CN118599744A - Halophilic microorganism for producing PHA using crude glycerol and its application - Google Patents

Abstract

The present invention relates to the field of recombinant microorganisms, and in particular to a halophilic microorganism for producing PHA using crude glycerol and its application. The halophilic microorganism overexpresses one or both of glycerol kinase or glycerol-3-phosphate dehydrogenase, and can produce metabolites (such as PHA) using crude glycerol, a cheap carbon source, thereby reducing production costs and avoiding the impact of crude glycerol on the environment.

Description

Halophilic microorganism for producing PHA using crude glycerol and its application

Technical Field

The present invention relates to the field of recombinant microorganisms, and in particular to a halophilic microorganism for producing PHA using crude glycerol and an application thereof.

Background Art

Polyhydroxyalkanoates (PHA) is a general term for a class of biosynthesized polymer polyesters. It is an energy storage substance synthesized by some microorganisms (mainly bacteria) under conditions of nutritional or metabolic imbalance. PHA has good biodegradability and biocompatibility, and is recognized as a green and environmentally friendly polymer material. Due to its diversity in types and properties, it can be applied to bulk plastics, medical materials, biofuels and even feed, and has attracted widespread attention. PHA has a variety of types and properties and has broad application prospects. However, its large-scale production is restricted by its relatively high production cost.

The production of PHA involves microbial fermentation in large fermenters, followed by downstream processing, which involves separating the microbial biomass from the fermentation broth and then separating and extracting the intracellular products or supernatant products. The production of PHA often requires expensive raw materials as a carbon source, such as glucose, and the downstream processing of intracellular products is expensive, which leads to high production costs for PHA.

Constructing recombinant metabolic pathways to avoid the use of expensive related carbon sources is an important idea to reduce the cost of PHA production through metabolic engineering. Currently, there are many studies that use the unique metabolic pathways of bacteria to produce PHA using various cheap raw materials in order to reduce costs, such as cane sugar molasses, cassava and corn starch, lignocellulose hydrolysate, whey, crude glycerol, and carbon sources extracted from waste.

Crude glycerol is a by-product of biodiesel, and its main components include glycerol (30% to 80%), water, methanol, soap, free fatty acids, methyl esters, residual catalysts, and very small amounts of impurities such as peptides, proteins and phospholipids. Crude glycerol is an economic and environmental disadvantage of the biodiesel industry, so it is necessary to convert crude glycerol into high value-added products to improve the economic sustainability of the biodiesel industry and reduce the environmental impact of crude glycerol waste treatment. Crude glycerol is considered to be an attractive carbon source alternative. Now a very popular trend is to use crude glycerol in polymer synthesis technology as a substitute for pure glycerol, and it is often used in the production of bioplastics. Compared with glucose, glycerol-based PHB has similar thermal and mechanical properties.

Although direct conversion of crude glycerol to PHA has great potential, it still faces technical challenges in industrial application. For example, untreated crude glycerol has high pH and high salt content, which inhibit the growth of most microorganisms; crude glycerol has complex components and impurities affect the growth of strains; strains lack glycerol metabolic pathways and cannot utilize crude glycerol; the conversion rate of PHA produced from crude glycerol is low and the yield is low; etc.

Crude glycerol cannot be used by most strains to produce PHA due to its high pH and high salinity, or the yield of PHA is low. For example, the non-patent literature: Valorization of waste glycerol for the production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer by Cupriavidus necator and extraction in a sustainable manner (Gahlawat G, Soni SK. Bioresour Technol, 2017, 243: 492-501) discloses that C. necator DSM 545 can synthesize PHB using crude glycerol as a substrate. Although the proportion can reach 59.8% of the biomass, the biomass is only 5.7 g/L. For example, the non-patent literature: Bioconversion of crude glycerol to polyhydroxyalkanoate by Bacillus thuringiensis under non-limiting nitrogen conditions (Kumar P, Ray S, Patel SKS, et al. Internat J Biol Macromol, 2015, 78: 9-16.) discloses that Bacillus thuringiensis EGU45 can also directly use crude glycerol to produce PHA, but it can only reach 3.16 g/L of biomass and 1.83 g/L of PHAs accumulation.

Summary of the invention

The present invention is based on the fact that halophilic microorganisms can efficiently produce PHA under high pH and high salinity conditions, and that halophilic microbial strains have a glycerol metabolic pathway in their cells. The present invention uses gene editing technology to enhance the expression intensity of proteins related to the glycerol metabolic pathway of the strain (such as glycerol kinase (GYK) and glycerol-3-phosphate dehydrogenase (GPDH)), thereby increasing the dry weight and product yield of the strain, and further enhances the expression of proteins related to the glycerol metabolic pathway by using a strong promoter to further increase the dry weight and product yield of the strain. This not only allows the use of cheap carbon sources instead of glucose to produce PHA, greatly reducing production costs, but also can alleviate the problem of excess crude glycerol, which is beneficial to economic sustainability while avoiding the impact of crude glycerol on the environment. Specifically:

In a first aspect of the present invention, there is provided an engineered halophilic microorganism for producing metabolites using crude glycerol, wherein the halophilic microorganism overexpresses glycerol kinase and/or glycerol-3-phosphate dehydrogenase.

The crude glycerol is a by-product of biodiesel.

The halophilic microorganisms are moderately halophilic microorganisms.

Preferably, the moderately halophilic microorganisms include Chromohalobacter, Halomonas, Halovibrio, Vibrio, Cobetia or Kushneria;

Preferably, the Halomonas genus includes Halomonas bluephagenesis or its derivatives, Halomonas campaniensis or its derivatives, Halomonas desiderata or its derivatives, Halomonas cupida or its derivatives, Halomonas smymensis or its derivatives, Halomonas levan or its derivatives, Halomonas lutescens or its derivatives, Halomonas elongata or its derivatives, Halomonas venusta or its derivatives or Halomonas alkaliantarctica or its derivatives.

In a specific embodiment of the present invention, the Halomonas genus includes but is not limited to Halomonas bluephagenesis TD01, Halomonas campaniensis LS21, Halomonasaydingkolgenesis M1, Halomonas bluephagenesis WZY254 or Halomonas bluephagenesis WZY278.

Preferably, the glycerol kinase or glycerol-3-phosphate dehydrogenase is independently endogenous or exogenous.

Preferably, the glycerol kinase or glycerol-3-phosphate dehydrogenase is independently derived from Halomonas.

In a specific embodiment of the present invention, the glycerol kinase or glycerol-3-phosphate dehydrogenase is endogenous.

Preferably, the glycerol kinase and/or glycerol-3-phosphate dehydrogenase is regulated by a promoter.

Preferably, the glycerol kinase is regulated by its own promoter, by a constitutive promoter, or by an inducible promoter.

Preferably, the glycerol-3-phosphate dehydrogenase is regulated by its own promoter, by a constitutive promoter, or by an inducible promoter.

