade the target gene.

[0105] The Halomonas include one or more of Halomonas bluephagenesis or its derivatives, Halomonas campaniensis or its derivatives, and Halomonas aydingkolgenesis or its derivatives; preferably, the Halomonas include one or more of Halomonas bluephagenesis TD01, Halomonas bluephagenesis TD1.0, Halomonas bluephagenesis TD-ADEL-58, Halomonas bluephagenesis TDADELP, Halomonas bluephagenesis TD68-194, Halomonas bluephagenesis 68d-G51-G43, Halomonas bluephagenesis 68F or Halomonas bluephagenesis 68FP, Halomonas campaniensis LS21 or Halomonas aydingkolgenesis M1.

[0106] Halomonas bluephagenesis TD1.0 is a strain obtained by integrating the Mmp1 RNA polymerase expression unit into the genome of Halomonas bluephagenesis TD01.

[0107] In a second aspect, the present invention provides a method for constructing the *Haloxymonas* strain described in the first aspect above, wherein the *Haloxymonas* strain expresses or overexpresses acetolactate synthase and acetolactate decarboxylase, and wherein the *Haloxymonas* strain knocks out or knocks down PHA hydratase and acetyl-CoA.

[0108] The expression or overexpression of acetolactate synthase and acetolactate decarboxylase in *Halomonas* involves introducing alsS and alsD into *Halomonas*. Preferably, alsS and alsD are introduced into *Halomonas* via plasmids. The sequences of the plasmids containing the alsS and alsD genes comprise one or more of SEQ ID NO: 1, 2, 3, 10, 11, 12, 13, 17, or 18.

[0109] Preferably, the construction method further includes any one or a combination of two of the following:

[0110] a) Downregulate or block the acetoin degradation pathway in the aforementioned Halomonas;

[0111] b) Downregulate or block the acetoin endogenous transport system in the aforementioned Halomonas;

[0112] c) Increase the membrane permeability of the aforementioned Halomonas bacteria;

[0113] d) Enhance the precursor supply for acetoin synthesis in the aforementioned Halomonas.

[0114] e) Reduce the consumption of precursors for acetoin synthesis in the aforementioned *Halomonas*; and / or,

[0115] f) Adjust the expression levels of acetolactate synthase and / or acetolactate decarboxylase.

[0116] The a) downregulation or blocking of the acetoin degradation pathway in the Halomonas includes knocking out or knocking down the encoding gene of the enzyme in the acetoin degradation pathway. Preferably, the enzyme in the acetoin degradation pathway includes one or two of 2,3-butanediol dehydrogenase or acetoin-utilizing protease. More preferably, one or more of butA, bdhA or acuC are knocked out or knocked down.

[0117] The plasmid used for knockout includes upstream and downstream homologous arm sequences of the gene site to be knocked out. The plasmid can be a suicide plasmid or a plasmid carrying sgRNA. The knockout is performed by introducing the plasmid into *Halomonas*.

[0118] Preferably, the plasmid that knocks out the phaB gene contains the upstream homologous arm sequence comprising nucleotides 1-500 of SEQ ID NO: 31, and the downstream homologous arm sequence comprising nucleotides 500-1000 of SEQ ID NO: 31.

[0119] Preferably, the plasmid that knocks out the butA gene contains upstream homologous arm sequences including nucleotides 1-984 of SEQ ID NO: 9 and downstream homologous arm sequences including nucleotides 985-1984 of SEQ ID NO: 9.

[0120] Preferably, the plasmid that knocks out the acuC gene contains upstream homologous arm sequences comprising nucleotides 1-500 of SEQ ID NO: 29 and downstream homologous arm sequences comprising nucleotides 501-1000 of SEQ ID NO: 29.

[0121] The plasmid that knocks out butA contains SEQ ID NO: 9.

[0122] The plasmid that knocks out acuC contains SEQ ID NO: 29.

[0123] b) Downregulating or blocking the acetoin endocytotransport system in the *Halomonas* species includes knocking out or knocking down the gene encoding the enzyme of the acetoin endocytotransport system; preferably, the enzyme of the acetoin endocytotransport system includes one or more of four-carbon dicarboxylic acid (4CDI) binding protein, four-carbon dicarboxylic acid (4CDI) TRAP transporter small permease protein, or four-carbon dicarboxylic acid (4CDI) TRAP transporter large permease protein; more preferably, knocking out or knocking down one or more of DctP, DctQ, or DctM.

[0124] Preferably, the plasmid that knocks out the DctP gene contains the upstream homologous arm sequence comprising nucleotides 1-500 of SEQ ID NO: 21, and the downstream homologous arm sequence comprising nucleotides 501-1000 of SEQ ID NO: 21.

[0125] Preferably, the plasmid that knocks out the DctP gene contains SEQ ID NO: 21.

[0126] Preferably, the plasmid that knocks out the DctQ gene contains upstream homologous arm sequences comprising nucleotides 1-500 of SEQ ID NO: 22, and downstream homologous arm sequences comprising nucleotides 501-1000 of SEQ ID NO: 22.

[0127] Preferably, the plasmid that knocks out the DctQ gene contains SEQ ID NO: 22.

[0128] Preferably, the plasmid that knocks out the DctM gene contains upstream homologous arm sequences including nucleotides 1-514 of SEQ ID NO: 23 and downstream homologous arm sequences including nucleotides 515-1014 of SEQ ID NO: 23.

[0129] Preferably, the plasmid that knocks out the DctM gene contains SEQ ID NO: 23.

[0130] Preferably, the plasmid that knocks out the DctPQM gene contains the upstream homologous arm sequence comprising nucleotides 1-514 of SEQ ID NO: 24, and the downstream homologous arm sequence comprising nucleotides 515-1014 of SEQ ID NO: 24.

[0131] Preferably, the plasmid that knocks out the DctPQM gene contains SEQ ID NO: 24.

[0132] c) Increasing the membrane permeability of the Halomonas includes knocking out or knocking down outer membrane protein-coding genes, preferably knocking out or knocking down one or both of lpxM or lpxL.

[0133] Preferably, the sgRNA that targets 1pxM is used to knock out the 1pxM gene. More preferably, the sgRNA or its target sequence includes nucleotides 36-55 of SEQ ID NO: 19.

[0134] Preferably, the plasmid that knocks out the lpxM gene contains the upstream homologous arm sequence comprising nucleotides 153-652 of SEQ ID NO: 19, and the downstream homologous arm sequence comprising nucleotides 653-1156 of SEQ ID NO: 19.

[0135] Preferably, the plasmid that knocks out the lpxM gene contains SEQ ID NO: 19.

[0136] Preferably, the knockout of the lpxL gene uses an sgRNA that targets plxL; more preferably, the sgRNA or its target sequence includes nucleotides 36-55 of SEQ ID NO: 20.

[0137] Preferably, the plasmid that knocks out the lpxL gene contains the upstream homologous arm sequence comprising nucleotides 153-652 of SEQ ID NO: 20, and the downstream homologous arm sequence comprising nucleotides 653-1156 of SEQ ID NO: 20.

[0138] Preferably, the plasmid that knocks out the lpxL gene contains SEQ ID NO: 20.

[0139] The d) enhancing the precursor supply for acetoin synthesis in the *Haloxylon ammodendron* comprises introducing a glucose invertase-encoding gene into the *Haloxylon ammodendron*, and / or knocking out or downsetting a phosphoenolpyruvate carboxylase-encoding gene; preferably, introducing gluC into the *Haloxylon ammodendron*, and / or knocking out or downsetting ppc.

[0140] Preferably, a plasmid containing the gluC gene is introduced into Halomonas bacteria.

[0141] Preferably, the sgRNA or its target sequence contained in the plasmid containing the gluC gene includes nucleotides 36-55 of SEQ ID NO: 7.

[0142] Preferably, the plasmid containing the gluC gene contains upstream homologous arms comprising nucleotides 153-654 of SEQ ID NO: 7, and downstream homologous arm sequences comprising nucleotides 1796-2295 of SEQ ID NO: 7.

[0143] Preferably, the sequence of the plasmid containing the gluC gene includes SEQ ID NO: 7.

[0144] Preferably, the plasmid that knocks out the ppc gene contains upstream homologous arms comprising nucleotides 1-1000 of SEQ ID NO: 8, and downstream homologous arm sequences comprising nucleotides 1001-2001 of SEQ ID NO: 8.

[0145] Preferably, the plasmid that knocks out the ppc gene contains SEQ ID NO: 8.

[0146] The aforementioned e) reducing the consumption of precursors for acetoin synthesis in the *Haloxymonas* includes knocking out or reducing one or more of pta, pyc, ldh, nagD, or ilvC.

[0147] Preferably, the plasmid that knocks out the pta gene contains upstream homologous arm sequences comprising nucleotides 1-1000 of SEQ ID NO: 4 and downstream homologous arm sequences comprising nucleotides 1001-2000 of SEQ ID NO: 4.

[0148] Preferably, the plasmid that knocks out pta contains SEQ ID NO: 4.

[0149] The ldh includes ldhA1 and / or ldhA2.

[0150] Preferably, the ldhA1 gene is knocked out using an sgRNA that targets ldhA1. More preferably, the sgRNA or its target sequence includes nucleotides 36-55 of SEQ ID NO: 5.

[0151] Preferably, the plasmid that knocks out the ldhA1 gene contains upstream homologous arm sequences comprising nucleotides 154-653 of SEQ ID NO: 5 and downstream homologous arm sequences comprising nucleotides 654-1153 of SEQ ID NO: 5.

[0152] Preferably, the plasmid that knocks out the ldhA1 gene contains SEQ ID NO: 5.

[0153] Preferably, the ldhA2 gene is knocked out using an sgRNA that targets ldhA2. More preferably, the sgRNA or its target sequence includes nucleotides 36-55 of SEQ ID NO: 6.

[0154] Preferably, the upstream homologous arm sequence of the plasmid that knocks out the ldhA2 gene includes nucleotides 154-653 of SEQ ID NO: 6, and the downstream homologous arm sequence includes nucleotides 654-1153 of SEQ ID NO: 6.

[0155] Preferably, the plasmid that knocks out the ldhA2 gene contains SEQ ID NO: 6.

[0156] Preferably, the plasmid that knocks out the ilvC gene contains the upstream homologous arm sequence comprising nucleotides 1-700 of SEQ ID NO: 28, and the downstream homologous arm sequence comprising nucleotides 701-1400 of SEQ ID NO: 28.

[0157] Preferably, the plasmid that knocks out the ilvC gene contains SEQ ID NO: 28.

[0158] f) Adjusting the expression levels of acetolactate synthase and / or acetolactate decarboxylase includes adjusting the promoter strength of the two enzymes.

[0159] Preferably, the promoters that regulate acetolactate synthase and acetolactate decarboxylase are such that the promoter strength regulating acetolactate synthase is weaker than the promoter strength regulating acetolactate decarboxylase.

[0160] In one specific embodiment of the present invention, the construction method includes introducing one or more plasmids from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 19, 20, 21, 22, 23, 24, 28 or 29 into Halomonas.

[0161] A third aspect of the present invention provides a method for producing acetoin, the method comprising culturing the *Haloxylon ammodendron* strain described in the first aspect or the *Haloxylon ammodendron* strain obtained by the construction method described in the second aspect. Preferably, a low-salt, low-oxygen method is used for producing acetoin.

[0162] The fermentation medium used to culture Halomonas contains inorganic salts, and the concentration of the inorganic salts in the medium is any value between 10 and 60 g / L. Preferably, the concentration of the inorganic salts in the medium is any value between 10 and 50 g / L, such as 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 55 g / L, and 60 g / L.

[0163] The inorganic salts mentioned include one or more of sodium chloride, potassium chloride, calcium chloride, barium chloride, potassium dihydrogen phosphate, ferrous sulfate, calcium carbonate, ammonium sulfate, or potassium nitrate.

[0164] The fermentation medium also includes carbon sources, nitrogen sources, and other substances suitable for the growth, reproduction, or production of metabolites by Halomonas.

[0165] The conditions for culturing the aforementioned Halomonas include a shaking speed, which is any value between 10 and 500 rpm. Preferably, the shaking speed is any value between 20 and 300 rpm, such as 10 rpm, 20 rpm, 50 rpm, 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm, 200 rpm, 250 rpm, 300 rpm, 350 rpm, 400 rpm, 450 rpm, and 500 rpm.

[0166] The temperature for culturing the aforementioned Halomonas includes 30℃-40℃, for example 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃ or 40℃.

[0167] The culture process does not require sterilization.

[0168] In a fourth aspect, the present invention provides a method for the co-production of acetoin and PHA, the method comprising culturing Halomonas bacteria, wherein the Halomonas bacteria express or overexpress acetolactate synthase and acetolactate decarboxylase.

[0169] The method includes a two-stage feeding method, which includes a first stage of feeding carbon source and a high proportion of nitrogen source (to promote acetoin synthesis), and a second stage of stopping the addition of nitrogen source (to limit the nitrogen source uptake of the cells and stimulate the cells to synthesize PHA).

[0170] The first stage of feeding promotes acetoin synthesis, the second stage of feeding promotes PHA synthesis, the acetoin is secreted extracellularly, and the PHA accumulates intracellularly.

[0171] The PHA includes homopolymers or copolymers of the monomers constituting PHA, such as one or more of P3HP, PHB, PHBHP, P(HA-LA), PHV, P34HB, PHBV, PHBV4HB, PHBHx, PHBHHp, PHO, PHN, PHD, P3HB4HB3HV, or P3HB4HB5HV. In P(HA-LA), LA represents lactic acid, and HA represents the PHA monomer, including but not limited to one or more of 3-hydroxybutyric acid, 4-hydroxybutyric acid, 3-hydroxyvalerate, 3-hydroxypropionic acid, 5-hydroxyvalerate, 3-hydroxyhexanoic acid, 6-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 7-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, or 3-hydroxydodecanoic acid.

[0172] The "overexpression" described in this invention refers to upregulating the expression of a target gene to a level higher than its natural (e.g., original strain) expression level, or causing a gene that was not originally expressed to be expressed. This can be achieved, for example, by increasing the expression intensity of the promoter or by introducing a copy number of the gene.

[0173] The abbreviation and full name of this application are shown in Table 1.

[0174]

[0175]

[0176] The above only summarizes some aspects of the present invention and is not, and should not be considered as limiting the present invention in any way.

[0177] All patents and publications mentioned in this application are incorporated herein by reference in their entirety. Those skilled in the art will recognize that certain modifications can be made to this invention without departing from its spirit or scope. The following embodiments further illustrate the invention in detail and should not be considered as limiting the scope of the invention or the specific methods described herein. Attached Figure Description

[0178] Figure 1 Acetonitrile and PHA synthesis pathway.