Preferably, the constitutive promoter includes but is not limited to P _porin promoter or its mutants (for example, those described in the literature: Shen R, Yin J, Ye JW, Xiang RJ, Ning ZY, Huang WZ, Chen GQ. Promoter Engineering for Enhanced P(3HB-co-4HB) Production by Halomonas bluephagenesis. ACS SynthBiol. 2018 Aug 17; 7(8): 1897-1906. doi: 10.1021/acssynbio.8b00102. Epub 2018 Jul31. PMID: 30024739., for example, P _porin203 , P _porin221 , P _porin194 , P _porin278 , P _porin68 , P _porin58 , P _porin42 or P _porin140 ), P _Sp6 promoter or its variants.

The P _porin promoter mutant comprises a high-strength promoter, a medium-strength promoter and a low-strength promoter.

Wherein, the low-strength promoters include P _porin221 , P _porin203 , P _porin194 and the like.

Among them, the medium strength promoters include P _porin42 , P _porin278 , P _porin58 , P _porin68 and the like.

Wherein, the high-strength promoter includes P _porin140 .

Preferably, the inducible promoter may be an IPTG (isopropyl-β-D-thiogalactoside) inducible T7 promoter or an AHL (homoserine lactone) inducible promoter. Preferably, the inducer includes but is not limited to IPTG or AHL.

Preferably, the inducible promoter is selected from P _frm promoter or its mutant, P _luc promoter or its variant, P _lac promoter or its variant, P _trp promoter or its variant, P _tac promoter or its variant, phage promoter or its variant, P _araBAD promoter or its variant or P _phaP promoter or its variant.

Preferably, the promoters regulating glycerol kinase or glycerol-3-phosphate dehydrogenase are the same or different.

In a specific embodiment of the present invention, the promoter is the glycerol kinase promoter or the glycerol-3-phosphate dehydrogenase promoter.

Preferably, the sequence of the glycerol kinase promoter itself comprises the nucleotide sequence shown in SEQ ID NO: 1, or comprises a nucleotide sequence having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 1.

SEQ ID NO: 1

GCATGATGTCTAATCTAAGGCACAGCATGGTCGTCGCTGAGCTAACGGTAAAGGAGAG CAGTTTCATACTTGAGCATTTGGGGCGGCTGGAGTAGCC

Preferably, the sequence of the glycerol-3-phosphate dehydrogenase promoter itself comprises the nucleotide sequence shown in SEQ ID NO: 2, or comprises a nucleotide sequence having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 2.

SEQ ID NO: 2

TCCTTTTAGGGCGGGCTTCGGCCCGCTCTATTAATGCTCAGAAACTAGGTTTGCTGACAACAGCAAAGCTTGCCGATCTACATAGGCTTATTGCTTTGTTGTCATGTTGAGACCGCTACCTAAACACGGGGCTTTTTCTTTGTTAATTGGAACCGCT

In a specific embodiment of the present invention, the promoter regulating glycerol kinase and/or glycerol 3-phosphate dehydrogenase is a constitutive promoter, and the constitutive promoter is P _Porin promoter or a mutant thereof, preferably P _porin140 ;

Preferably, the sequence of P _porin140 comprises the nucleotide sequence as shown in SEQ ID NO: 3 or 4, or comprises a nucleotide sequence having more than 90% homology with the nucleotide sequence as shown in SEQ ID NO: 3 or 4.

SEQ ID NO: 3

CCGTAGGGCAAATAGGGTCATGCCTCCACACCGCTCGTCACATCCTGTTGCGTTCACTGGAATCCCAGTATAAGATTTGACCTGCGAGCAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGAGTTACTAGAGAAAGAGGAGAAATACTAG

SEQ ID NO: 4

TGAGAGGGCTTCACTCAAGAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGCGCGCCCAGCTGATGCTCCACACCGCTCGTCACATCCTGTTGCGTTCACTGGAATCCCAGTATAAGATTTGACCTGCGAGCAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGAGTTACTAGAGAAAGAGGAGAAATACTAG

In a specific embodiment of the present invention, the promoter for regulating glycerol kinase is P _porin140 , and the sequence of P _porin140 is shown in SEQ ID NO:3.

In a specific embodiment of the present invention, the promoter regulating glycerol-3-phosphate dehydrogenase is P _porin140 , and the sequence of P _porin140 is shown in SEQ ID NO:4.

Preferably, the glycerol kinase and/or glycerol-3-phosphate dehydrogenase is expressed on a plasmid or on a chromosome.

Preferably, the carbon source used in culturing the halophilic microorganism to produce metabolites includes crude glycerol, which is a by-product of biodiesel.

Preferably, the biodiesel is obtained by esterifying vegetable oil, animal oil, waste oil or microbial oil with alcohol.

Further preferably, the crude glycerol is the substance remaining after extracting biodiesel after esterification. More preferably, the substance preferably includes fatty acid methyl ester or fatty acid ethyl ester.

Preferably, the extraction is extraction.

Preferably, in terms of mass ratio, the crude glycerol comprises glycerol, further preferably comprises any value of 30%-80% glycerol, more preferably comprises any value of 70%-80% glycerol; for example, comprises 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80% glycerol.

Preferably, the crude glycerol further comprises one or more of water, methanol, soap, free fatty acids, methyl esters, catalysts, peptides, proteins or phospholipids.

Preferably, the carbon source also includes one or more of glucose, sucrose, glucosamine, glucosamine, 4-hydroxybutyric acid, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol, 1,12-dodecanediol, fructose, xylose, cellulose, lactose, lactic acid, lauric acid, acetic acid, caproic acid, propionic acid, valeric acid, caproic acid, butyric acid, palm oil or oleic acid.

In a specific embodiment of the present invention, the carbon source is crude glycerol, and the concentration of crude glycerol in the culture medium is greater than 1 g/L, preferably any value in the range of 10-300 g/L, and further preferably any value in the range of 10-50 g/L, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or 300 g/L.

Preferably, the culture medium for culturing the halophilic microorganism also includes inorganic salts. Further preferably, the inorganic salts include one or more of potassium salts, zinc salts, magnesium salts, cobalt salts, sodium salts, calcium salts or phosphates, such as potassium dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, cobalt chloride, sodium chloride, potassium chloride, magnesium carbonate, calcium carbonate, sodium carbonate, etc.

Further preferably, the salt concentration in the culture medium for culturing the halophilic microorganism is any value in the range of 2.5-200 g/L, and further preferably is 50-120 g/L, for example 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 64, 65, 66, 66.2, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 g/L.

Preferably, the pH value of the culture medium for culturing the halophilic microorganism is any value between 6 and 11, for example, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5 or 11.

Preferably, the culture medium for culturing the halophilic microorganism also includes a nitrogen source. Further preferably, the nitrogen source includes an organic nitrogen source and/or an inorganic nitrogen source, and more preferably, the organic nitrogen source includes but is not limited to one or more of peanut cake powder, soybean cake powder, cottonseed cake powder, corn steep liquor, yeast powder, fish meal, silkworm pupa powder, peptone, bran, waste mycelium or amino acids (also including amino acid salts, such as one or more of potassium salts, sodium salts, calcium salts, magnesium salts or zinc salts). More preferably, the inorganic nitrogen source includes but is not limited to one or more of ammonium salts (such as ammonium sulfate), nitrates or ammonia water.