[0179] Figure 2 : Schematic diagram of the construction process of recombinant halomonas in the example.

[0180] Figure 3 Four metabolic engineering strategies to increase acetoin production. Detailed Implementation

[0181] The present invention will be described in detail below by way of examples.

[0182] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0183] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0184] Escherichia coli was grown on LB medium containing: 10 g / L sodium chloride, 10 g / L peptone (OXID, catalog number LP0042), and 5 g / L yeast extract (OXID, catalog number LP0021).

[0185] Unless otherwise specified in the examples, the growth temperature for both Escherichia coli and Halomonas bluephagenesis TD01 was 37°C and 200 rpm.

[0186] The Halomonas bluephagenesis TD01 used in the examples is derived from the literature Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01 (Tan et al, Bioresource Technology, 2011); this bacterium is available to the public from Tsinghua University.

[0187] The Halomonas campaniensis LS21 used in the examples is derived from the literature A seawater-based open and continuous process for polyhydroxyalkanoates production by recombinant Halomonas campaniensis LS21 grown in mixed substrates (Yue et al, Biotechnology for Biofuels, 2014); this bacterium is available to the public from Tsinghua University.

[0188] Halomonas aydingkolgenesis M1 is a self-flocculating halomonas bacterium, as described in patent application publication number CN111593006A; the bacterium can be obtained from Tsinghua University.

[0189] The Halomonas bluephagenesis TD1.0 used in the examples is a strain obtained by integrating the Mmp1 RNA polymerase expression unit into the genome of Halomonas bluephagenesis TD01 CGMCCNo.4353, which is Halomonas sp. TD1.0 described in patent application publication number CN113621639A; the public can obtain this strain from Tsinghua University.

[0190] The Halomonas bluephagenesis TD1.0△phaC used in the examples is derived from the literature CRISPR / Cas9 editing genome of extremophile Halomonas spp. (Qin et al, Metabolic Engineering, 2018); this bacterium is available to the public from Tsinghua University.

[0191] The genome editing methods in the examples are divided into CRISPR editing methods and homologous recombination methods based on the suicide plasmid pRE112.

[0192] For CRISPR genome editing methods, see the literature CRISPR / Cas9 editing genome of extremophile Halomonas spp (Qin et al, Metabolic Engineering, 2018).

[0193] For the homologous recombination method of suicide plasmid pRE112, please refer to the literature Development of Halomonas TD01 as a host for open production of chemicals (Fu et al, Metabolic Engineering, 2014).

[0194] For the genome editing sites described in this application, please refer to the literature CRISPR / Cas9 editing genome of extremophile Halomonas spp (Qin et al, Metabolic Engineering, 2018).

[0195] Biosynthesis of diverseα,ω-diol-derived polyhydroxyalkanoates by engineered Halomonas bluephagenesis (Yan et al, Metabolic Engineering, 2022).

[0196] 60LB medium: 20-60 g / L glucose, 60 g / L sodium chloride, 5 g / L yeast extract, 10 g / L tryptone.

[0197] Unless otherwise specified in the examples, the acetoin fermentation medium is:

[0198] 40 g / L glucose, 50 g / L sodium chloride, 1-10 g / L yeast extract, 1-5 g / L urea, 1.5-5.2 g / L potassium dihydrogen phosphate, 0.2-0.4 g / L magnesium sulfate, 8.5-10 g / L disodium hydrogen phosphate, 7-15 ml / L component III, and 1-5 ml / L component IV.

[0199] Component III: 5 g / L ferric ammonium citrate, 2 g / L calcium chloride dihydrate, 41.7 ml concentrated hydrochloric acid (12 mol / L), and water to a final volume of 1000 ml.

[0200] Component IV: 100 mg / L zinc sulfate heptahydrate, 30 mg / L manganese chloride tetrahydrate, 300 mg / L boric acid, 200 mg / L cobalt chloride hexahydrate, 10 mg / L anhydrous copper sulfate, 20 mg / L nickel chloride hexahydrate, and 30 mg / L sodium molybdate dihydrate.

[0201] All of the above culture media can be prepared using standard preparation methods.

[0202] Methods for detecting acetokines:

[0203] Take 1 mL of fermentation broth, centrifuge at 12000 rpm for 10 min, and collect the supernatant. Determine the acetoin content in the supernatant using high performance liquid chromatography. The detection conditions are: Aminex HPX-87H column (7.8 × 300 mm, bio-rad), mobile phase is 5 mM dilute sulfuric acid solution, column temperature is 65℃, flow rate is 0.4 mL / min, injection volume is 10 μl, differential detector, and retention time is 25.6 min.

[0204] The embodiments described in this application are a detailed description of the construction method and application of Halomonas bacillus for producing acetoin. They are illustrative rather than limiting. Several embodiments can be listed according to the defined scope. Therefore, changes and modifications without departing from the overall concept of the present invention should be within the protection scope of the present invention.

[0205] Example 1: Construction of expression plasmid overexpressing acetoin synthesis gene cluster

[0206] The alsSD and alsDS gene clusters were synthesized by overexpressing acetoin using a high-copy expression plasmid. Both alsS and alsD genes are derived from Bacillales and synthesized after codon optimization adapted to Halomonas bluephagenesis TD01. Specifically, the plasmid containing the inducible promoter mmp1, the inducible promoter plux, and the expression genes was constructed by inserting DNA fragments such as the inducible promoter mmp1, the inducible promoter plux, and the alsDS and alsSD gene clusters into the original expression plasmid pSEVA341 (containing the chloramphenicol resistance gene) or pSEVA321 using the Gibson method. The protein encoded by the alsS gene is the sequence shown in WP_003244057.1 (SEQ ID NO: 25) in the NCBI database, and the protein encoded by the alsD gene is the sequence shown in WP_003244457.1 (SEQ ID NO: 26) in the NCBI database.

[0207] Mutant strains containing the target plasmid were screened using primers designed by colony PCR and confirmed by gene sequencing. Colony PCR is a routine procedure.

[0208] Finally, through colony PCR and gene sequencing, it was confirmed that the alsDS plasmid driven by the inducible promoter mmp1 was successfully inserted into the expression plasmid pSEVA341 and named pZH126; the alsSD plasmid driven by the inducible promoter mmp1 was successfully inserted into the expression plasmid pSEVA341 and named pZH146; the alsD plasmid driven by the inducible promoter mmp1 was successfully inserted into the expression plasmid pSEVA341 and named pZH160.

[0209] To obtain a recombinant strain Halomonas bluephagenesis TD1.0△phaCB with phaB knocked out (gene sequence SEQ ID NO: 30), the phaB gene was knocked out using a suicide plasmid-mediated editing method based on the recombinant strain Halomonas bluephagenesis TD1.0△phaC.

[0210] The constructed high-copy expression plasmids pZH126, pZH146, and pZH160 were transformed into recombinant strains Halomonas bluephagenesis TD1.0, Halomonas campaniensis LS21, Halomonassaydingkolgenesis M1, Halomonas bluephagenesis TD1.0△phaC, and Halomonasbluephagenesis TD1.0△phaCB via Escherichia coli S17-1 conjugation. Fermentation tests showed that the strains overexpressed the acetoin synthesis gene cluster, enabling strains that originally did not produce acetoin to acquire the ability to produce acetoin.

[0211] The specific implementation process is as follows:

[0212] (1) Construction of recombinant strain Halomonas bluephagenesis TD1.0△phaCB

[0213] The plasmid construction method for the homologous recombination editing method used in the suicide plasmid is as follows: DNA fragments of the upstream and downstream homologous arms are inserted into the original expression plasmid pRE112 (containing the chloramphenicol resistance gene) using the Gibson method. The sequence of the inserted plasmid is SEQ ID NO: 31, arranged in the following order: upstream homologous arm (nucleotides 1-500) and downstream homologous arm (nucleotides 501-1000).

[0214] The pRE112 suicide plasmid expressing the homologous recombination template was transformed into the recombinant strain Halomonas bluephagenesis TD1.0△phaC via E. coli S17-1 conjugation. In the first round, transformants integrated into the genome were selected by chloramphenicol resistance screening. In the second round, the plasmid containing the homing endonuclease expression module was transformed into the pHelp plasmid via E. coli S17-1 conjugation. The plasmids were screened on chloramphenicol and antibiotic-free plates, respectively. Single clones that did not grow on chloramphenicol-added plates but grew on antibiotic-free plates were further identified.

[0215] Gene-edited mutant strains were screened using primers designed for colony PCR and confirmed by gene sequencing. Colony PCR is a routine procedure.

[0216] Finally, colony PCR and gene sequencing confirmed that the phaB gene sequence had been knocked out in the genome of Halomonas bluephagenesis TD1.0△phaC.

[0217] (2) Construction of high-copy plasmids pZH126, pZH146, and pZH160, and low-copy plasmids pZH176 and pZH342

[0218] The plasmid construction method for efficient expression of the target gene is as follows: DNA fragments such as the inducible promoter mmp1, the inducible promoter plux, and the alsDS and alsSD gene clusters are inserted into the original expression plasmid pSEVA341 (containing the chloramphenicol resistance gene) or pSEVA321 using the Gibson method.

[0219] The sequence of expression plasmid pZH126 is SEQ ID NO: 1, arranged in the following order: upstream homologous arm P mmp1 The promoter sequence (nucleotides 1-165), synthetic RBS sequence 1 (nucleotides 166-192), alsD gene sequence (nucleotides 193-959), synthetic RBS sequence 2 (nucleotides 960-1002), and alsS gene sequence (nucleotides 1003-2715) are all present. The expression plasmid pZH176 is identical to the expression plasmid pZH126 except for its backbone, which is derived from the original plasmid pSEVA321.

[0220] The sequence of expression plasmid pZH146 is SEQ ID NO: 2, arranged in the following order: upstream homologous arm P mmp1 Promoter sequence (nucleotides 1-165), synthetic RBS sequence 1 (nucleotides 166-192), alsS gene sequence (nucleotides 193-1904), synthetic RBS sequence 2 (nucleotides 1905-1947), alsD gene sequence (nucleotides 1948-2715).

[0221] The sequence of expression plasmid pZH160 is SEQ ID NO: 3, arranged in the following order: upstream homologous arm P mmp1 Promoter sequence (nucleotides 1-165), synthetic RBS sequence 1 (nucleotides 166-192), alsD gene sequence (nucleotides 193-959), terminator (nucleotides 960-1114), luxR gene sequence (nucleotides 1115-1867), synthetic RBS sequence 2 and P lux Promoter sequence (nucleotides 2000-2097), alsS gene sequence (nucleotides 2056-3810). The expression plasmid pZH342 is identical to the expression plasmid pZH160 except for the backbone, which is derived from the original plasmid pSEVA321.

[0222] Mutant strains containing the target plasmid were screened using primers designed by colony PCR and confirmed by gene sequencing. Colony PCR is a routine procedure.

[0223] Finally, through colony PCR and gene sequencing, it was confirmed that the plasmid pZH126 was named alsDS driven by the inducible promoter mmp1, pZH146 was named alsSD driven by the inducible promoter mmp1, and pZH160 was named alsS driven by the inducible promoter plux. Similarly, the plasmid pZH176 was named alsDS driven by the inducible promoter mmp1, and pZH342 was named alsS driven by the inducible promoter plux.

[0224] (2) Shake-flask fermentation was used to verify the acetoin production levels of recombinant bacteria Halomonas bluephagenesis TD1.0, Halomonas campaniensis LS21, Halomonas aydingkolgenesis M1, Halomonas bluephagenesis TD1.0△phaCB, and Halomonas bluephagenesis TD1.0△phaCB conjugated with expression plasmids pZH126, pZH146, and pZH160.

[0225] The specific steps for producing acetoin by shake-flask fermentation of recombinant strains are as follows:

[0226] The recombinant strain was inoculated into 20 mL of LB60 medium and cultured for 12-16 h. Then, it was transferred to a new 20 mL LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture.

[0227] 2.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of acetoin fermentation medium, and a shake-flask experiment was conducted. The shaker temperature was 37℃, and the rotation speed was 200 rpm. After 4 h of culture, different doses of IPTG were added to induce the expression of the acetoin synthesis gene alsDS or alsD by the mmp1 promoter, and different doses of AHL were added to induce the expression of the acetoin synthesis gene alsS by the plux promoter. After 48 h of culture, the acetoin content was measured. Each experiment was performed in triplicate, and the results were averaged. The results are shown in Table 2-6 below.

[0228] a. Shake-flask experiment on plasmid expression for acetoin production in WT and modified Halomonas bacteria.

[0229] The blank expression plasmid pSEVA341 and the high expression plasmid pZH126 were introduced into recombinant strains Halomonas bluephagenesis TD1.0, Halomonas campaniensis LS21, and Halomonas aydingkolgenesis M1. Different doses of IPTG were used to induce the expression of the acetoin synthesis gene cluster alsDS in the mmp1 promoter, and the acetoin content was detected.

[0230] Table 2. Comparison of acetoin synthesis levels among several Halomonas species via plasmid overexpression of the alsDS gene cluster.

[0231]

[0232]

[0233] The results showed that wild-type Halomonas bluephagenesis TD01, Halomonas campaniensis LS21, recombinant strains Halomonas bluephagenesis TD1.0, Halomonas aydingkolgenesis M1, recombinant strain Halomonas bluephagenesis TD1.0-pSEVA341 with blank plasmid, or recombinant strains Halomonas bluephagenesis TD1.0-pZH126, Halomonas campaniensis LS21-pZH126, and Halomonas aydingkolgenesis M1-pZH126 with expression plasmid could not produce acetoin without the addition of an inducer. This indicates that wild-type Halomonas bluephagenesis TD01, Halomonas campaniensis LS21, and recombinant strain Halomonas aydingkolgenesis M1 cannot synthesize acetoin naturally. Without the addition of the inducer IPTG, the recombinant strains *Halomonas bluephagenesis* TD1.0-pZH126, *Halomonas campaniensis* LS21-pZH126, and *Halomonas aydingkolgenesis* M1-pZH126 could not produce acetoin, indicating that plasmid expression was either not leaked or the acetoin yield from leaked expression was very low. When the concentration of the inducer IPTG reached 100 mg / L, the acetoin yields reached 2.16 g / L, 1.37 g / L, and 1.09 g / L, respectively. These results demonstrate that overexpression of the exogenous acetoin synthesis gene cluster alsDS using a high-copy plasmid can produce acetoin in *Halomonas bluephagenesis* TD1.0, *Halomonas campaniensis* LS21, and *Halomonas aydingkolgenesis* M1, with *Halomonas bluephagenesis* TD1.0 serving as the starting strain for subsequent experiments.