In a specific embodiment of the present invention, the culture medium for culturing the halophilic microorganisms includes a carbon source, a nitrogen source, inorganic salts and trace elements.

Preferably, the trace elements include one or more of ammonium ferric citrate, calcium chloride, zinc sulfate heptahydrate, manganese chloride, boric acid, cobalt chloride hexahydrate, copper sulfate pentahydrate, nickel chloride hexahydrate or sodium molybdate dihydrate.

Preferably, the metabolite comprises PHA.

The second aspect of the present invention provides a method for preparing the halophilic microorganism described in the first aspect, the preparation method comprising introducing a nucleotide sequence encoding glycerol kinase and/or a nucleotide sequence encoding glycerol-3-phosphate dehydrogenase into the halophilic microorganism.

Preferably, the nucleotide sequence encoding glycerol kinase comprises the nucleotide sequence shown in SEQ ID NO: 5, or comprises a nucleotide sequence having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 5.

SEQ ID NO: 5:

TTGGATGATAATAAAAGCGAAACAAAACATACAAAATCGAAAGAACCTACAACAAAGTGAACGTGATATGTCCTCCTTTATTCTCGCCATCGATCAAGGTACGACCAGCTCGCGGCCCATTTTATTTGACCGTCAGGGCCAAATAGAGGCGGTTGCCCAACAGGAATTCCCTCAGCACTTCCCCCAAGACGGGTGGATAGAGCATGATCCCGAAAATATTTGGGAGACTGTCATCGCCACTTGTCGCGAGGTGCTCGAAAAGGCGAGTATT TCGCCAGAGCAGATTGATGGAATTGGCATTACTAACCAGCGTGAAACCACAGTGGTGTGGGATAGAGAGACGGGTAAGCCTCTCTACAATGCGATTGTTTGGCAAGACCGGCGCACC TCTGAACTTTGTCAAACACTCCGCGACCAAGGGCATACAGAGGCAGTACAAGCTAAGACAGGCCTCTTAATAGACCCCTATTTTTCAGCCACCAAACTAGCGTGGATACTCGATAACGTAGAAGGAGCCCGCGAACGAGCCCAACGCGGTGAGCTACTCTTTGGCACTATCGATACCTTCTTGATTTGGCGATTAACCAATGGCACGCAACATGTTACTGATGCCACCAACGCATCACGAACAGCCCATTTTTAATATCCATAA TCAAACATGGGACGATGAGCTGTTAGCTTTGTTTAACGTTCCTGCCAACATGCTGCCCGAGGTAAAAGATTCCAGCGATGATTTTGGCACCACTGACGCGCATTGGTTAGGGGCGCCCATACCTA TTGCCGGTGTGGCAGGGGATCAGCAGGCCGCGCTAGTGGGTCAAGCGTGTTTTCAACCCGGAATGGGCAAAAGCACCTATGGCACCGGGTTGTTTTATGATCGTTAACACTGGGGATACACCGTCGGTATCACGAAATCGCCTATTAACGACCATTGGATACCGAATTAATGGAAAACCGACCTATGCAATGGAAGGCAGTATCTTCGTTGCTGGGGCGACTGTGCAGTGGCTACGCGATGGGTTGAACCTGTTTGCA GATGCGTCAGAAACAGAGGCGCTTGCTAAAGAAACCCGCAGCGGACATAGCGTTTACTTGGTACCTGCTTTACTGGCTTGGGTGCACCTCATTGGGATCCTAAAGCGCGCGGGGCAATTTTTGGCCTTAC TCGGGATACCGGCATTGCCGAAATTGTTGCGGCGGGCCTCCAGGCGGTGTGTTACCAAACCCGCGACCTGCAGCACTGCATGAACGACGATATGGAAGCCACGCCAGGTAACCTACGTGTCGATGGTGGCATGGTGAAGAACAGCTGGGTGATGCAGTTTTTGGCTGATATGCTCGACGTCCAGGTAGATAGACCCACAATTCTTGAGACGACGGCGCTCGGAGCCGCTTACTTGGCAGGCTTGCGTTTTGGGTTGGTA TGACACGCTTGAAGAAATTGAGCAACTCTGGCGCTGCGAGAAGAGCTTTACACCTAAAATGGAAGAATCTACTCGGGAGGCGTTATACCAGGGTTGGTTAGACGCCGTGAATCGCGTGCGTTCCAATTAA

Preferably, the nucleotide sequence encoding glycerol-3-phosphate dehydrogenase includes the nucleotide sequence shown in SEQ ID NO: 6, or comprises a nucleotide sequence having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 6.

SEQ ID NO: 6

ATGAAACTGCGTAACCGGAATATCGAGAAATTGCACACTGACGACTTTGATGCACTAATCATCGGTGGCGGCATTAACGGTGCAGCAACCGCAGCCGCGCTTGCAGGCAAGGGTGCTAAAGTCGCCTTAATAGACCGCGGTGATTTTGCGGGAAGCACCAGTATGCACTCCTCCAACTTAGTTTGGGGAGGCATTAAATATATGGAAAGTAAGGATTTCGCATTAGTACGCAAACTGTGTAAAAGTCGTAATCATCT GATTAAAAGTTACCCTTCTACGGTTCAGGAAATTCGCTTCTTACCACTATCAGTAAAGGCTTCCGCCATTCACCGAAATATTTATGGGCGGGTACCTGGCTTTACTGGCTCATGGGGAACGGTTTTACCAAGCTTGCCCCGCTTACTATCTCCAAAG AATATTAAGCAAGAAGAGCCGATTATTGATATCGAAGGCTCAGTAGGTGGGTTTGAATACTCAGATGCCTACTTACACGATAACGATGCCAGATTTGTATTTAATTTTGTTCGTCATTCGCTTAATTACGGGGCGATTGCAGCCAATTATGTTGAGTCTCTTGGTGCTGAACGTGAAGGACAGCACTGGGTAACGAAAGCGCGCAATGTAATGGATGGCAGCACGTTTAATATCCGTTCCAAAGTATTAATTAAT GCTGCTGGCCCCTGGGTGGATCAGCACAACGCTCTCACCGGCGAAAAAAACCACCCATCAGCATCTTTATTCAAAGGGCATTCACCTGATAGTTCCGCAACTGACGGAAACTAAGCGGGTACTTGCGTTTTTTGCCGACGATGGGCGACTGTTCTTTGTC ATTCCCATGGGTAATCGCACTTGCATCGGCACCACTGATACGCATATGGAGCACCCAGAGGTAGATGTCACAGCTGAAGACATCGAGTTTGTATTGGAAAATATCAATAAGCGCCTAACGTTAGAAAAGCCGCTTACCCAAGACGATATTATTTCAACACGCTGCGGTGTTCGGCCGCTAGCTATCAAAGCGGATCAAGGTAGCGATCGCGATTTTCTCCAACTATCGCGTAAGCATGTGATCGATACCAATGAAGATA GCGCCCACATTAGTATTTTTGGCGGTAAATTAACCGACTGCTTGAACGTAGGTGATGAAATTGCAGAGGAAGTGGTTCGACTAGGCGTTAATCTCAGCGATATTAACTATCGCTGGTATGGTGAGCCGCCAGAACCCGTTAAACAGCAGTTCATG GACCAGGCTAAACGGATGAACCTAGATGCGATGACCGCGCCAACATCCACCGAGCCGCTCTCCTCGCGACTATGGCGTCGCTATGCAGAACAGGCCATGCAAATGCTAGAAAAAATCCGCCAGGATCCTGCAGAAGCCGATATTCTCATTGAAGGTACCGAGTATATTCGCTGTGAACTTGAGCATGCCCGAGATCACGAAATGATTACGCAGTTGGAGGACTTCCTGCGCCGAAGAGCCAAAGTCTCGCTGGTTGTTC ACCATGAACAGCTGCGCCACTCTAAGGGGCTGAAAGAAGCGTGTCGCGTTCTGTTCGGAGACCAAGCGGAAGAGCGTTTTGCCACCTACTTTGCTGAAAATCGTGATACTTCACAGCCTTATACTGCACCGGCAGAACCACAGCACACTCACTGA

Preferably, the introducing into the halophilic microorganism comprises using sgRNA, and further preferably comprises using an upstream homology arm and/or a downstream homology arm.

In a specific embodiment of the present invention, the preparation method comprises introducing the expression vector into a halophilic microorganism.

Preferably, the expression vector comprises a nucleotide sequence encoding one or both of glycerol kinase and glycerol-3-phosphate dehydrogenase.

Preferably, the expression vector comprises a promoter. The definition of the promoter is the same as that of the first aspect of the present invention.

The expression vector can replicate, transcribe and translate in the host cell. Therefore, it also contains other conventional expression elements, such as terminator, restriction site and the like.

The terminator sequence includes a nucleotide sequence as shown in any one of SEQ ID NO: 7 or 8, or includes a nucleotide sequence having more than 90% homology with the nucleotide sequence as shown in any one of SEQ ID NO: 7 or 8.

SEQ ID NO: 7

GAGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT

SEQ ID NO: 8

TGATAAGAAAATAATAAAAAAGCCGGATTAATAATCTGGCTTTTTATATTTCTCTTGACCC TTGGGGTTATCATTGGGCTTACTAGTGAGCTC

Preferably, the expression vector can be expressed in a free state in the halophilic microorganism, or one or both of the nucleotide sequences encoding glycerol kinase or glycerol-3-phosphate dehydrogenase can be inserted into the chromosome of the halophilic microorganism for expression.

In a specific embodiment of the present invention, the expression vector inserts the nucleotide sequence encoding glycerol kinase and the nucleotide sequence encoding glycerol-3-phosphate dehydrogenase into the chromosome of a halophilic microorganism for expression, preferably inserted into the genome of the halophilic microorganism by gene editing (further preferably by CRISPR/Cas9 technology), preferably inserted into one or more of the G3 site (SEQ ID NO: 15), G4 site (SEQ ID NO: 9), G7 site (SEQ ID NO: 16) or G51 site (SEQ ID NO: 17) of the halophilic microorganism.

SEQ ID NO: 9

GATGGCAACGCCCTCTGTAATACCTACGCATGAATTACTCCTTTAGAGCGTGTAAATCGCCAGCACAACGCCCTTATGAAAAGACGCTTAAATCGAGCGTCTGATTAAAGGCCGATAGCCCAAAGCAGGCTCGAGCAATCAGCCTTCCACCCAAGCTATTTTCGTTAATTCTTTACCCCAGAAAAACTCCACAAATACAAAATATAGTTAACTTTTGAGGCGATGCAAATCCACCTTCCGAGCACATCGATCATTCACCTAGCTA GATGAGACAGGCAGCTAATCATGTGCATCATCATTAAAAATTAACCGAGTAGCTTGCGGCACTTCATCTCACCTTTCATTTCCTTATGCTGAACACGGTAAGCTTGGCGATAACACCGCTACTGATGGCTAAAAATCAACG

SEQ ID NO: 15

TAACCATCTAATGATCAGCGAGTCATGCTGGGATGGTTAAACAAGCTGTTAATTAGTCTATTTCTCCAATAGTGGCGCGGCTAAACTTGGACTAAGGCCGCAGTTTTGACGCTAAAACTAACCGAGACGCTACTCATGCAGCCAACTGCACGCTTTTCATTGTCGCCGGCTCTTCCTGAGACCCACACTCAACACAGCCATGGAGCTTGCTGTTGTGGTTCACCA

SEQ ID NO: 16

AGGTAGTCGCATTATCTTCTTTTGTGCTTTCGCCTTATGGCTCTAGGCGCTTGGATGCTCGTGATATTTTCCTACTCATTGGTCGGAAGATTCGAGACTATCTTCGATGTACACCATTACGGGGGTTGTCACGGGGTGATATAGAGTGTATCGCGCAAAGTTCACAAATGTGTCACGGGGACTCTAACGATAGGGCGATGTCTGCATTAACGCCTCCCCTCATTTCAATAAGTGTGTTTCAGCAAGGAGAGCGCCCATGGCG ACTACACGCCGTAAAACTC

SEQ ID NO: 17

GTACGCAGTAATGCGCGCACTCAGTGCACGACTCTTGTTTAATTAATCAAAAGTAAGAAACTAGTTCTTAAAAACTAACCACATTGTTAAATTCTGTCATAGCGACTCAGTGTAAAAACTCATTCAACTAATCGCCTACAACAAAGACAAGCTTTTATATACCAGCTTTAAAACCGGGCAACTAAATATCACCCAAGGCTTTAAATTAAACAATAAGAAACAATTATCGCATTAATAATTAACGCTAAAAATCATGAATTAATTAAACTGCTA AAAAGTGTTTCTTTAACACTTTGATTTTTAATTGACTAATAAAAACCATTAAGGAAGCACTAATGCTACTTAAAACGCCCGCTAGCGATTAGCGGTCATCGC

Preferably, the target site sequence of the sgRNA targeting the G3 site comprises the nucleotide sequence shown in SEQ ID NO: 18, or comprises a nucleotide sequence having more than 90% homology with SEQ ID NO: 18.

Preferably, the target site sequence of the sgRNA targeting the G4 site comprises the nucleotide sequence shown in SEQ ID NO: 19, or comprises a nucleotide sequence having more than 90% homology with SEQ ID NO: 19.

Preferably, the target site sequence of the sgRNA targeting the G7 site comprises the nucleotide sequence shown in SEQ ID NO: 20, or comprises a nucleotide sequence having more than 90% homology with SEQ ID NO: 20.

Preferably, the target site sequence of the sgRNA targeting the G51 site comprises the nucleotide sequence shown in SEQ ID NO: 21, or comprises a nucleotide sequence having more than 90% homology with SEQ ID NO: 21.

SEQ ID NO: 18: AATAGTGGCGCGGCTAAACT

SEQ ID NO: 19: TTCACCTAGCTAGATGAGAC

SEQ ID NO: 20: ACACCATTACGGGGGTGTCA

SEQ ID NO: 21: TTGGGTGATATTTAGTTGCC

In one embodiment of the present invention, the insertion is performed at the G4 site of the halophilic microorganism.