[0234] Table 3. Acetoin content of recombinant strain Halomonas bluephagenesis TD1.0 after overexpression of the acetoin synthesis gene cluster on the conjugation plasmid pZH146.

[0235] Halomonas bluephagenesis TD1.0-pZH146 0 0 Halomonas bluephagenesis TD1.0-pZH146 6.25 0.97 Halomonas bluephagenesis TD1.0-pZH146 12.5 1.06 Halomonas bluephagenesis TD1.0-pZH146 25 1.54 Halomonas bluephagenesis TD1.0-pZH146 50 1.96 Halomonas bluephagenesis TD1.0-pZH146 100 1.86 Halomonas bluephagenesis TD1.0-pZH146 200 1.52

[0236] The results showed that when the recombinant strain Halomonas bluephagenesis TD1.0-pZH146 was induced to express the alsSD gene cluster with different concentrations of IPTG, the acetoin yields were 0.97, 1.06, 1.54, 1.96, 1.86, and 1.52 g / L, respectively. The acetoin yield reached its maximum when the IPTG concentration was 50 mg / L, but the yield was lower than that of the Halomonas bluephagenesis TD1.0-pZH126 strain.

[0237] b. Shake-flask experiment to precisely regulate synthase expression in Halomonas bluephagenesis TD1.0 using high-copy expression plasmids.

[0238] The micro-regulation expression plasmid pZH160 was introduced into the recombinant strain Halomonas bluephagenesis TD1.0. Different doses of IPTG were used to induce the expression of the acetoin synthesis gene alsD by the mmp1 promoter, and different doses of AHL were used to induce the expression of the acetoin synthesis gene alsS by the plux promoter. The acetoin content was then detected.

[0239] Table 4. Acetoin content produced by strain TD1.0 after micro-regulation of acetoin synthesis gene cluster expression.

[0240]

[0241] The results showed that expressing the two acetoin synthases, alsD and alsS, separately using the micro-regulatory plasmid (pZH160) in the recombinant strain Halomonas bluephagenesis TD1.0 resulted in a maximum acetoin yield of 3.13 g / L. This expression efficiency was better than that achieved by using the single regulatory plasmid (pZH126) to express the alsDS gene cluster, increasing the yield by 44.91%. These results demonstrate that by finely regulating the efficient expression of each acetoin synthase, acetoin production in Halomonas bluephagenesis TD1.0 can be increased.

[0242] c. Shake-flask experiment of acetoin production by plasmid expression in Halomonas bluephagenesis TD1.0△phaCB.

[0243] The above shake-flask experiments demonstrated the co-production of acetoin and PHB in the recombinant strain Halomonas bluephagenesis TD1.0-pZH160. The acetoin and PHB synthetic pathways are described below. Figure 1The highest yield of acetoin was obtained at 3.13 g / L, but the final CDW of the strain was 10.45 g / L and the PHB content was 68.62%. Compared with the control that did not produce acetoin, the CDW and PHB content of the strain decreased by 19.14% and 23.36%, respectively.

[0244] To further increase the yield of acetoin, this embodiment attempts to produce acetoin based on recombinant strains Halomonas bluephagenesis TD1.0△phaC and Halomonas bluephagenesis TD1.0△phaCB.

[0245] The high-expression plasmid pZH126 was introduced into the recombinant strains Halomonas bluephagenesis TD1.0△phaC and Halomonas bluephagenesis TD1.0△phaCB. The expression of the acetoin synthesis gene cluster alsDS by the mmp1 promoter was induced using different doses of IPTG, and the acetoin content was detected.

[0246] Table 5. Ethiopogonin production by plasmid expression in Halomonas bluephagenesis TD1.0△phaC and Halomonas bluephagenesis TD1.0△phaCB

[0247]

[0248] The results showed that by using the regulatory plasmid (pZH126) to express the gene cluster alsDS in the recombinant strains Halomonas bluephagenesis TD1.0△phaC and Halomonas bluephagenesis TD1.0△phaCB, the highest yields of acetoin reached 3.46 g / L and 4.16 g / L, respectively, which were 60.19% and 92.59% higher than the highest yield in the recombinant strain Halomonas bluephagenesis TD1.0-pZH126. Compared with Halomonas bluephagenesis TD1.0△phaC-pZH126, the yield of acetoin in the recombinant strain Halomonas bluephagenesis TD1.0△phaCB-pZH126 was increased by 20.23%. The results showed that knocking out the key genes phaC and phaCB for PHA synthesis reduced one pathway in the recombinant strain Halomonas bluephagenesis TD1.0△phaCB that consumes pyruvate, the precursor of acetoin synthesis. The increase in pyruvate improved the yield of acetoin. Moreover, knocking out phaCB enhanced the carbon flow towards acetoin synthesis more than knocking out phaC. Therefore, the recombinant strain Halomonas bluephagenesis TD1.0△phaCB was selected as the starting strain for subsequent experiments.

[0249] d. Shake-flask experiment of micro-regulating plasmid expression for acetoin production in Halomonas bluephagenesis TD1.0△phaCB.

[0250] The high-expression microregulatory plasmid pZH160 was introduced into the recombinant strain Halomonas bluephagenesis TD1.0△phaCB. Different doses of IPTG were used to induce the expression of the acetoin synthesis gene alsD by the mmp1 promoter, and different doses of AHL were used to induce the expression of the acetoin synthesis gene alsS by the plux promoter. The acetoin content was then detected.

[0251] Table 6 shows the acetoin production rate of plasmid expression in Halomonas bluephagenesis TD1.0△phaCB.

[0252]

[0253] The results showed that using the micro-regulation plasmid (pZH160) to express the gene cluster alsDS in the recombinant strain Halomonas bluephagenesis TD1.0△phaCB, the highest acetoin yield reached 4.76 g / L, which was 52.08% higher than the highest yield in the recombinant strain Halomonas bluephagenesis TD1.0-pZH160, and 14.42% higher than the highest yield in the recombinant strain Halomonas bluephagenesis TD1.0△phaCB-pZH126. These results indicate that fine-tuning still contributes to increased acetoin yield.

[0254] The following examples further demonstrate the construction of strains that produce acetoin. A schematic diagram of the construction process can be found in [link to diagram]. Figure 2 .

[0255] Example 2: Construction of a recombinant strain that reduces the consumption of acetoin precursors

[0256] Pyruvate is a precursor to acetoin synthesis. To reduce the consumption of pyruvate, the precursor of acetoin synthesis, in other metabolic pathways, the key genes for the synthesis of acetic acid from pyruvate, phosphoacetyltransferase pta, lactate dehydrogenase ldhA1 and ldhA2, which are key genes for the direct synthesis of lactate from pyruvate, were knocked out on the recombinant strain Halomonas bluephagenesis TD1.0△phaCB. The key gene for the synthesis of (R)-2,3-dihydroxyisovalerate, keto acid reductase ilvC, which consumes acetolactate, was also knocked out. Recombinant strains TDZH113, TDZH114, TDZH115, and TDZH120 were constructed.

[0257] Fermentation tests revealed that the recombinant strains TDZH113, TDZH114, TDZH115, and TDZH120 exhibited enhanced acetoin synthesis capabilities.

[0258] The specific implementation process is as follows:

[0259] (1) Construction of recombinant strain TDZH113

[0260] The plasmid construction method for the homologous recombination editing method of the suicide plasmid is as follows: DNA fragments of the upstream and downstream homologous arms are inserted into the original expression plasmid pRE112 (containing the chloramphenicol resistance gene) using the Gibson method. The sequence of the inserted plasmid is SEQ ID NO: 4, arranged in the following order: upstream homologous arm (nucleotides 1-1000) and downstream homologous arm (nucleotides 1001-2000).

[0261] The pRE112 suicide plasmid expressing the homologous recombination template was transformed into the recombinant strain Halomonas bluephagenesis TD1.0△phaCB via E. coli S17-1 conjugation. In the first round, transformants integrated into the genome were selected by chloramphenicol resistance screening. In the second round, the plasmid containing the homing endonuclease expression module was transformed into the pHelp plasmid via E. coli S17-1 conjugation. The plasmids were screened on chloramphenicol and antibiotic-free plates, respectively. Single clones that did not grow on chloramphenicol-added plates but grew on antibiotic-free plates were further identified.

[0262] Gene-edited mutant strains were screened using primers designed for colony PCR and confirmed by gene sequencing. Colony PCR is a routine procedure.

[0263] Finally, through colony PCR and gene sequencing, it was confirmed that the pta gene sequence had been knocked out in the genome of Halomonas bluephagenesis TD1.0△phaCB, and the recombinant bacteria constructed was named TDZH113.

[0264] (2) Construction of recombinant strain TDZH114

[0265] The genome was edited using the CRISPR genome editing method. Specifically, the plasmid containing sgRNA and the recombinant template was constructed by inserting the sgRNA expression module, upstream and downstream homologous arms, and other DNA fragments into the original expression plasmid pSEVA241 (containing kanamycin and spectinomycin resistance genes) using the Gibson method. The sequence of the inserted plasmid is SEQ ID NO: 5, arranged in the following order: sgRNA expression module (nucleotides 1-153), upstream homologous arm (nucleotides 154-653), and downstream homologous arm (nucleotides 654-1153).

[0266] The pSEVA241 plasmid expressing sgRNA and recombinant template and the pQ08 plasmid expressing Cas9 were transformed into the recombinant bacterium Halomonas bluephagenesis TD1.0△phaCB via Escherichia coli S17-1 conjugation.

[0267] Gene-edited mutant strains were screened using primers designed for colony PCR and confirmed by gene sequencing. Colony PCR is a routine procedure.

[0268] Finally, through colony PCR and gene sequencing, it was confirmed that the ldhA1 gene sequence had been knocked out in the genome of the recombinant strain Halomonas bluephagenesis TD1.0△phaCB, and the constructed recombinant strain was named TDZH114.

[0269] (3) Construction of recombinant strain TDZH115

[0270] The genome was edited using the CRISPR genome editing method. Specifically, the plasmid containing sgRNA and the recombinant template was constructed by inserting the sgRNA expression module, upstream and downstream homologous arms, and other DNA fragments into the original expression plasmid pSEVA241 (containing kanamycin and spectinomycin resistance genes) using the Gibson method. The sequence of the inserted plasmid is SEQ ID NO: 6, arranged in the following order: sgRNA expression module (nucleotides 1-153), upstream homologous arm (nucleotides 154-653), and downstream homologous arm (nucleotides 654-1153).

[0271] The pSEVA241 plasmid expressing sgRNA and recombinant template and the pQ08 plasmid expressing Cas9 were transformed into the recombinant bacterium Halomonas bluephagenesis TD1.0△phaCB via Escherichia coli S17-1 conjugation.

[0272] Gene-edited mutant strains were screened using primers designed for colony PCR and confirmed by gene sequencing. Colony PCR is a routine procedure.

[0273] Finally, through colony PCR and gene sequencing, it was confirmed that the ldhA2 gene sequence had been knocked out in the genome of the recombinant strain Halomonas bluephagenesis TD1.0△phaCB, and the constructed recombinant strain was named TDZH115.

[0274] (4) Construction of recombinant strain TDZH120

[0275] The plasmid construction method for the homologous recombination editing method of the suicide plasmid is as follows: DNA fragments of the upstream and downstream homologous arms are inserted into the original expression plasmid pRE112 (containing the chloramphenicol resistance gene) using the Gibson method. The sequence of the inserted plasmid is SEQ ID NO: 28, arranged in the following order: upstream homologous arm (nucleotides 1-700) and downstream homologous arm (nucleotides 701-1400).

[0276] The pRE112 suicide plasmid expressing the homologous recombination template was transformed into the recombinant strain Halomonas bluephagenesis TD1.0△phaCB via E. coli S17-1 conjugation. In the first round, transformants integrated into the genome were selected by chloramphenicol resistance screening. In the second round, the plasmid containing the homing endonuclease expression module was transformed into the pHelp plasmid via E. coli S17-1 conjugation. The plasmids were screened on chloramphenicol and antibiotic-free plates, respectively. Single clones that did not grow on chloramphenicol-added plates but grew on antibiotic-free plates were further identified.

[0277] Gene-edited mutant strains were screened using primers designed for colony PCR and confirmed by gene sequencing. Colony PCR is a routine procedure.

[0278] Finally, through colony PCR and gene sequencing, it was confirmed that the ilvC gene sequence had been knocked out in the genome of Halomonas bluephagenesis TD1.0△phaCB, and the recombinant bacteria constructed was named TDZH120.

[0279] (5) Shake-flask fermentation was used to verify the acetoin production levels of recombinant bacteria TDZH113, TDZH114, TDZH115, and TDZH120 combined with the expression plasmid pZH126.

[0280] The specific steps for producing acetoin by shake-flask fermentation of recombinant strains are as follows:

[0281] Recombinant strains TDZH113-pZH126, TDZH114-pZH126, TDZH115-pZH126, and TDZH120-pZH126 were inoculated into 20 mL of LB60 medium and cultured for 12-16 h. Then, they were transferred to a new 20 mL LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain seed culture.

[0282] 2.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of acetoin fermentation medium for a shake-flask experiment. The shaker temperature was 37℃ and the rotation speed was 200 rpm. After 4 h of culture, 50 mg / L IPTG was added to induce the expression of the acetoin synthesis gene by the mmp1 promoter. After 48 h of culture, the acetoin content was measured. Each experiment was performed in triplicate, and the results were averaged. The results are shown in Table 7.

[0283] Table 7. Enhancing the supply of acetoin precursors and increasing the acetoin content produced by recombinant strains.

[0284]

[0285] The results showed that knocking out the key gene phosphotransacetase pta, which is crucial for the synthesis of acetic acid from pyruvate, increased the acetoin yield of recombinant strain TDZH113-pZH126 by 14.66%; knocking out the key genes lactate dehydrogenases ldhA1 and ldhA2, which are crucial for the direct synthesis of lactate from pyruvate, increased the acetoin yield of recombinant strains TDZH114-pZH126 and TDZH115-pZH126 by 50% and 26.68%, respectively; and knocking out the key gene ketoate reductase ilvC, which is crucial for the synthesis of (R)-2,3-dihydroxyisovalerate from acetolactate, increased the acetoin yield of recombinant strain TDZH120-pZH126 by 19.23%.

[0286] These results indicate that knocking out key genes for pyruvate to other byproducts, such as pta, ldhA1, ldhA2, and ilvC, enhances the supply of pyruvate, a precursor for acetoin synthesis, and can improve acetoin production levels.