Preferably, the expression vector further comprises an sgRNA expression module.

Preferably, the sgRNA expression module comprises the nucleotide sequence shown in SEQ ID NO: 10, or comprises a nucleotide sequence having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 10.

SEQ ID NO: 10

TTGACAGCTAGCTCAGTCCTAGGTATAATACTAGTTTCACCTAGCTAGATGAGACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGAACCCGGGGTACC

Preferably, the expression vector further comprises an upstream homology arm and/or a downstream homology arm sequence.

Preferably, the upstream homology arm sequence includes the nucleotide sequence shown in SEQ ID NO: 11, or includes a nucleotide sequence having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 11.

Preferably, the downstream homology arm sequence includes the nucleotide sequence shown in SEQ ID NO: 12, or includes a nucleotide sequence having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 12.

SEQ ID NO: 11

GTGCGCTTGCCACAGCTTCGCGCTCGTTCTGTGGCTCCGGCAGCATCGACGAATAGTAGGCGATTCGCTGGAAACGGTCGGTCGGCTTGACGTTCCCTGGCAGCGGTGTATCGCTGCTTGGGTTTGAAAAGTCTAATTCTTCCAGCAGTGCCAATTGCTGATCGTAAGGCGGATCATTGGTCATGATTCTAAATTCTCTACCGTGGTGAATCATGGATTCACCATCGATATATTCAATGATCGCTGAATC CCCAGTAGCATCTTCAATTGCCAAATGTACGGTTGCCTTACTACCCCGCGCCTCCGCCATGACAATTTGTACATCTTCGAGTATGTCGAGTGCTTCTTCGACAGTGGCTGCGTTGTCGATCACATATTGCCCCCACAAACCTGCCTGCATTCCTGGTTTGCCAGGGTCACGCGGGCCAAAGTCGGTGGCCGTGAGATAGAGCATGTGGAACGCCAGCCCCTTTTCGTTGAATCCATCGGCAGCGCCGATG CCATAAACCGTGGTGACCATGCTGGCGTACTTCGACGTCCATGTTGCTGCGTTCTCCTCCACGACGACTTCCGCGCCCAGCATGCCCCCATTTCGCTCCATGCCACGGGGGAAGACAGTAATAACCGGATCTGTCGATTCGGGCCAGTCCATTGTGCGGGCAACATAAATGCCCTGCTCGTTGTCATTCCATAAGTACCTAGAACACGCCCATGCTGCATTGGCCATTACCATCGCACCAATCACGATGGCAACGCCCCCTCTGTAATA CCTACGCATGAATTACTCCTTTAGAGCGTGTAAATCGCCAGCACAACGCCCTTATGAAAAGACGCTAAATCGAGCGTCTGATTAAAGGCCGATAGCCCAAAGCAGGCTCGAGCAATCAGCCTTCCACCCAAGCTATTTTCGTTAATTCTTTACCCCAGAAAAACTCCACAAATACAAAATATAGTTAACTTTTGAGGCGATGCAAATCCACCTTCCGAGCACATCGATCA

SEQ ID NO: 12

CTCACCTTTCATTTCCTTATGCTGAACACGGTAAGCTTGGCGATAACACCGCTACTGATGGCTAAAAATCAACGATGACTTCCCATTGGATACTTGGCCCTGGGGCCATTGGTCGCCTGTTGGCGCATTCGCTTTCACCCATTACTGACATCACCTTGATTGGCCGACGTGCATTGCCTGAACAGCAACGCCTGACCACCCTGAAGGTGAAGAACGAACTCAGCGGCTAGCGAGCGTTACGGTTGCGGAG CTAGCCTCTCACTCACTGCCTGTGCCTGGGTTTGTGCATATCACCACCAAAGCCATGGCCGCCGAGGCGGCGCTTGCCAGCATTGCCGATGTGGTTGCGCCCACGACCCCGCTGGTGCTGTGGCAGAACGGTTTTGGCACAGCCACGGCTGACTGATAGGTGGCCAGGGCCAGTGCTGTGTGCGACCACGACTCAAGGCGCTTACTTAACCGGCAGTGATGGCGTGGTTCACGCCGGGCGCGGCCGA CGTTTATTGGTGATCTCAACAATCAACGCGCTGCGCTAGCAAAAACACTGGCGCAGACGTTAGGTGAGGCAGGCTTTACCGCCACGCCGGTAGACGATATTCGCCAACGCCTGTGGCAAAAGCTGGCGGTTAATGCGGCGATCAACCCATTGGTGGCGCTCAATGGCGTGCGCAATGGTGAGCTGCGCGGTGATGCTTATGCAGTCCGTTTGGCAGCGGTGGTAAAGGAAGTCGCGGCGATTTTAAAGCA GGAAAACATTGCACCACCGAATGGTGGCGAAGGTGAAGACGCGTGGTTGGCGCTTGTGTGGCAGGTGGTGGAGAACACCGCTAACAATAAGGCCTCGATGTTGCAGGATGTTGAGGCCAGACGCACCACCGAGCGCGGGGCGATTTTAGGGCCGTTGATTGATAGCGCCGAGCGCCATGGGTTGCCGTGTGGGTTGTTGAAGGAGCTTGATAGCGAATTGGCTAAATTGGAGGCGAAGTTTAGGGAACA

Preferably, the expression vector may be a prokaryotic expression vector or a eukaryotic expression vector, preferably a prokaryotic expression vector. Preferably, the expression vector is a plasmid (eg, pSE series plasmid, preferably pSEVA341).

In a specific embodiment of the present invention, the expression vector comprises, from the 5' end to the 3' end, in sequence: an sgRNA expression module, an upstream homology arm, a glycerol kinase gene promoter, a glycerol kinase gene, a terminator, a glycerol-3-phosphate dehydrogenase gene promoter, a glycerol-3-phosphate dehydrogenase gene, a terminator, and a downstream homology arm. Preferably, the sequence of the expression vector comprises a nucleotide sequence as shown in SEQ ID NO: 13, or comprises a nucleotide sequence having more than 90% homology to the nucleotide sequence as shown in SEQ ID NO: 13.