[0287] Example 3: Construction of a recombinant strain that enhances the supply of acetoin precursors

[0288] Pyruvate is a precursor for acetoin synthesis. To enhance the supply of acetoin precursors, the gluC gene (glucose invertase) from Bacillales was overexpressed on the recombinant strain Halomonasbluephagenesis TD1.0△phaCB, and the phosphoenolpyruvate carboxylase gene ppc, a key gene for the synthesis of oxaloacetate from phosphoenolpyruvate, was knocked out, thus constructing recombinant strains TDZH117 and TDZH118.

[0289] Fermentation tests revealed that the recombinant strains TDZH117 and TDZH118 exhibited enhanced acetoin synthesis capabilities.

[0290] The specific implementation process is as follows:

[0291] (1) Construction of recombinant strain TDZH117

[0292] The genome was edited using the CRISPR genome editing method. Specifically, the plasmid containing sgRNA and a recombination template was constructed by connecting upstream and downstream homologous arms, the sgRNA expression module, and P... porin The promoter module, gluC gene, and other DNA fragments were inserted into the original expression plasmid pSEVA241 (containing kanamycin and spectinomycin resistance genes) using the Gibson method. The sequence of the inserted plasmid is SEQ ID NO: 7, arranged as follows: sgRNA expression module (nucleotides 1-152), upstream homologous arm (nucleotides 153-654), P... porin The promoter module (nucleotides 655-925), the gluC gene (nucleotides 926-1795), and the downstream homologous arm (nucleotides 1796-2295) are all present. The protein sequence encoded by the gluC gene is SEQ ID NO: 27.

[0293] The pSEVA241 plasmid expressing sgRNA and recombinant template and the pQ08 plasmid expressing Cas9 were transformed into the recombinant bacterium Halomonas bluephagenesis TD1.0△phaCB via Escherichia coli S17-1 conjugation.

[0294] Gene-edited mutant strains were screened using primers designed for colony PCR and confirmed by gene sequencing. Colony PCR is a routine procedure.

[0295] Finally, colony PCR and gene sequencing confirmed that the gluC gene expression module, consisting of nucleotides 153-2295 of SEQ ID NO: 7, was inserted at site G51 in the genome of the recombinant strain Halomonas bluephagenesis TD1.0△phaCB. The constructed recombinant strain was named TDZH117.

[0296] (2) Construction of recombinant strain TDZH118

[0297] The plasmid construction method for the homologous recombination editing method used in the suicide plasmid is as follows: DNA fragments of the upstream and downstream homologous arms are inserted into the original expression plasmid pRE112 (containing the chloramphenicol resistance gene) using the Gibson method. The sequence of the inserted plasmid is SEQ ID NO: 8, arranged in the following order: upstream homologous arm (nucleotides 1-1000) and downstream homologous arm (nucleotides 1001-2001).

[0298] The pRE112 suicide plasmid expressing the homologous recombination template was transformed into recombinant bacteria Halomonas bluephagenesis TD1.0 and Halomonas bluephagenesis TD1.0△phaCB via E. coli S17-1 conjugation. In the first round, transformants integrated into the genome were selected by chloramphenicol resistance screening. In the second round, the pHelp plasmid with a homing endonuclease expression module was transformed into E. coli S17-1 conjugation. The plasmids were screened on chloramphenicol and antibiotic-free plates, respectively. Single clones that did not grow on chloramphenicol-added plates but grew on antibiotic-free plates were further identified.

[0299] Gene-edited mutant strains were screened using primers designed for colony PCR and confirmed by gene sequencing. Colony PCR is a routine procedure.

[0300] Finally, through colony PCR and gene sequencing, it was confirmed that the ppc gene sequence had been knocked out in the genomes of Halomonas bluephagenesis TD1.0 and Halomonas bluephagenesis TD1.0△phaCB. The recombinant strain with the ppc gene knocked out in the genome of Halomonas bluephagenesis TD1.0△phaCB was named TDZH118.

[0301] (3) Shake-flask fermentation was used to verify the acetoin production level of recombinant bacteria TDZH117 and TDZH118 combined with expression plasmid pZH126.

[0302] The specific steps for producing acetoin by shake-flask fermentation of recombinant strains are as follows:

[0303] The recombinant strain was inoculated into 20 mL of LB60 medium and cultured for 12-16 h. Then, it was transferred to a new 20 mL LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture.

[0304] 2.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of acetoin fermentation medium, and a shake-flask experiment was conducted. The shaker temperature was 37℃ and the rotation speed was 200 rpm. After 4 h of culture, 50 mg / L IPTG was added to induce the expression of the acetoin synthesis gene by the mmp1 promoter. After 48 h of culture, the acetoin content was measured. Each experiment was performed in triplicate, and the results were averaged. The results are shown in Table 8.

[0305] Table 8. Content of acetoin produced by recombinant strains supplying enhanced acetoin synthesis precursors.

[0306]

[0307] The results showed that overexpression of the Bacillus-derived gluC gene (encoding glucose invertase) increased the acetoin yield of recombinant strain TDZH117-pZH126 by 33.65%; knockout of the key gene phosphoenolpyruvate carboxylase ppc, which is crucial for the synthesis of oxaloacetate from phosphoenolpyruvate, increased the acetoin yield of recombinant strain TDZH118-pZH126 by 38.7%.

[0308] These results indicate that knocking out the key gene ppc for pyruvate to other byproducts or overexpressing the glucose-related gene gluC can enhance the supply of pyruvate, a precursor to acetoin synthesis, and thus improve acetoin production levels.

[0309] Example 4: Construction of recombinant strains with deleted acetoin degradation pathway

[0310] Acetoin can also serve as an energy source for microorganisms, including carbon and nitrogen. To prevent the degradation of synthesized acetoin within cells, the butA gene in the acetoin degradation pathway was knocked out, resulting in the engineered bacteria TDZH108 and TDZH116. The recombinant bacteria constructed by knocking out the acetoin-consuming gene, the acuC gene (encoding a protease), was named TDZH119.

[0311] Fermentation tests showed that the recombinant bacteria constructed improved the production capacity of acetoin.

[0312] The specific implementation process is as follows:

[0313] (1) Construction of recombinant bacteria TDZH108 and TDZH116

[0314] The plasmid construction method for the homologous recombination editing method used in the suicide plasmid is as follows: DNA fragments of the upstream and downstream homologous arms are inserted into the original expression plasmid pRE112 (containing the chloramphenicol resistance gene) using the Gibson method. The sequence of the inserted plasmid is SEQ ID NO: 9, arranged sequentially as the upstream homologous arm (nucleotides 1-984) and the downstream homologous arm (nucleotides 985-1984).

[0315] The pRE112 suicide plasmid expressing the homologous recombination template was transformed into recombinant bacteria Halomonas bluephagenesis TD1.0 and Halomonas bluephagenesis TD1.0△phaCB via E. coli S17-1 conjugation. In the first round, transformants integrated into the genome were selected by chloramphenicol resistance screening. In the second round, the pHelp plasmid with a homing endonuclease expression module was transformed into E. coli S17-1 conjugation. The plasmids were screened on chloramphenicol and antibiotic-free plates, respectively. Single clones that did not grow on chloramphenicol-added plates but grew on antibiotic-free plates were further identified.

[0316] Gene-edited mutant strains were screened using primers designed for colony PCR and confirmed by gene sequencing. Colony PCR is a routine procedure.

[0317] Finally, through colony PCR and gene sequencing, it was confirmed that the butA gene sequence had been knocked out in the genomes of Halomonas bluephagenesis TD1.0 and Halomonas bluephagenesis TD1.0△phaCB, and the recombinant bacteria constructed were named TDZH108 and TDZH116.

[0318] (2) Construction of recombinant strain TDZH119

[0319] The plasmid construction method for the homologous recombination editing method used in the suicide plasmid is as follows: DNA fragments of the upstream and downstream homologous arms are inserted into the original expression plasmid pRE112 (containing the chloramphenicol resistance gene) using the Gibson method. The sequence of the inserted plasmid is SEQ ID NO: 29, arranged in the following order: upstream homologous arm (nucleotides 1-500) and downstream homologous arm (nucleotides 501-1000).

[0320] The pRE112 suicide plasmid expressing the homologous recombination template was transformed into the recombinant strain Halomonas bluephagenesis TD1.0△phaCB via E. coli S17-1 conjugation. In the first round, transformants integrated into the genome were selected by chloramphenicol resistance screening. In the second round, the plasmid containing the homing endonuclease expression module was transformed into the pHelp plasmid via E. coli S17-1 conjugation. The plasmids were screened on chloramphenicol and antibiotic-free plates, respectively. Single clones that did not grow on chloramphenicol-added plates but grew on antibiotic-free plates were further identified.

[0321] Gene-edited mutant strains were screened using primers designed for colony PCR and confirmed by gene sequencing. Colony PCR is a routine procedure.

[0322] Finally, after colony PCR and gene sequencing, it was confirmed that the acuC gene sequence had been knocked out in the genome of Halomonas bluephagenesis TD1.0△phaCB, and the recombinant bacteria constructed was named TDZH119.

[0323] (3) Shake-flask fermentation was used to verify the acetoin production level of recombinant bacteria TDZH108, TDZH116, and TDZH119 conjugated to expression plasmid pZH126.

[0324] The specific steps for producing acetoin by shake-flask fermentation of recombinant strains are as follows:

[0325] The recombinant strain was inoculated into 20 mL of LB60 medium and cultured for 12-16 h. Then, it was transferred to a new 20 mL LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture.

[0326] 2.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of acetoin fermentation medium, and a shake-flask experiment was conducted. The shaker temperature was 37℃ and the rotation speed was 200 rpm. After 4 hours of incubation, 50 mg / L of IPTG inducer was added. After 48 hours of incubation, the acetoin content was measured. The experiment was performed in triplicate, and the results were averaged.

[0327] Table 9 shows the content of acetoin produced by plasmid expression in recombinant bacteria TDZH108, TDZH116, and TDZH119.

[0328]

[0329] The results showed that the recombinant strain TDZH108-pZH126 produced 3.23 g / L of acetoin, a 75.54% increase compared to Halomonas bluephagenesis TD1.0-pZH126. The recombinant strain TDZH116-pZH126 produced 7.72 g / L of acetoin, an 85.58% increase compared to Halomonas bluephagenesis TD1.0△phaCB-pZH126. The recombinant strain TDZH119-pZH126 produced 6.12 g / L of acetoin, a 47.12% increase compared to Halomonas bluephagenesis TD1.0△phaCB-pZH126. This example demonstrates that by knocking out the butA and acuC genes, blocking the acetoin degradation pathway, the acetoin production level can be improved.

[0330] Example 5: Production of acetoin using Halomonas under low-salt conditions

[0331] Because the cultivation of halophilic bacteria requires the use of high-concentration sodium chloride (60 g / L NaCl), the treatment of high-salt wastewater is very complicated, increasing the production cost of the product. This application proposes that acetoin can be produced using halophilic bacteria that are tolerant to low salt, thereby reducing production costs. The basal strain Halomonas bluephagenesis TD1.0△phaCB used has had the key gene phaCB for PHA synthesis knocked out, resulting in almost no PHA formation in the cultured cells. Since PHA and other affinity substances can enhance the tolerance of halophilic bacteria to high salt, the absence of PHA allows for the production of acetoin under low-salt conditions.

[0332] a. Shake-flask fermentation to verify the acetoin production level of recombinant strain Halomonas bluephagenesis TD1.0ΔphaCB bound to the high-expression plasmid pZH126 under low-salt conditions.

[0333] The specific steps for producing acetoin by shake-flask fermentation of recombinant strains are as follows:

[0334] The recombinant strain was inoculated into 20 mL of LB60 medium and cultured for 12-16 h. Then, it was transferred to a new 20 mL LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture.

[0335] 2.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of acetoin fermentation medium. Four NaCl concentrations were set at 50 g / L, 40 g / L, 30 g / L, and 20 g / L for shake-flask experiments. The shaker temperature was 37℃ and the rotation speed was 200 rpm. 50 mg / L of IPTG inducer was added after 4 h of culture. After 48 h of culture, the acetoin content was measured. Each experiment was performed in triplicate, and the results were averaged. The results are shown in Table 10.

[0336] Table 10. Content of acetoin produced by Halomonas under low-salt conditions.

[0337]

[0338] The results showed that the yields of CDW, acetoin, and 2,3-BDO of the recombinant strain Halomonas bluephagenesis TD1.0△phaCB varied under different salt concentrations. Comparing the acetoin yields at NaCl concentrations of 20 g / L and 50 g / L, the acetoin yield increased by 78.61% when the salt concentration in the culture medium was reduced.

[0339] These results indicate that the recombinant bacterium Halomonas bluephagenesis TD1.0△phaCB, due to its loss of the ability to synthesize PHA and the lack of intracellular affinity substances, is more suitable for growth in a lower salt concentration environment. This also promotes changes in the intracellular environment, improves the conditions for acetoin synthesis, and these factors can increase the level of acetoin production.

[0340] b. Shake-flask fermentation to verify the acetoin production level of recombinant strains TDZH108 and TDZH116 conjugated with the high-expression plasmid pZH126 under low-salt conditions.

[0341] The specific steps for producing acetoin by shake-flask fermentation of recombinant strains are as follows:

[0342] The recombinant strain was inoculated into 20 mL of LB60 medium and cultured for 12-16 h. Then, it was transferred to a new 20 mL LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture.

[0343] 2.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of acetoin fermentation medium. A shake-flask experiment was conducted with a NaCl concentration of 20 g / L. The shaker temperature was 37℃ and the rotation speed was 200 rpm. After 4 hours of cultivation, 50 mg / L of IPTG inducer was added. After 48 hours of cultivation, the acetoin content was measured. Each experiment was performed in triplicate, and the results were averaged. The results are shown in Table 11 below.

[0344] Table 11. Content of acetoin produced by recombinant bacteria TDZH108 and TDZH116 under low-salt conditions.

[0345]

[0346] The results showed that when recombinant bacteria TDZH108 and TDZH116 conjugated with the expression plasmid pZH126, the acetoin production of both bacteria changed in a medium with a salt concentration of 20 g / L. Comparing the acetoin production at NaCl concentrations of 20 g / L and 50 g / L, when the salt concentration in the medium was reduced, the acetoin production of recombinant bacteria TDZH108 decreased by 4.95%, while the acetoin production of recombinant bacteria TDZH116 increased by 30.18%.

[0347] These results indicate that the recombinant strain TDZH108 retains its halophilic characteristics and thrives in high-salt environments. Lowering the salt concentration is detrimental to acetoin production, leading to a decrease in yield. Conversely, the recombinant strain TDZH116, lacking the ability to synthesize PHA and possessing a deficiency in intracellular affinity substances, is unable to withstand higher salt stress. This makes it more suitable for growth in lower-salt environments, promoting changes in the intracellular environment, improving acetoin synthesis conditions, and enhancing the activity of acetoin synthase. These factors contribute to the increased acetoin production levels of the strain.