SEQ ID NO: 13

TTGACAGCTAGCTCAGTCCTAGGTATAATACTAGTTTCACCTAGCTAGATGAGACGTTTTAGAGCTAGAAATAGCAA

GTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGAACCCGGGGTACCG

TGCGCTTGCCACAGCTTCGCGCTCGTTCTGTGGCTCCGGCAGCATCGACGAATAGTAGGCGATTCGCTGGAAACGG

TCGGTCGGCTTGACGTTCCCTGGCAGCGGTGTATCGCTGCTTGGGTTTGAAAAGTCTAATTCTTCCAGCAGTGCCA

ATTGCTGATCGTAAGGCGGATCATTGGTCATGATTCTAAATTCTCTACCGTGGTGAATCATGGATTCACCATCGATAT

ATTCAATGATCGCTGAATCCCCAGTAGCATCTTCAATTGCCAAATGTACGGTTGCCTTACTACCCCGCGCCTCCGCC

ATGACAATTTGTACATCTTCGAGTATGTCGAGTGCTTCTTCGACAGTGGCTGCGTTGTCGATCACATATTGCCCCCA

CAAACCTGCCTGCATTCCTGGTTTGCCAGGGTCACGCGGGCCAAAGTCGGTGGCCGTGAGATAGAGCATGTGGAA

CGCCAGCCCCTTTTCGTTGAATCCATCGGCAGCGCCGATGCCATAAACCGTGGTGACCATGCTGGCGTACTTCGAC

GTCCATGTTGCTGCGTTCTCCTCCACGACGACTTCCGCGCCCAGCATGCCCCCATTTCGCTCCATGCCACGGGGGA

AGACAGTAATAACCGGATCTGTCGATTCGGGCCAGTCCATTGTGCGGGCAACATAAATGCCCTGCTCGTTGTCATTC

CATAAGTACCTAGAACACGCCCCATGCTGCATTGGCCATTACCATCGCACCAATCACGATGGCAACGCCCCCTCTGTA

ATACCTACGCATGAATTACTCCTTTAGAGCGTGTAAATCGCCAGCACAACGCCCTTATGAAAAGACGCTTAAATCGA

GCGTCTGATTAAAGCCCGATAGCCCAAAGCAGGCTCGAGCAATCAGCCTTCCACCCAAGCTATTTTCGTTAATTCTT

TACCCCAGAAAAACTCCACAAATACAAAATATAGTTAACTTTTGAGGCGATGCAAATCCACCTTCCGAGCACATCG

ATCAGCATGATGTCTAATCTAAGGCACAGCATGGTCGTCGCTGAGCTAACGGTAAAGGAGAGCAGTTTCATACTTG

AGCATTTGGGGCGGCTGGAGTAGCCTTGGATGATAATAAAAGCGAAACAAAACATACAAAATCGAAAGAACCTACA

ACAAAGTGAACGTGATATGTCCTCCTTTATTCTCGCCATCGATCAAGGTACGACCAGCTCGCGCGCCATTTTATTTG

ACCGTCAGGGCCAAATAGAGGCGGTTGCCCAACAGGAATTCCCTCAGCACTTCCCCCAAGACGGGTGGATAGAGC

ATGATCCCGAAAATATTTGGGAGACTGTCATCGCCACTTGTCGCGAGGTGCTCGAAAAGGCGAGTATTTCGCCAGA

GCAGATTGATGGAATTGGCATTACTAACCAGCGTGAAACCACAGTGGTGTGGGATAGAGAGACGGGTAAGCCTCT

CTACAATGCGATTGTTTGGCAAGACCGGCGCACCTCTGAACTTTGTCAAACACTCCGCGACCAAGGGCATACAGA

GGCAGTACAAGCTAAGACAGGCCTCTTAATAGACCCCTATTTTTCAGCCACCAAACTAGCGTGGATACTCGATAAC

GTAGAAGGAGCCCGCGAACGAGCCCAACGCGGTGAGCTACTCTTTGGCACTATCGATACCTTCTTGATTTGGCGAT

TAACCAATGGCACGCAACATGTTACTGATGCCACCAACGCATCACGAACAGCCATTTTTAATATCCATAATCAAACA

TGGGACGATGAGCTGTTAGCTTTGTTTAACGTTCCTGCCAACATGCTGCCCGAGGTAAAAGATTCCAGCGATGATT

TTGGCACCACTGACGCGCATTGGTTAGGGGCGCCCATACCTATTGCCGGTGTGGCAGGGGATCAGCAGGCCGCGCT

AGTGGGTCAAGCGTGTTTTCAACCGGAATGGGCAAAAGCACCTATGGCACCGGGTTGTTTTATGATCGTTAACACT

GGGGATACACCGTCGGTATCACGAAATCGCCTATTAACGACCATTGGATACCGAATTAATGGAAAACCGACCTATGC

AATGGAAGGCAGTATCTTCGTTGCTGGGGCGACTGTGCAGTGGCTACGCGATGGGTTGAACCTGTTTGCAGATGCG

TCAGAAACAGAGGCGCTTGCTAAAGAAACCCGCAGCGGACATAGCGTTTACTTGGTACCTGCTTTTACTGGCTTGG

GTGCACCTCATTGGGATCCTAAAGCGCGCGGGGCAATTTTTGGCCTTACTCGGGATACCGGCATTGCCGAAATTGTT

GCGGCGGGCCTCCAGGCGGTGTGTTACCAAACCCGCGACCTGCAGCACTGCATGAACGACGATATGGAAGCCACG

CCAGGTAACCTACGTGTCGATGGTGGCATGGTGAAGAACAGCTGGGTGATGCAGTTTTTGGCTGATATGCTCGACG

TCCAGGTAGATAGACCCACAATTCTTGAGACGACGGCGCTCGGAGCCGCTTACTTGGCAGGCTTGCGTTTGGGTTG

GTATGACACGCTTGAAGAAATTGAGCAACTCTGGCGCTGCGAGAAGAGCTTTACACCTAAAATGGAAGAATCTAC

TCGGGAGGCGTTATACCAGGGTTGGTTAGACGCCGTGAATCGCGTGCGTTCCAATTAAGAGCTGCTAACAAAGCCC

GAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCT

TGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTCCTTTTAGGGCGGGCTTCGGCCCGCTCTATTAATGCT

CAGAAACTAGGTTTGCTGACAACAGCAAAGCTTGCCGATCTACATAGGCTTATTGCTTTGTTGTCATGTTGAGACC

GCTACCTAAACACGGGGCTTTTTCTTTGTTAATTGGAACCGCTATGAAACTGCGTAACCGGAATATCGAGAAATTGC

ACACTGACGACTTTGATGCACTAATCATCGGTGGCGGCATTAACGGTGCAGCAACCGCAGCCGCGCTTGCAGGCA