[0348] Example 6: Construction of recombinant strains overexpressing the acetoin synthesis gene cluster

[0349] Based on the strain derived from *Halomonas bluephagenesis* TD1.0, acetoin synthesis gene clusters were overexpressed in two ways. Specifically, the alsSD gene cluster driven by the inducible promoter mmp1 and the constitutive strong promoter porin were integrated into the G4 site of the *Halomonas bluephagenesis* TD1.0 genome, respectively, to construct recombinant strains named TDZH125 and TDZH141. The alsSD gene cluster driven by the inducible promoter mmp1 and the constitutive strong promoter porin were integrated into the G4 site of the *Halomonas bluephagenesis* TD1.0 ΔphaCB genome, to construct recombinant strains named TDZH133 and TDZH142. Based on recombinant strain TDZH125, a portion of the sequence between the coding sequences of the alsS and alsD genes was replaced with different constitutive promoters P. porin68 P porin194 P porin203By designing artificial ribosome binding site (RBS) sequences and screening for appropriate alsD gene expression levels while fixing alsS gene expression, the constructed strains were named TDZH126, TDZH127, and TDZH128, respectively.

[0350] Based on the recombinant strain TDZH116, acetoin synthesis gene clusters were overexpressed in two ways. Specifically, the alsSD gene cluster driven by the inducible promoter mmp1 and the constitutive strong promoter porin were integrated into the G4 site of the TDZH116 genome, respectively, to construct recombinant strains named TDZH244 and TDZH245. Based on the recombinant strain TDZH245, the constitutive strong promoter P was integrated into the G43 site of its genome. porin The alsSD gene cluster was used to construct a recombinant bacterium named TDZH285.

[0351] The constructed recombinant strains TDZH125, TDZH126, TDZH127, TDZH128, TDZH133, TDZH239, TDZH244, TDZH245, and TDZH285, after fermentation testing, improved acetoin production capacity.

[0352] The specific implementation process is as follows:

[0353] (1) Construction of recombinant bacteria TDZH125, TDZH133, TDZH239, and TDZH244

[0354] The genome was edited using the CRISPR genome editing method. Specifically, the plasmid containing sgRNA and a recombination template was constructed by connecting upstream and downstream homologous arms, the sgRNA expression module, and P... mmp1 The promoter, alsD, alsS genes and other DNA fragments were inserted into the original expression plasmid pSEVA241 (containing kanamycin and spectinomycin resistance genes) using the Gibson method.

[0355] The sequence of the inserted plasmid is SEQ ID NO: 10, arranged in the following order: sgRNA expression module (nucleotides 1-152), upstream homologous arm (nucleotides 153-1152), P mmp1 Promoter (nucleotides 1153-1344), alsSD gene cluster (nucleotides 1345-3881), downstream homologous arm (nucleotides 3882-4881).

[0356] The pSEVA241 plasmid expressing sgRNA and recombinant template and the pQ08 plasmid expressing Cas9 were transformed into recombinant bacteria Halomonas bluephagenesis TD1.0, Halomonasbluephagenesis TD1.0△phaCB, TDZH108 and TDZH116 via Escherichia coli S17-1 conjugation.

[0357] Colony PCR was used to design primers for screening mutant strains with gene insertions, and the results were confirmed by gene sequencing. Colony PCR is a routine procedure.

[0358] Finally, colony PCR and gene sequencing confirmed that the P nucleotide at positions 1153-3881 of SEQ ID NO: 10 was inserted into the G4 site of the recombinant bacteria Halomonas bluephagenesis TD1.0, Halomonas bluephagenesis TD1.0△phaCB, TDZH108, and TDZH116. mmp1 The promoter and alsSD gene cluster sequences were determined. The recombinant bacteria constructed were named TDZH125, TDZH133, TDZH239, and TDZH244. Then, by continuously and repeatedly passaged the successfully genome-edited strains in liquid medium and streaking them onto spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates, strains with CRISPR plasmid loss were identified, facilitating the next round of genome editing.

[0359] (2) Construction of recombinant bacteria TDZH126, TDZH127, and TDZH128

[0360] The genome was edited using the CRISPR genome editing method. Specifically, the plasmid containing sgRNA and recombination template was constructed by inserting DNA fragments, including upstream and downstream homologous arms, sgRNA expression module, alsS gene module driven by the porin promoter, and alsD gene module driven by the mmp1 promoter, into the original expression plasmid pSEVA241 (containing kanamycin and spectinomycin resistance genes) using the Gibson method.

[0361] The sequence of the inserted plasmid is SEQ ID NO: 11, arranged in the following order: sgRNA expression module (nucleotides 1-152), upstream homologous arm (nucleotides 153-1152), mmp1 promoter and artificially designed RBS (nucleotides 1153-1344), alsD gene (nucleotides 1345-2112), P porin The promoter and artificially designed RBS (nucleotides 2113-2390, where nucleotides 2325-2327 are replaced with GAC) is Pporin68 The promoter of the alsS gene (nucleotides 2391-4116) and its downstream homologous arm (nucleotides 4117-5116) are shown. P is the gene whose nucleotides 2325-2327 are replaced with GTA and whose nucleotides 2329-2332 are replaced with CTAA. porin194 Promoter. The promoter is P, where nucleotides 2325-2327 of SEQ ID NO: 11 are replaced with GTA and nucleotides 2329-2332 are replaced with CCCT. porin203 Promoter.

[0362] The pSEVA241 plasmid expressing sgRNA and recombinant template and the pQ08 plasmid expressing Cas9 were transformed into the recombinant bacterium Halomonas bluephagenesis TD1.0 via Escherichia coli S17-1 conjugation.

[0363] Colony PCR was used to design primers for screening mutant strains with gene insertions, and the results were confirmed by gene sequencing. Colony PCR is a routine procedure.

[0364] Finally, colony PCR and gene sequencing confirmed that the recombinant strain *Halomonas bluephagenesis* TD1.0 had inserted the *mmp1-alsD-porin-alsS* gene sequence (nucleotides 1153-4116 of SEQ ID NO: 11) into the G4 site. The recombinant strain with nucleotides 2325-2327 containing GAC was named TDZH126, and the recombinant strains with nucleotides 2329-2332 containing CTAA and CCCT were named TDZH127 and TDZH128, respectively. Then, by continuously and repeatedly passaged the successfully genome-edited strains in liquid culture medium and streaking them on spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates, strains with CRISPR plasmid loss were identified, facilitating the next round of genome editing.

[0365] (3) Construction of recombinant strain TDZH245

[0366] The genome was edited using the CRISPR genome editing method. Specifically, the plasmid containing sgRNA and recombinant template was constructed by inserting DNA fragments such as upstream and downstream homologous arms, sgRNA expression module, and alsDS gene module driven by the porin promoter into the original expression plasmid pSEVA241 (containing kanamycin and spectinomycin resistance genes) using the Gibson method.

[0367] The sequence of the inserted plasmid is SEQ ID NO: 12, arranged as follows: sgRNA expression module (nucleotides 1-152), upstream homologous arm (nucleotides 153-1152), P porin Promoter and artificially designed RBS (nucleotides 1153-1425), alsDS gene cluster (nucleotides 1426-3949), downstream homologous arm (nucleotides 3950-4962).

[0368] The pSEVA241 plasmid expressing sgRNA and recombinant template and the pQ08 plasmid expressing Cas9 were transformed into recombinant bacteria TDZH116 via E. coli S17-1 conjugation.

[0369] Colony PCR was used to design primers for screening mutant strains with gene insertions, and the results were confirmed by gene sequencing. Colony PCR is a routine procedure.

[0370] Finally, colony PCR and gene sequencing confirmed that the P nucleotide at positions 1153-4962 of SEQ ID NO: 12 was inserted into the G4 site of the recombinant strain TDZH116 genome. porin -alsDS gene sequence. The constructed recombinant bacteria were named TDZH245. Then, by continuously and repeatedly passaged the genome-edited strains in liquid culture medium and streaking them on spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates, strains with CRISPR plasmid loss were identified, facilitating the next round of genome editing.

[0371] (4) Construction of recombinant strain TDZH285

[0372] The genome was edited using the CRISPR genome editing method. Specifically, the plasmid containing sgRNA and a recombination template was constructed by connecting upstream and downstream homologous arms, the sgRNA expression module, and P... porin DNA fragments, including the promoter-driven alsDS gene module, were inserted into the original expression plasmid pSEVA241 (containing kanamycin and spectinomycin resistance genes) using the Gibson method.

[0373] The sequence of the inserted plasmid is SEQ ID NO: 13, arranged in the following order: sgRNA expression module (nucleotides 1-152), upstream homologous arm (nucleotides 153-1152), P porin Promoter and artificially designed RBS (nucleotides 1153-1613), alsDS gene cluster (nucleotides 1614-4300), downstream homologous arm (nucleotides 4301-5300).

[0374] The pSEVA241 plasmid expressing sgRNA and recombinant template and the pQ08 plasmid expressing Cas9 were transformed into recombinant bacteria TDZH245 via E. coli S17-1 conjugation.

[0375] Colony PCR was used to design primers for screening mutant strains with gene insertions, and the results were confirmed by gene sequencing. Colony PCR is a routine procedure.

[0376] Finally, colony PCR and gene sequencing confirmed that the P nucleotide at positions 1153-4300 of SEQ ID NO: 13 was inserted into the G43 site of the recombinant strain TDZH245 genome. porin The -alsDS gene sequence was used. The recombinant strain was named TDZH285. Then, by continuously and repeatedly passaged the genome-edited strain in liquid medium and streaking it on spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates, strains with CRISPR plasmid loss were identified, facilitating the next round of genome editing.

[0377] (5) Shake-flask fermentation was used to verify the acetoin production levels of the inducible recombinant strains TDZH125, TDZH126, TDZH127, TDZH128, TDZH133, TDZH239, and TDZH244.

[0378] The specific steps for producing acetoin by shake-flask fermentation of recombinant strains are as follows:

[0379] The recombinant strain was inoculated into 20 mL of LB60 medium and cultured for 12-16 h. Then, it was transferred to a new 20 mL LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture.

[0380] 2.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of acetoin fermentation medium, and a shake-flask experiment was conducted. The shaker temperature was 37℃ and the rotation speed was 200 rpm. After 4 h of culture, 200 mg / L of IPTG inducer was added. After 48 h of culture, the acetoin content was measured. Each experiment was performed in triplicate, and the results were averaged. The results are shown in Table 12.

[0381] Table 12. Acetaminophen content produced by recombinant strains after overexpression of the acetocin synthesis gene cluster.

[0382] TDZH125 200 3.12 TDZH126 200 2.16 TDZH127 200 1.60 [[ID=*15]]TDZH128 200 1.35 TDZH133 200 3.52 TDZH239 200 3.27 TDZH244 200 4.15

[0383] The results showed that P was integrated into the Halomonas bluephagenesis TD1.0 genome. mmp1The recombinant strain TDZH125 of -alsS achieved an acetoin yield of 3.12 g / L. Based on the recombinant strain TDZH125, the promoter of the alsS gene was replaced with the constitutive promoter P. porin68 P porin194 P porin203 The acetoin production of recombinant strains TDZH126, TDZH127, and TDZH128 decreased with decreasing promoter strength of the alsS gene, reaching 2.16 g / L, 1.60 g / L, and 1.35 g / L, respectively, representing reductions of 30.77%, 48.72%, and 56.73% compared to recombinant strain TDZH125. P was integrated into the genome of Halomonasbluephagenesis TD1.0ΔphaCB. mmp1 The recombinant strain TDZH133 of -alsDS achieved an acetoin yield of 3.52 g / L, a 12.82% increase compared to the recombinant strain TDZH125. P was integrated into the TDZH108 genome. mmp1 The recombinant strain TDZH239 of -alsDS achieved an acetoin yield of 3.27 g / L, which was 4.81% higher than that of the recombinant strain TDZH125. P was integrated into the TDZH116 genome. mmp1 The recombinant strain TDZH244 of -alsDS achieved an acetoin yield of 4.15 g / L, which was 33.01% higher than that of the recombinant strain TDZH125.

[0384] These results demonstrate that by integrating P into the genome... mmp1 The recombinant bacteria obtained by the -alsDS expression module can stably produce acetoin, but the alsS gene is replaced with P. porin After the promoter, acetoin production increases with P porin The promoter strength decreased, indicating that the constitutive promoter is weaker than the inductive promoter P. mmp1 Integrating 1-2 copies of the alsDS gene cluster into the genome requires replacement with the strongest P. porin Additionally, strains that knock out phaCB and butA showed improved genome integration performance. mmp1 The -alsDS expression module increases acetoin production.

[0385] (6) Shake-flask fermentation was used to verify the acetoin production level of the constitutive recombinant strains TDZH245 and TDZH285.

[0386] The specific steps for producing acetoin by shake-flask fermentation of recombinant strains are as follows:

[0387] The recombinant strain was inoculated into 20 mL of LB60 medium and cultured for 12-16 h. Then, it was transferred to a new 20 mL LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture.

[0388] 2.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of acetoin fermentation medium, and a shake-flask experiment was conducted. The shaker temperature was 37℃ and the rotation speed was 200 rpm. After 48 h of incubation, the acetoin content was measured. Each experiment was performed in triplicate, and the results were averaged. The results are shown in Table 13.

[0389] Table 13. Acetaminophen content produced by recombinant strains after overexpression of the acetocin synthesis gene cluster.

[0390] Halomonas bluephagenesis TD01 0 TDZH245 4.04 TDZH285 10.69

[0391] The results showed that one copy of P was integrated into the genome of TDZH116. porin The recombinant bacterium TDZH245, constructed using the -alsDS expression module, synthesized 4.04 g / L acetoin. Based on the recombinant bacterium TDZH245, two copies of P were integrated... porin After using the -alsDS expression module, the acetoin yield of recombinant strain TDZH285 increased by 164.60%.

[0392] These results demonstrate that increasing the copy number of the acetoin synthesis gene cluster in Halomonas can improve acetoin production levels.

[0393] (7) Shake-flask fermentation was used to verify the acetoin production level of constitutive recombinant bacteria TDZH245 and TDZH285 conjugated to expression plasmid pZH126, in order to guide subsequent genome integration.

[0394] The specific steps for producing acetoin by shake-flask fermentation of recombinant strains are as follows:

[0395] The recombinant strain was inoculated into 20 mL of LB60 medium and cultured for 12-16 h. Then, it was transferred to a new 20 mL LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture.

[0396] 2.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of acetoin fermentation medium, and a shake-flask experiment was conducted. The shaker temperature was 37℃ and the rotation speed was 200 rpm. After 48 h of incubation, the acetoin content was measured. Each experiment was performed in triplicate, and the results were averaged. The results are shown in Table 14.

[0397] Table 14. Acetaminophen content produced by recombinant strains after overexpression of the acetocin synthesis gene cluster.

[0398] Halomonas bluephagenesis TD01 0 0 TDZH245 0 4.04 TDZH285 0 10.69 TDZH245-pZH126 0 4.16 TDZH245-pZH126 6.25 10.70 TDZH245-pZH126 12.5 11.55 25 11.88 TDZH245-pZH126 50 13.12 TDZH245-pZH126 100 11.69 TDZH245-pZH126 0 10.17 TDZH285-pZH126 6.25 13.12 TDZH285-pZH126 12.5 11.28 TDZH285-pZH126 25 10.80 TDZH285-pZH126 50 10.30 TDZH285-pZH126 100 9.73

[0399] The results showed that one copy of P was integrated into the genome of TDZH116. porin The recombinant strain TDZH245, constructed using the -alsDS expression module, was fermented with the high-copy expression plasmid pZH126. When the IPTG inducer concentrations were 0, 6.25, 12.5, 25, 50, and 100 mg / L, the acetoin yields reached 4.16 g / L, 10.70 g / L, 11.55 g / L, 11.88 g / L, 13.12 g / L, and 11.69 g / L, respectively. The highest yield was achieved at an inducer concentration of 50 mg / L, representing a 224.75% increase compared to the recombinant strain TDZH245. The integration of two copies of P... porin The recombinant strain TDZH285, constructed using the -alsDS expression module, was conjugated with the high-copy expression plasmid pZH126 for fermentation experiments. When the IPTG inducer concentrations were 0, 6.25, 12.5, 25, 50, and 100 mg / L, the acetoin yields reached 10.17 g / L, 13.12 g / L, 11.28 g / L, 10.80 g / L, 10.30 g / L, and 9.73 g / L, respectively. The highest yield was achieved at an inducer concentration of 6.25 mg / L, representing a 22.73% increase compared to the recombinant strain TDZH285.

[0400] Recombinant bacteria TDZH245 and TDZH285, carrying the same plasmid, were induced to express the acetoin gene cluster. When the induction doses were 50 mg / L and 6.25 mg / L, respectively, the acetoin yield was 13.12 g / L, which was also the maximum yield. These results indicate that in addition to increasing the copy number of the acetoin synthesis gene cluster, fine regulation of the expression levels of alsD and alsS genes can improve the acetoin production level.

[0401] Example 7: Construction of a recombinant strain with finely regulated expression of the acetoin synthesis gene cluster

[0402] Based on strain TDZH285, constitutive promoters optimally expressing the acetoin synthesis genes alsS and alsD were screened and integrated into the G51 and GY2 sites of the TDZH285 genome. Specifically, TDZH285 was conjugated with the microregulatory plasmid pZH342 (sequence SEQ ID NO: 3) derived from the low-copy plasmid pSEVA321, and the IPTG-inducible promoter P... mmp1 Inducible expression of acetolactate decarboxylase gene alsD, AHL inducible promoter P plux To induce expression of the acetolactate synthase gene alsS, the regulatory strengths of two inducible promoters were identified for optimal yield, while simultaneously, a constitutive promoter P was selected from a set of promoter strengths ranging from low to high. porinDriven by GFP expression plasmid pSEVA321-P porinX -GFP and mCherry expression plasmid pSEVA321-P porinY -mCherry, with the inductive promoter P mmp1 -GFP-P plux In the -mCherry expression plasmid, constitutive promoters P with corresponding strengths were screened out. porinX P porinY P driven by promoters with different configurations porinX -alsD-P porinY The -alsS gene sequence was integrated into the G51 site of TDZH285. The recombinant strain was named TDZH307. Based on the recombinant strain TDZH307, a constitutive promoter-driven P... porinX -alsD-P porinY The -alsS gene fragment was integrated into the GY2 site of the TDZH307 genome, and the constructed strain was named TDZH308.

[0403] The constructed recombinant strains TDZH307 and TDZH308, after fermentation testing, improved the acetoin production capacity.

[0404] The specific implementation process is as follows:

[0405] (1) Constructing characterization plasmids for screening promoters

[0406] Construction method of constitutive expression plasmid: From the porin promoter library in the Halomonas bluephagenesis TD01 genetic toolbox, a set of porin promoters from low to high concentrations were selected to construct a constitutive GFP characterization plasmid. Specifically, plasmids containing different P... porin Promoter: P porin197 P porin259 P porin194 P porin70 P porin60 P porin58 P porin185 P porin138 P porin141 P porin3 DNA fragments such as the GFP gene or mCherry gene are inserted into the original expression plasmid pSEVA321 (containing the carbamomycin resistance gene) using the Gibson method.

[0407] pSEVA321-P porinX The sequence of the constitutive expression plasmid for -GFP is SEQ ID NO: 14, which consists of the porin promoter, the artificially designed RBS (nucleotides 1-176), and the GFP gene (nucleotides 177-893).

[0408] pSEVA321-P porinY The sequence of the -mCherry constitutive expression plasmid is SEQ ID NO: 15, which consists of the porin promoter, the artificially designed RBS (nucleotides 1-176), and the mCherry gene (nucleotides 177-887).

[0409] Among them, the plasmid with nucleotides CACC at positions 53-56 is P. porin197 The promoter, in the constructed plasmid, has nucleotides 53-56 as TAGC, which is P. porin259 The promoter, in the constructed plasmid, has nucleotides 53-56 as CTAA, which is P. porin194 The promoter, in the constructed plasmid, has nucleotides CAC at positions 49-51, which is P. porin70 The promoter, in the constructed plasmid, has nucleotides CAT at positions 49-51, which is P. porin60 The promoter, in the constructed plasmid, has nucleotides 49-51 of ATA, which is P. porin58 The promoter, in the constructed plasmid, has nucleotides 53-56 as ACTC, which is P. porin185 The promoter, in the constructed plasmid, has nucleotides 53-56 as AGAC, which is P. porin138 The promoter, in the constructed plasmid, has nucleotides 53-56 of ATAC, which is P. porin141 The promoter, in the constructed plasmid, nucleotides 49-51 of TGA are P. porin3 Promoter.

[0410] Inducible expression plasmid pSEVA321-P mmp1 -sfGFP-P plux The sequence of -mCherry is SEQ ID NO: 16, arranged as P. mmp1 Promoter and artificially designed RBS1 (nucleotides 1-185), sfGFP gene (nucleotides 186-902), terminator (nucleotides 903-1057), luxR gene and its promoter (nucleotides 1058-1893), insulator (nucleotides 1894-1942), artificially designed RBS2 and P plux Promoter (nucleotides 1943-2040). mCherry gene (nucleotides 2041-2748), the constructed plasmid was named pZH316.

[0411] Colony PCR was used to design primers, screen for the correct expression plasmids, and confirm their selection via gene sequencing. Colony PCR is a routine procedure.

[0412] The three plasmids pSEVA321-porinX-GFP, pSEVA321-porinX-mCherry, and pZH316 were transformed into recombinant bacteria TDZH285 via E. coli S17-1 conjugation, and the corresponding promoter strength data were obtained by shake-flask experiments.

[0413] (2) Data on acetoin production obtained from shake-flask fermentation experiment of recombinant strain TDZH285-pZH342.

[0414] Based on the recombinant strain TDZH285, shake-flask fermentation was used to screen for constitutive promoters optimally expressing the acetoin synthesis genes alsS and alsD. The low-copy microregulatory plasmid pZH342 was introduced into the recombinant strain TDZH285, and shake-flask data were used to screen for finely regulated promoters.

[0415] a. Immobilize alsD expression in recombinant strain TDZH285-pZH342 and screen for suitable alsS induction strength.

[0416] The specific steps for producing acetoin by shake-flask fermentation of recombinant strains are as follows:

[0417] The recombinant strain was inoculated into 20 mL of LB60 medium and cultured for 12-16 h. Then, it was transferred to a new 20 mL LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture.

[0418] 2.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of acetoin fermentation medium for a shake-flask experiment. The shaker temperature was 37℃ and the rotation speed was 200 rpm. After 4 hours of incubation, 2 x 10⁻⁶ mg / L of the culture medium was added to all shake flasks. -2 g / L of IPTG as an inducer, while 10 g / L was added to each of the 5 shake flasks. -8 10 -7 10 -6 10 -5 10 -4 g / L of inducer AHL. After 48 h of culture, the acetoin content was measured. Each experiment was conducted in triplicate, and the results were averaged. The results are shown in Table 15.

[0419] Table 15 shows the acetoin content produced by recombinant strains after expressing the acetoin synthesis gene cluster.

[0420] TDZH285-pZH126 <![CDATA[2×10 -2 ]]> <![CDATA[10 -8 ]]> 11.70 TDZH285-pZH342 <![CDATA[2×10 -2 ]]> <![CDATA[10 -7 ]]> 13.08 TDZH285-pZH342 <![CDATA[2×10 -2 ]]> <![CDATA[10 -6 ]]> 15.27 TDZH285-pZH342 <![CDATA[2×10 -2 ]]> <![CDATA[10 -5 ]]> 15.75 TDZH285-pZH342 <![CDATA[2×10 -2 ]]> <![CDATA[10 -4 ]]> 13.53

[0421] The results showed that when recombinant strain TDZH285 was conjugated with microregulatory plasmid pZH342, the acetoin yields after adding different concentrations of inducer AHL were 11.70, 13.08, 15.27, 15.75, and 13.53 g / L, respectively. The optimal concentration of inducer AHL was 10 g / L.-5 At a concentration of g / L, acetoin production reaches its maximum value.

[0422] The results indicate that by immobilizing alsD expression in plasmids, the optimal alsS induction intensity is 10. -5 Using g / L of inducer AHL, and by fixing the expression of alsS, suitable alsD expression induction intensity can be screened.

[0423] b. The recombinant strain TDZH285-pZH342 uses a dual inducer to regulate the alsDS gene cluster, and screens for suitable induction strength.

[0424] The specific steps for producing acetoin by shake-flask fermentation of recombinant strains are as follows:

[0425] The recombinant strain was inoculated into 20 mL of LB60 medium and cultured for 12-16 h. Then, it was transferred to a new 20 mL LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture.

[0426] 2.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of acetoin fermentation medium for a shake-flask experiment. The shaker temperature was 37 °C and the rotation speed was 200 rpm. After 4 hours of incubation, 2 × 10⁻⁶ mg / L of the culture medium was added to all shake flasks. -3 2×10 -2 g / L, 2×10 -1 Inducer IPTG, 10 -7 10 -6 10 -5 g / L of inducer AHL. After 48 h of culture, the acetoin content was measured. Each experiment was conducted in triplicate, and the results were averaged. The results are shown in Table 16.

[0427] Table 16. Acetoin content produced by recombinant strains after micro-regulation of acetoin synthesis gene cluster expression.

[0428] TDZH285-pZH342 0 0 9.71 TDZH285 0 0 8.39 TDZH285-pZH342 <![CDATA[2×10 -3 ]]> <![CDATA[10 -7 ]]> 8.62 TDZH285-pZH342 <![CDATA[2×10 -3 ]]> <![CDATA[10 -6 ]]> 9.60 TDZH285-pZH342 <![CDATA[2×10 -3 ]]> <![CDATA[10 -5 ]]> 11.93 TDZH285-pZH342 <![CDATA[2×10 -2 ]]> <![CDATA[10 -7 ]]> 11.49 TDZH285-pZH342 <![CDATA[2×10 -2 ]]> <![CDATA[10 -6 ]]> 13.12 TDZH285-pZH342 <![CDATA[2×10 -2 ]]> <![CDATA[10 -5 ]]> 15.93 TDZH285-pZH342 <![CDATA[2×10 -1 ]]> <![CDATA[10 -7 ]]> 11.35 TDZH285-pZH342 <![CDATA[2×10 -1 ]]> <![CDATA[10 -6 ]]> 12.86 TDZH285-pZH342 <![CDATA[2×10 -1 ]]> <![CDATA[10 -5 ]]> 11.4

[0429] The results showed that the recombinant strain TDZH285, when conjugated with the microregulatory plasmid pZH342, exhibited better performance when 2×10⁻⁶ plasmids were added. -2 g / L inducer IPTG and 10 g / L -5 After adding g / L of inducer AHL, the maximum yield of acetoin reached 15.93 g / L, which was 89.86% higher than the control without inducer.

[0430] The results indicate that fine-tuning the expression of acetoin synthase in plasmids can improve acetoin production. To obtain a stable expression strain, an induction dose of 2 × 10⁻⁶ was selected. -2 g / L IPTG and 10 -5P at g / L AHL mmp1 and P plux Increasing promoter strength, replacing it with a constitutive promoter, and then integrating it into the genome of recombinant bacteria TDZH285 can optimize the expression of acetoin synthase.

[0431] (3) Obtaining corresponding data on fluorescence expression intensities of inducible and constitutive promoters through shake-flask fermentation experiments.

[0432] a. Inducible promoters drive fluorescent protein expression intensity

[0433] The specific steps for determining the fluorescence intensity during shake-flask fermentation of recombinant strains are as follows:

[0434] The conjugation strain TDZH285-pZH316 was inoculated into 20 mL of LB60 medium and cultured for 12-16 h. Then, it was transferred to a new 20 mL of LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture.

[0435] 2.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of acetoin fermentation medium for a shake-flask experiment. The shaker temperature was 37 °C and the rotation speed was 200 rpm. After 4 h of culture, different doses of the inducers IPTG and AHL were added according to the protocol. After 48 h of culture, the fluorescence intensity of GFP and mCherry was detected by flow cytometry. Each experiment was performed in triplicate, and the results were averaged. The results are shown in Table 17.

[0436] Table 17 Fluorescence intensity of recombinant strains after expression of GFP and mCherry genes in induced plasmids.

[0437]

[0438]

[0439] The results showed that the recombinant bacterial TDZH285 conjugated with the fluorescent expression plasmid pZH316, when 2×10⁻⁶ g of each was added, resulted in a positive effect. -2 g / L inducer IPTG and 10 g / L -5 After inducing with g / L of AHL, the fluorescence intensity of sfGFP was 22486.67, and the fluorescence intensity of mCherry was 1649.67.