AGGGTGCTAAAGTCGCCTTAATAGACCGCGGTGATTTTGCGGGAAGCACCAGTATGCACTCCTCCAACTTAGTTTG

GGGAGGCATTAAATATATGGAAAGTAAGGATTTCGCATTAGTACGCAAACTGTGTAAAAGTCGTAATCATCTGATTA

AAAGTTACCCTTCTACGGTTCAGGAAAATTCGCTTTCTTACCACTATCAGTAAAGGCTTCCGCCATTCACCGAAATAT

TTATGGGCGGGTACCTGGCTTTACTGGCTCATGGGGAACGGTTTTACCAAGCTTGCCCCGCTTACTATCTCCAAAGAA

TATTAAGCAAGAAGAGCCGATTATTGATATCGAAGGCTCAGTAGGTGGGTTTGAATACTCAGATGCCTACTTACACG

ATAACGATGCCAGATTTGTATTTAATTTTGTTCGTCATTCGCTTAATTACGGGGCGATTGCAGCCAATTATGTTGAGT

CTCTTGGTGCTGAACGTGAAGGACAGCACTGGGTAACGAAAGCGCGCAATGTAATGGATGGCAGCACGTTTAATA

TCCGTTCCAAAGTATTAATTAATGCTGCTGGCCCCTGGGTGGATCAGCACAACGCTCTCACCGGCGAAAAAAACCAC

CCATCAGCATCTTTATTCAAAGGGCATTCACCTGATAGTTCCGCAACTGACGGAAACTAAGCGGGTACTTGCGTTTT

TTGCCGACGATGGGCGACTGTTCTTTGTCATTCCCATGGGTAATCGCACTTGCATCGGCACCACTGATACGCATATG

GAGCACCCAGAGGTAGATGTCACAGCTGAAGACATCGAGTTTGTATTGGAAAATATCAATAAGCGCCTAACGTTAG

AAAAGCCGCTTACCCAAGACGATATTATTTCAACACGCTGCGGTGTTCGGCCGCTAGCTATCAAAGCGGATCAAGG

TAGCGATCGCGATTTTCTCCAACTATCGCGTAAGCATGTGATCGATACCAATGAAGATAGCGCCCACATTAGTATTTT

TGGCGGTAAATTAACCGACTGCTTGAACGTAGGTGATGAAATTGCAGAGGAAGTGGTTCGACTAGGCGTTAATCTC

AGCGATATTAACTATCGCTGGTATGGTGAGCCGCCAGAACCCGTTAAACAGCAGTTCATGGACCAGGCTAAACGGA

TGAACCTAGATGCGATGACCGCGCCAACATCCACCGAGCCGCTCTCCTCGCGACTATGGCGTCGCTATGCAGAACA

GGCCATGCAAATGCTAGAAAAAATCCGCCAGGATCCTGCAGAAGCCGATATTTCATTGAAGGTACCGAGTATATTC

GCTGTGAACTTGAGCATGCCCGAGATCACGAAATGATTACGCAGTTGGAGGACTTCCTGCGCCGAAGAGCCAAAG

TCTCGCTGGTTGTTCACCATGAACAGCTGCGCCACTCTAAGGGGCTGAAAGAAGCGTGTCGCGTTCTGTTCGGAG

ACCAAGCGGAAGAGCGTTTTGCCACCTACTTTGCTGAAAATCGTGATACTTCACAGCCTTATACTGCACCGGCAGA

ACCACAGCACACTCACTGATGATAAGAAATAATAAAAAAGCCGGATTAATAATCTGGCTTTTTATATTCTCTTGACC

CTTGGGGTTATCATTGGGCTTACTAGTGAGCTCCTCACCTTCATTTCCTTATGCTGAACACGGTAAGCTTGGCGATA

ACACCGCTACTGATGGCTAAAAATCAACGATGACTTCCCATTGGATACTTGGCCCTGGGGCCATTGGTCGCCTGTTG

GCGCATTCGCTTTCACCCATTACTGACATCACCTTGATTGGCCGACGTGCATTGCCTGAACAGCAACGCCTGACCA

CCCCTGAAGGTGAAGAACGAACTCAGCGGCTAGCGAGCGTTACGGTTGCGGAGCTAGCCTCTCACTCACTGCCTG

TGCCTGGGTTTGTGCATATCACCACCAAAGCCATGGCCGCCGAGGCGGCGCTTGCCAGCATTGCCGATGTGGTTGC

GCCCACGACCCCGCTGGTGCTGTGGCAGAACGGTTTTTTGGCACAGCCACGGCTGACTGATAGGTGGCCAGGGCC

AGTGCTGTGTGCGACCACGACTCAAGGCGCTTACTTAACCGGCAGTGATGGCGTGGTTCACGCCGGGCGCGGGCC

GACGTTTATTGGTGATCTCAACAATCAACGCGCTGCGCTAGCAAAAACACTGGCGCAGACGTTAGGTGAGGCAGG

CTTTACCGCCACGCCGGTAGACGATATTCGCCAACGCCTGTGGCAAAAGCTGGCGGTTAATGCGGCGATCAACCCA

TTGGTGGCGCTCAATGGCGTGCGCAATGGTGAGCTGCGCGGTGATGCTTATGCAGTCCGTTTGGCAGCGGTGGTAA

AGGAAGTCGCGGCGATTTTAAAGCAGGAAAACATTGCACCACCGAATGGTGGCGAAGGTGAAGACGCGTGGTTG

GCGCTTGTGTGGCAGGTGGTGGAGAACACCGCTAACAATAAGGCCTCGATGTTGCAGGATGTTGAGGCCAGACGC

ACCACCGAGCGCGGGGCGATTTTAGGGCCGTTGATTGATAGCGCCGAGCGCCATGGGTTGCCGTGTGGGTTGTTGA

AGGAGCTTGATAGCGAATTGGCTAAATTGGAGGCGAAGTTTTTAGGGAACA

In a specific embodiment of the present invention, the expression vector comprises, from the 5' end to the 3' end, in sequence: an sgRNA expression module, an upstream homology arm, a P _porin140 promoter, a glycerol kinase gene, a terminator, a P _porin140 promoter, a glycerol-3-phosphate dehydrogenase gene, a terminator, and a downstream homology arm. Preferably, the sequence of the expression vector comprises a nucleotide sequence as shown in SEQ ID NO: 14, or comprises a nucleotide sequence having more than 90% homology to the nucleotide sequence shown in SEQ ID NO: 14.