[0440] The results indicate that the recombinant strain TDZH285 requires a significantly higher expression level of acetyllactone decarboxylase alsD than acetyllactone synthase alsS, which can improve acetoin production. To obtain a stable engineered strain, [the following was observed] from P... porinConstituent promoters with fluorescence intensities of approximately 22486.67 for sfGFP and approximately 1649.67 for mCherry were screened from the promoter library and then integrated into the genome of recombinant bacteria TDZH285, which can optimize the expression of acetoin synthase.

[0441] b. Constitutive promoters drive the expression intensity of the fluorescent protein sfGFP.

[0442] The specific steps for determining the fluorescence intensity during shake-flask fermentation of recombinant strains are as follows:

[0443] The conjugated strain TDZH285-pSEVA321-P porinX -GFP was inoculated into 20 mL of LB60 medium and cultured for 12-16 h. Then, it was transferred to a new 20 mL of LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture.

[0444] 2.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of acetoin fermentation medium, and a shake-flask experiment was conducted. The shaker temperature was 37℃ and the rotation speed was 200 rpm. After 48 h of culture, the fluorescence intensity of sfGFP was detected by flow cytometry. Each experiment was performed in triplicate, and the average value was taken. The results are shown in Table 18.

[0445] Table 18 Fluorescence intensity of recombinant strains after expression of GFP gene on constitutive plasmids.

[0446] <![CDATA[TDZH285-pSEVA321-P porin197 -GFP]]> 106.33 <![CDATA[TDZH285-pSEVA321-P porin259 -GFP]]> 616.45 <![CDATA[TDZH285-pSEVA321-P porin194 -GFP]]> 954.00 <![CDATA[TDZH285-pSEVA321-P porin70 -GFP]]> 1822.67 <![CDATA[TDZH285-pSEVA321-P porin60 -GFP]]> 3812.33 <![CDATA[TDZH285-pSEVA321-P porin58 -GFP]]> 6334.67 <![CDATA[TDZH285-pSEVA321-P porin185 -GFP]]> 23014.67 <![CDATA[TDZH285-pSEVA321-P porin141 -GFP]]> 35651.33 <![CDATA[TDZH285-pSEVA321-P porin3 -GFP]]> 53440.67

[0447] The results showed that the recombinant bacterium TDZH285 conjugated with the fluorescent expression plasmid TDZH285-pSEVA321-P porinX -GFP, after shake-flask fermentation, the constitutive promoter of sfGFP with a fluorescence intensity close to 22486.67 is P. porin185 The fluorescence intensity was 23014.67.

[0448] The results indicate that the induced promoter P mmp1 -GFP is added at 2×10 -2 g / L inducer IPTG, GFP fluorescence expression intensity and constitutive promoter P porin185 -GFP fluorescence expression intensities are similar, so P can be selected. porin185 This will serve as the promoter for the next step of integrating the acetolactate decarboxylase alsD gene.

[0449] c. Constitutive promoters drive the expression intensity of the fluorescent protein mCherry.

[0450] The specific steps for determining the fluorescence intensity during shake-flask fermentation of recombinant strains are as follows:

[0451] The conjugated strain TDZH285-pSEVA321-P porinY -mCherry was inoculated into 20 mL of LB60 medium and cultured for 12-16 h. Then, it was transferred to a new 20 mL of LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture.

[0452] 2.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of acetoin fermentation medium, and a shake-flask experiment was conducted. The shaker temperature was 37℃ and the rotation speed was 200 rpm. After 48 h of culture, the mCherry fluorescence intensity was detected by flow cytometry. Each experiment was performed in triplicate, and the average value was taken. The results are shown in Table 19.

[0453] Table 19 Fluorescence intensity of recombinant strains after expression of the mCherry gene on the constitutive plasmid.

[0454]

[0455]

[0456] The results showed that the recombinant bacterium TDZH285 conjugated with the fluorescent expression plasmid TDZH285-pSEVA321-P porinX -mCherry, after shake-flask fermentation, showed a fluorescence intensity close to 1649.67. The constitutive promoter of mCherry is P. porin60 The fluorescence intensity was 1740.00.

[0457] The results indicate that the induced promoter P plux -mCherry adds 10 -5 At a concentration of g / L inducer AHL, the mCherry fluorescence expression intensity was correlated with the constitutive promoter P. porin60 -mCherry has similar fluorescence expression intensity, so P can be selected. porin60 This will serve as the promoter for the next step of integrating the acetolactate synthase alsS gene.

[0458] (4) Construction of recombinant strain TDZH307

[0459] The genome was edited using the CRISPR genome editing method. Specifically, the plasmid containing sgRNA and a recombination template was constructed by connecting upstream and downstream homologous arms, the sgRNA expression module, and P... porin185 Promoter, alsD gene, P porin60 The promoter, alsS gene and other DNA fragments were inserted into the original expression plasmid pSEVA241 (containing kanamycin and spectinomycin resistance genes) using the Gibson method.

[0460] The sequence of the inserted plasmid is SEQ ID NO: 17, arranged in the following order: sgRNA expression module (nucleotides 1-152), upstream homologous arm (nucleotides 153-1152), P porin185 Promoter and artificially synthesized RBS1 (nucleotides 1153-1328), alsD gene (nucleotides 1329-2096), P porin60 The promoter and artificially synthesized RBS sequence 2 (nucleotides 2097-2186), alsS gene (nucleotides 2187-3905), and downstream homologous arm (nucleotides 3906-4885).

[0461] The pSEVA241 plasmid expressing sgRNA and recombinant template and the pQ08 plasmid expressing Cas9 were transformed into recombinant bacteria TDZH285 via E. coli S17-1 conjugation.

[0462] Colony PCR was used to design primers for screening mutant strains with gene insertions, and the results were confirmed by gene sequencing. Colony PCR is a routine procedure.

[0463] Finally, colony PCR and gene sequencing confirmed that the alsSD gene sequence (nucleotides 1153-3905 of SEQ ID NO: 17) was inserted into the G51 site of the recombinant strain TDZH285. The constructed recombinant strain was named TDZH307. Then, by continuously and repeatedly passaged the successfully genome-edited strains in liquid culture medium and streaking them on spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates, strains with CRISPR plasmid loss were identified, facilitating the next round of genome editing.

[0464] (5) Construction of recombinant strain TDZH308

[0465] The genome was edited using the CRISPR genome editing method. Specifically, the plasmid containing sgRNA and a recombination template was constructed by connecting upstream and downstream homologous arms, the sgRNA expression module, and P... porin185 Promoter, alsD gene, P porin60 The promoter, alsS gene and other DNA fragments were inserted into the original expression plasmid pSEVA241 (containing kanamycin and spectinomycin resistance genes) using the Gibson method.

[0466] The sequence of the inserted plasmid is SEQ ID NO: 18, arranged in the following order: sgRNA expression module (nucleotides 1-152), upstream homologous arm (nucleotides 153-652), P porin185 Promoter and artificially synthesized RBS1 (nucleotides 653-828), alsD gene (nucleotides 829-1596), Pporin60 The promoter and artificially synthesized RBS sequence 2 (nucleotides 1597-1686), alsS gene (nucleotides 1687-3405), and downstream homologous arm (nucleotides 3406-3905).

[0467] The pSEVA241 plasmid expressing sgRNA and recombinant template and the pQ08 plasmid expressing Cas9 were transformed into recombinant bacteria TDZH307 via E. coli S17-1 conjugation.

[0468] Colony PCR was used to design primers for screening mutant strains with gene insertions, and the results were confirmed by gene sequencing. Colony PCR is a routine procedure.

[0469] Finally, colony PCR and gene sequencing confirmed that the alsSD gene sequence (nucleotides 653-3405 of SEQ ID NO: 18) was inserted into the GY2 site of the recombinant strain TDZH307. The constructed recombinant strain was named TDZH308. Then, by continuously and repeatedly passaged the successfully genome-edited strains in liquid culture medium and streaking them onto spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates, strains with CRISPR plasmid loss were identified, facilitating the next round of genome editing.

[0470] (6) Shake-flask fermentation was used to verify the acetoin production levels of recombinant strains TDZH245, TDZH285, TDZH307, and TDZH308.

[0471] The specific steps for producing acetoin by shake-flask fermentation of recombinant strains are as follows:

[0472] The recombinant strain was inoculated into 20 mL of LB60 medium and cultured for 12-16 h. Then, it was transferred to a new 20 mL LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture.

[0473] 2.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of acetoin fermentation medium. Different concentrations of glucose were added for shake-flask experiments. The shaker temperature was 37℃ and the rotation speed was 200 rpm. After 48 h of incubation, the acetoin content was measured. Each experiment was performed in triplicate, and the average result was taken. The results are shown in Table 20.

[0474] Table 20. Acetaminophen content produced by recombinant strains after overexpression of the acetocin synthesis gene cluster.

[0475]

[0476] The results showed that recombinant strains TDZH245 and TDZH285 synthesized 4.64 g / L and 10.30 g / L acetoin, respectively, in a medium containing 40 g / L glucose. Based on recombinant strain TDZH285, after integrating a third copy of alsDS into the genome, the constructed recombinant strain TDZH307 achieved an acetoin yield of 14.37 g / L in a medium containing 50 g / L glucose. Based on recombinant strain TDZH285, after inserting a fourth copy of alsSD, the constructed recombinant strain TDZH308 achieved an acetoin yield of 18.66 g / L in a medium containing 60 g / L glucose. The recombinant strains TDZH285, TDZH307, and TDZH308 increased the acetoin yield by 121.98%, 209.70%, and 302.16%, respectively, compared to TDZH245.

[0477] These results demonstrate that increasing the copy number of the acetoin synthesis gene cluster in the genome and optimizing the expression levels of the alsS and alsD genes can improve acetoin production.

[0478] Example 8: Construction of recombinant bacteria with knocked-out outer membrane protein to produce acetoin

[0479] Because halophilic bacteria have relatively thick outer membranes, their efficiency in exchanging matter and energy is affected. Knocking out the outer membrane proteins of the recombinant bacterium TDZH308 can enhance the glucose transport rate of halophilic bacteria and also improve their acetoin secretion capacity. Based on the recombinant bacterium TDZH308 constructed in Example 7, this patent knocks out the key outer membrane genes lpxM and lpxL to construct strains TDZH321 and TDZH322.

[0480] Fermentation tests showed that the recombinant strains TDZH321 and TDZH322 improved the synthesis and secretion of acetoin.

[0481] The specific implementation process is as follows:

[0482] (1) Construction of recombinant bacteria TDZH321 and TDZH322

[0483] The genome was edited using the CRISPR genome editing method. Specifically, the plasmid containing sgRNA and the recombinant template was constructed by inserting the sgRNA expression module, upstream and downstream homologous arms, and other DNA fragments into the original expression plasmid pSEVA241 (containing kanamycin and spectinomycin resistance genes) using the Gibson method. The sequence of the recombinant strain TDZH321 inserted into the plasmid is SEQ ID NO: 19, arranged in the following order: sgRNA expression module (nucleotides 1-152), upstream homologous arm (nucleotides 153-652), and downstream homologous arm (nucleotides 653-1156). The sequence of the recombinant strain TDZH321 inserted into the plasmid is SEQ ID NO: 20, arranged in the following order: sgRNA expression module (nucleotides 1-152), upstream homologous arm (nucleotides 153-652), and downstream homologous arm (nucleotides 653-1156).

[0484] The pSEVA241 plasmid expressing sgRNA and recombinant template and the pQ08 plasmid expressing Cas9 were transformed into recombinant bacteria TDZH308 via E. coli S17-1 conjugation.

[0485] Gene-edited mutant strains were screened using primers designed for colony PCR and confirmed by gene sequencing. Colony PCR is a routine procedure.

[0486] Finally, through colony PCR and gene sequencing, it was confirmed that the key outer membrane genes lpxM and lpxL sequences that had been knocked out in the genome of the recombinant strain TDZH308 were identified. The recombinant strains were named TDZH321 and TDZH322, respectively.

[0487] (2) Shake-flask fermentation was used to verify the acetoin production levels of recombinant strains TDZH321 and TDZH322.

[0488] The specific steps for producing acetoin by shake-flask fermentation of recombinant strains are as follows:

[0489] The recombinant strain was inoculated into 20 mL of LB60 medium and cultured for 12-16 h. Then, it was transferred to a new 20 mL LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture.

[0490] 2.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of acetoin fermentation medium, and a shake-flask experiment was conducted. The shaker temperature was 37℃ and the rotation speed was 200 rpm. After 48 h of incubation, the acetoin content was measured. Each experiment was performed in triplicate, and the results were averaged. The results are shown in Table 21.

[0491] Table 21. Acetaminophen content produced by recombinant strains after overexpression of the acetocin synthesis gene cluster.

[0492] TDZH285-pZH342 19.60 TDZH308 22.55 TDZH321 22.75

[0493] The results showed that recombinant strains TDZH321 and TDZH322 produced 22.55 g / L and 22.75 g / L of acetoin, respectively, with yields increasing by 15.05% and 16.07% compared to recombinant strain TDZH308. This example demonstrates that knocking out lpxL and lpxM can reduce the permeability of the Halomonas cell membrane, enhance the exchange of intracellular and extracellular substances and energy, and improve the production and secretion levels of acetoin.

[0494] Example 9: Construction of recombinant strains with defects in the acetoin endocytosis transport system

[0495] Acetoni, as a small molecule compound, can be secreted extracellularly through the cell membrane. To improve the secretion capacity of acetoin, based on the recombinant bacterium TDZH308 constructed in Example 7, the key genes DctP, DctQ, DctM, and DctPQM in the acetoin endocytosis transport system were knocked out to construct strains TDZH365, TDZH366, TDZH367, and TDZH368.

[0496] Fermentation tests showed that the recombinant strains TDZH365, TDZH366, TDZH367, and TDZH368 improved the synthesis and secretion of acetoin.

[0497] The specific implementation process is as follows:

[0498] (1) Construction of recombinant bacteria TDZH365, TDZH366, TDZH367, and TDZH368

[0499] The plasmid construction method for the homologous recombination editing method used in the suicide plasmid is as follows: DNA fragments of the upstream and downstream homologous arms are inserted into the original expression plasmid pRE112 (containing the chloramphenicol resistance gene) using the Gibson method. The sequence of the TDZH365 inserted plasmid is SEQ ID NO: 21, arranged sequentially as upstream homologous arm (nucleotides 1-500) and downstream homologous arm (nucleotides 501-1000). The sequence of the TDZH366 inserted plasmid is SEQ ID NO: 22, arranged sequentially as upstream homologous arm (nucleotides 1-500) and downstream homologous arm (nucleotides 501-1000). The sequence of the TDZH367 inserted plasmid is SEQ ID NO: 23, arranged sequentially as upstream homologous arm (nucleotides 1-514) and downstream homologous arm (nucleotides 515-1014). The sequence of the TDZH368 inserted plasmid is SEQ ID NO: 24, arranged as follows: upstream homologous arm (nucleotides 1-514) and downstream homologous arm (nucleotides 515-1014).

[0500] The pRE112 suicide plasmid expressing the homologous recombination template was transformed into recombinant strain TDZH308 via E. coli S17-1 conjugation. In the first round, transformants integrated into the genome were selected by chloramphenicol resistance screening. In the second round, the pHelp plasmid with a homing endonuclease expression module was transformed into E. coli S17-1 conjugation. The plasmids were screened on chloramphenicol and antibiotic-free plates, respectively. Single clones that did not grow on chloramphenicol-added plates but grew on antibiotic-free plates were further identified.

[0501] Gene-edited mutant strains were screened using primers designed for colony PCR and confirmed by gene sequencing. Colony PCR is a routine procedure.

[0502] Finally, through colony PCR and gene sequencing, it was confirmed that the gene sequences DctP, DctQ, DctM, and DctPQM had been knocked out in the TDZH308 genome. The recombinant bacteria constructed were named TDZH365, TDZH366, TDZH367, and TDZH368. Then, by continuously and repeatedly passaged the genome-edited strains in liquid culture medium and streaking them on spectinomycin-resistant and non-resistant plates, strains with the loss of the pHelp plasmid were identified, facilitating the next round of genome editing.

[0503] (2) Shake-flask fermentation was used to verify the acetoin production levels of recombinant strains TDZH365, TDZH366, TDZH367, and TDZH368.

[0504] The specific steps for producing acetoin by shake-flask fermentation of recombinant strains are as follows:

[0505] The recombinant strain was inoculated into 20 mL of LB60 medium and cultured for 12-16 h. Then, it was transferred to a new 20 mL LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture.

[0506] 2.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of acetoin fermentation medium, and a shake-flask experiment was conducted. The shaker temperature was 37℃ and the rotation speed was 200 rpm. After 48 h of incubation, the acetoin content was measured. Each experiment was performed in triplicate, and the results were averaged. The results are shown in Table 22.

[0507] Table 22. Acetaminophen content produced by recombinant strains after overexpression of the acetocin synthesis gene cluster.

[0508] TDZH322 19.60 TDZH308 23.26 TDZH365 22.43 TDZH366 20.04 TDZH367 25.79

[0509] The results showed that recombinant strains TDZH365, TDZH366, TDZH367, and TDZH368 produced 23.26 g / L, 22.43 g / L, 20.04 g / L, and 25.79 g / L of acetoin, respectively, increasing yields by 18.67%, 14.44%, 2.24%, and 31.58%. This example demonstrates that knocking out DctP, DctQ, DctM, and DctPQM to disrupt the acetoin endotoxin transport system can improve acetoin production.

[0510] Metabolic engineering strategies, such as reducing precursor consumption, enhancing precursor supply, knocking out degradation genes, overexpressing acetoin synthesis gene clusters, knocking out outer membrane genes to increase membrane permeability, and modifying defective acetoin endocytosis transport systems, are used to modify bacteria to increase acetoin production. TDZH368 Figure 3 注:原内容中“TDZH128”疑似有重复,我按照原样翻译了。你可根据实际情况检查确认。 .

[0511] Example 10: Production of acetoin using *Halomycin* under hypoxic conditions

[0512] a. Shake-flask fermentation to verify the acetoin production level of recombinant strain Halomonas bluephagenesis TD1.0ΔphaCB conjugated with the high-expression plasmid pZH126 under hypoxic conditions.

[0513] The specific steps for producing acetoin by shake-flask fermentation of recombinant strains are as follows:

[0514] The recombinant strain was inoculated into 20 mL of LB60 medium and cultured for 12-16 h. Then, it was transferred to a new 20 mL LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture.

[0515] 2.5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of acetoin fermentation medium for a shake-flask experiment. The shaker temperature was 37℃, and the shaking speeds were set to 50 rpm, 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm, 200 rpm, and 250 rpm. 50 mg / L of IPTG inducer was added after 4 hours of incubation. After 48 hours of incubation, the acetoin content was measured. Each experiment was performed in triplicate, and the average results were taken. The results are shown in Table 23.

[0516] Table 23. Content of acetoin produced by *Halomonas* under hypoxic conditions.

[0517]

[0518]

[0519] The results showed that the acetoin yield of recombinant strain TDZH308 varied significantly when cultured on a shaker at different rotation speeds. The acetoin yields at rotation speeds of 50, 100, 110, 120, 130, 140, 150, 200, and 250 rpm were 6.37 g / L, 15.67 g / L, 16.27 g / L, 17.29 g / L, 18.53 g / L, 21.11 g / L, 20.69 g / L, 18.17 g / L, and 13.43 g / L, respectively. The acetoin yield at a rotation speed of 140 rpm was 16.18% higher than that at the normal rotation speed of 200 rpm.

[0520] These results indicate that when recombinant strain TDZH308 is cultured at low speeds, both the amplitude and rotation speed of the shaker affect the oxygen transfer efficiency in the culture medium. The reduced oxygen transfer efficiency places TDZH308 in a hypoxic state, causing the bacteria to allocate more energy and resources to acetoin synthesis, thus reducing their survival needs. Simultaneously, the lower shear force also improves the conditions for acetoin synthesis. These factors contribute to increased acetoin production levels.

[0521] From the above description, it should be noted and understood that various modifications and improvements can be made to the invention described in detail above without departing from the spirit and scope of the invention as claimed in the claims. Therefore, the scope of the claimed technical solutions is not limited to any specific exemplary teachings given.

[0522] The applicant declares that the above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention and should not be construed as limiting the specific implementation of the invention to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the inventive concept, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. An engineered halomonas bacterium ( Halomonas ), characterized in that, The *Halomonas* strain expresses acetolactate synthase and acetolactate decarboxylase, wherein the amino acid sequence of the acetolactate synthase is SEQ ID NO: 25, and the amino acid sequence of the acetolactate decarboxylase is SEQ ID NO:

26. Furthermore, the aforementioned *Halomonas*: A) Not expressed phaC and phaB ; or, B) Expression phaC and phaB It is not functional or its function is weakened; The aforementioned Halomonas ( Halomonas )for Halomonas bluephagenesis , Halomonas campaniensis , Halomonas aydingkolgenesis One or more of them.

2. The Halomonas bacterium according to claim 1, characterized in that, The aforementioned *Haloxymonas* species does not express enzymes of the acetoin degradation pathway, or the expressed enzymes of the acetoin degradation pathway are not functional or have weakened function. The enzymes involved in the acetoin degradation pathway include one or both of 2,3-butanediol dehydrogenase or acetoin-utilizing protease.

3. The Halomonas bacterium according to claim 1, characterized in that, Knock out or knock down butA、bdhA or acuC One or more of them.

4. The Halomonas bacterium according to claim 2, characterized in that, Downregulation or blocking of the acetoin endocytotransport system in the aforementioned Halomonas includes enzymes that do not express the acetoin endocytotransport system or enzymes that express the acetoin endocytotransport system that are not functional or have reduced function. The enzymes of the acetoin endocytotransport system include one or more of the following: tetracarbonyl carboxylic acid binding protein, tetracarbonyl carboxylic acid TRAP transporter small permease protein, or tetracarbonyl carboxylic acid TRAP transporter large permease protein.

5. The Halomonas bacterium according to claim 4, characterized in that, Knock out or knock down DctP , DctQ or DctM One or more of them.

6. The Halomonas bacterium according to claim 4, characterized in that, Increasing the membrane permeability of the *Haloxymonas* bacterium includes the *Haloxymonas* bacterium not expressing an outer membrane protein or expressing an outer membrane protein that is not functional or has weakened function, wherein the outer membrane protein includes one or both of lpxM or lpxL.

7. The Halomonas bacterium according to claim 6, characterized in that, Knock out or knock down lpxM or lpxL One or two of them.

8. The Halomonas bacillus according to any one of claims 1-7, characterized in that, Enhancing the precursor supply for acetoin synthesis in *Halomonas* includes expressing glucose invertase, and / or enhancing the precursor supply for acetoin synthesis in *Halomonas* includes not expressing phosphoenolpyruvate carboxylase or expressing phosphoenolpyruvate carboxylase that is nonfunctional or functionally weakened.

9. The Halomonas bacterium according to claim 8, characterized in that, Introduced into Halomonas gluC , and / or, knock out or knock down PPC .

10. The Halomonas bacterium according to claim 8, characterized in that, Reducing the consumption of precursors for acetoin synthesis in the aforementioned Halomonas includes not expressing one or more of phosphorylated acetyltransferase, pyruvate carboxylase, lactate dehydrogenase, pyruvate dehydrogenase, or keto acid reductase, or expressing one or more of phosphorylated acetyltransferase, pyruvate carboxylase, lactate dehydrogenase, pyruvate dehydrogenase, or keto acid reductase that is nonfunctional or has reduced function.

11. The Halomonas bacterium according to claim 10, characterized in that, Knock out or knock down pta , pyc , ldh , nagD or ilvC One or more of them.

12. The Halomonas bacterium according to claim 1, characterized in that, The acetolactate synthase and / or acetolactate decarboxylase mentioned are derived from the order Bacillus (Bacillus). Bacillales ).

13. The Halomonas bacterium according to claim 1, characterized in that, The acetolactate synthase and / or acetolactate decarboxylase mentioned are derived from Bacillus spp. ( Bacillus ).

14. The Halomonas bacterium according to claim 1, characterized in that, The *Halomonas* species contains a foreign gene, and the *Halomonas* species are knocked out or knocked down. phaC Encoding genes and phaB Encoding genes.

15. The Halomonas bacterium according to claim 14, characterized in that, The exogenous genes mentioned also include glucose invertase-encoding genes.

16. The *Halomonas* strain according to claim 14 or 15, characterized in that, The foreign gene is expressed on a plasmid and / or on a chromosome.

17. The Halomonas bacterium according to claim 16, characterized in that, The exogenous gene is one or more copies.

18. The Halomonas bacterium according to claim 16, characterized in that, The exogenous gene is regulated by the promoter.

19. The Halomonas bacterium according to claim 18, characterized in that, The promoters mentioned include constitutive promoters and / or inducible promoters.

20. The Halomonas bacterium according to claim 19, characterized in that, The constitutive promoters include wild-type P porin Or its mutants.

21. The Halomonas bacterium according to claim 20, characterized in that, The aforementioned P porin Mutants include P porin58 、 P porin42 、P porin68 、P porin140 、P porin278 、P porin194 、P porin211 、P porin221 、P porin203 、P porin197 、P porin259 、 P porin70 、P porin60 、P porin185 、P porin138 、P porin141 P porin3 .

22. The Halomonas bacterium according to claim 19, characterized in that, The inducible promoter includes P lux promoter or its mutant, P mmp promoter or its mutant, P tac promoter or its mutant, P trp Promoter or its mutant.

23. The Halomonas bacterium according to claim 22, characterized in that, The aforementioned P mmp Mutants include P mmp1 , P mmp3 、 P mmp9 .

24. The Halomonas bacterium according to claim 1, characterized in that, The aforementioned halomonas include Halomonas bluephagenesis TD01 Halomonas campaniensis LS21 or Halomonas aydingkolgenesis One or more of M1.

25. A method for constructing *Haloxylon ammodendron* according to any one of claims 1-24, characterized in that, The *Haloxylonus* strain expresses acetolactate synthase and acetolactate decarboxylase, and the *Haloxylonus* strain is knocked out or knocked down. phaC and phaB .

26. The construction method according to claim 25, characterized in that, The construction method includes any one or a combination of two of the following: a) Downregulate or block the acetoin degradation pathway in the *Haloxymonas*, wherein the *Haloxymonas* does not express an enzyme of the acetoin degradation pathway or the expressed enzyme of the acetoin degradation pathway is nonfunctional or has weakened function, and the enzyme of the acetoin degradation pathway includes one or both of 2,3-butanediol dehydrogenase or acetoin-utilizing protease. b) Downregulate or block the acetoin endocytotransport system in the *Haloxymonas*, wherein downregulating or blocking the acetoin endocytotransport system in the *Haloxymonas* includes enzymes that do not express the acetoin endocytotransport system or enzymes that express the acetoin endocytotransport system that are not functional or have weakened function, wherein the enzymes of the acetoin endocytotransport system include one or more of four-carbon dicarboxylic acid binding protein, four-carbon dicarboxylic acid TRAP transporter small permease protein or four-carbon dicarboxylic acid TRAP transporter large permease protein. c) Increasing the membrane permeability of the *Haloxymonas*, which includes the *Haloxymonas* not expressing an outer membrane protein or expressing an outer membrane protein that is not functional or has weakened function, wherein the outer membrane protein includes one or both of lpxM or lpxL. d) Enhancing the precursor supply for acetoin synthesis in the *Haloxymonas*, wherein enhancing the precursor supply for acetoin synthesis in the *Haloxymonas* includes expressing glucose invertase, and / or, enhancing the precursor supply for acetoin synthesis in the *Haloxymonas* includes not expressing phosphoenolpyruvate carboxylase or expressing phosphoenolpyruvate carboxylase that is not functional or has weakened function. e) Reducing the consumption of precursors for acetoin synthesis in *Halomonas*, including not expressing one or more of phosphorylated acetyltransferases, pyruvate carboxylases, lactate dehydrogenases, pyruvate dehydrogenases, or keto acid reductases; or, the expressed phosphorylated acetyltransferases, pyruvate carboxylases, lactate dehydrogenases, pyruvate dehydrogenases, or keto acid reductases are nonfunctional or functionally weakened; and / or, f) Adjusting the expression levels of acetolactate synthase and / or acetolactate decarboxylase, which is achieved by adjusting the promoter strength of the two enzymes.

27. The construction method according to claim 25, characterized in that, Introduce one or more sequences from SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 19, 20, 21, 22, 23, 24, 28 or 29 into Halomonas.

28. The construction method according to claim 25, characterized in that, Introduce one or more plasmids from SEQ ID NO: 1, 2 or 3 into Halomonas.

29. A method for producing acetoin, characterized in that, The method includes culturing *Haloxylon ammodendron* as described in any one of claims 1-24 or *Haloxylon ammodendron* obtained by the construction method as described in any one of claims 25-28.

30. The method according to claim 29, characterized in that, The fermentation medium used to culture Halomonas contains inorganic salts, and the concentration of the inorganic salts in the medium is 10-60 g / L.

31. The method according to claim 30, characterized in that, The concentration of the inorganic salt in the culture medium is 10-50 g / L.

Images