SEQ ID NO: 14

TTGACAGCTAGCTCAGTCCTAGGTATAATACTAGTTTCACCTAGCTAGATGAGACGTTTTAGAGCTAGAAATAGCAA

GTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGAACCCGGGGTACCG

TGCGCTTGCCACAGCTTCGCGCTCGTTCTGTGGCTCCGGCAGCATCGACGAATAGTAGGCGATTCGCTGGAAACGG

TCGGTCGGCTTGACGTTCCCTGGCAGCGGTGTATCGCTGCTTGGGTTTGAAAAGTCTAATTCTTCCAGCAGTGCCA

ATTGCTGATCGTAAGGCGGATCATTGGTCATGATTCTAAATTCTCTACCGTGGTGAATCATGGATTCACCATCGATAT

ATTCAATGATCGCTGAATCCCCAGTAGCATCTTCAATTGCCAAATGTACGGTTGCCTTACTACCCCGCGCCTCCGCC

ATGACAATTTGTACATCTTCGAGTATGTCGAGTGCTTCTTCGACAGTGGCTGCGTTGTCGATCACATATTGCCCCCA

CAAACCTGCCTGCATTCCTGGTTTGCCAGGGTCACGCGGGCCAAAGTCGGTGGCCGTGAGATAGAGCATGTGGAA

CGCCAGCCCCTTTTCGTTGAATCCATCGGCAGCGCCGATGCCATAAACCGTGGTGACCATGCTGGCGTACTTCGAC

GTCCATGTTGCTGCGTTCTCCTCCACGACGACTTCCGCGCCCAGCATGCCCCCATTTCGCTCCATGCCACGGGGGA

AGACAGTAATAACCGGATCTGTCGATTCGGGCCAGTCCATTGTGCGGGCAACATAAATGCCCTGCTCGTTGTCATTC

CATAAGTACCTAGAACACGCCCCATGCTGCATTGGCCATTACCATCGCACCAATCACGATGGCAACGCCCCCTCTGTA

ATACCTACGCATGAATTACTCCTTTAGAGCGTGTAAATCGCCAGCACAACGCCCTTATGAAAAGACGCTTAAATCGA

GCGTCTGATTAAAGCCCGATAGCCCAAAGCAGGCTCGAGCAATCAGCCTTCCACCCAAGCTATTTTCGTTAATTCTT

TACCCCAGAAAAACTCCACAAATACAAAATATAGTTAACTTTTGAGGCGATGCAAATCCACCTTCCGAGCACATCG

ATCACCGTAGGGCAAATAGGGTCATGCCTCCACACCGCTCGTCACATCCTGTTGCGTTCACTGGAATCCCAGTATAA

GATTTGACCTGCGAGCAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTA

CAAATAATTTTGTTTAAGAGTTACTAGAGAAAGAGGAGAAATACTAGTTGGATGATAATAAAGCGAAACAAAACAT

ACAAAATCGAAAGAACCTACAACAAAGTGAACGTGATATGTCCTCCTTTATTCTCGCCATCGATCAAGGTACGACC

AGCTCGCGGCCATTTATTTGACCGTCAGGGCCAAATAGAGGCGGTTGCCCAACAGGAATTCCCTCAGCACTTCC

CCCAAGACGGGTGGATAGAGCATGATCCCGAAAATATTTGGGAGACTGTCATCGCCACTTGTCGCGAGGTGCTCGA

AAAGGGCGAGTATTTCGCCAGAGCAGATTGATGGAATTGGCATTACTAACCAGCGTGAAACCACAGTGGTGTGGGAT

AGAGAGACGGGTAAGCCTCTCTACAATGCGATTGTTTGGCAAGACCGGCGCACCTCTGAACTTTGTCAAACACTC

CGCGACCAAGGGCATACAGAGGCAGTACAAGCTAAGACAGGCCTCTTAATAGACCCCTATTTTTCAGCCACCAAA

CTAGCGTGGATACTCGATAACGTAGAAGGAGCCCGCGAACGAGCCCAACGCGGTGAGCTACTCTTTGGCACTATC

GATACCTTCTTGATTTGGCGATTAACCAATGGCACGCAACATGTTACTGATGCCACCAACGCATCACGAACAGCCAT

TTTTAATATCCATAATCAAACATGGGACGATGAGCTGTTAGCTTTGTTTAACGTTCCTGCCAACATGCTGCCCGAGG

TAAAAGATTCCAGCGATGATTTTGGCACCACTGACGCGCATTGGTTAGGGGCGCCCATACCTATTGCCGGTGTGGC

AGGGGATCAGCAGGCCGCGCTAGTGGGTCAAGCGTGTTTTCAACCCGGAATGGGCAAAAGCACCTATGGCACCGG

GTGTTTTTATGATCGTTAACACTGGGGATACACCGTCGGTATCACGAAATCGCCTATTAACGACCATTGGATACCGAA

TTAATGGAAAACCGACCTATGCAATGGAAGGCAGTATCTTCGTTGCTGGGGCGACTGTGCAGTGGCTACGCGATGG

GTTGAACCTGTTTGCAGATGCGTCAGAAACAGAGGCGCTTGCTAAAGAAACCCGCAGCGGACATAGCGTTTACTT

GGTACCTGCTTTACTGGCTTGGGTGCACCTCATTGGGATCCTAAAGCGCGCGGGGCAATTTTTGGCCTTACTCGG

GATACCGGCATTGCCGAAATTGTTGCGGCGGGCCTCCAGGCGGTGTGTTACCAAACCCGCGACCTGCAGCACTGC

ATGAACGACGATATGGAAGCCACGCCAGGTAACCTACGTGTCGATGGTGGCATGGTGAAGAACAGCTGGGTGATG

CAGTTTTTGGCTGATATGCTCGACGTCCAGGTAGATAGACCCACAATTCTTGAGACGACGGCGCTCGGAGCCGCTT

ACTTGGCAGGCTTGCGTTTGGGTTGGTATGACACGCTTGAAGAAATTGAGCAACTCTGGCGCTGCGAGAAGAGCT

TTACACCTAAAATGGAAGAATCTACTCGGGAGGCGTTATACCAGGGTTGGTTAGACGCCGTGAATCGCGTGCGTTC

CAATTAAGAGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAA

CCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGAGAGGGCTTC

ACTCAAGAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGCGCGCCCAGCTGATGCCTCCACACCGCTCGTCA

CATCCTGTTGCGTTCACTGGAATCCCAGTATAAGATTTGACCTGCGAGCAAGCTGTCACCGGATGTGCTTTCCGGTC

TGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGAGTTACTAGAGAAAGAGGAGAAATACT

AGATGAAACTGCGTAACCGGAATATCGAGAAATTGCACACTGACGACTTTGATGCACTAATCATCGGTGGCGGCAT

TAACGGTGCAGCAACCGCAGCCGCCTTGCAGGCAAGGGTGCTAAAGTCGCCTTAATAGACCGCGGTGATTTTGC

GGGAAGCACCAGTATGCACTCCTCCAACTTAGTTTGGGGAGGCATTAAATATATGGAAAGTAAGGATTTCGCATTAG

TACGCAAACTGTGTAAAAGTCGTAATCATCTGATTAAAAGTTACCCTTCTACGGTTCAGGAAATTCGCTTTTCTTACC

ACTATCAGTAAAGGCTTCGCCATTCACCGAAATATTTATGGGCGGGTACCTGGCTTTACTGGCTCATGGGGAACGG

TTTTACCAAAGCTGCCCCGCTTACTATCTCCAAAGAATATTAAGCAAGAAGAGCCGATTATTGATATCGAAGGCTCAG

TAGGGTGGGTTTGAATACTCAGATGCCTACTTACACGATAACGATGCCAGATTTGTATTTAATTTTGTTCGTCATTCGC

TTAATTACGGGGCGATTGCAGCCAATTATGTTGAGTCTCTTGGTGCTGAACGTGAAGGACAGCACTGGGTAACGAA

AGCGCGCAATGTAATGGATGGCAGCACGTTTAATATCCGTTCCAAAGTATTAATTAATGCTGCTGGCCCCTGGGTGG

ATCAGCACAACGCTCTCACCGGCGAAAAAAACCACCCATCAGCATCTTTATTCAAAGGGCATTCACCTGATAGTTCC

GCAACTGACGGAAACTAAGCGGGTACTTGCGTTTTTTGCCGACGATGGGCGACTGTTCTTTGTCATTCCCATGGGT

AATCGCACTTGCATCGGCACCACTGATACGCATATGGAGCACCCAGAGGTAGATGTCACAGCTGAAGACATCGAGT

TTGTATTGGAAAATATCAATAAGCGCCTAACGTTAGAAAAGCCGCTTACCCAAGACGATATTATTTCAACACGCTGC

GGTGTTCGGCCGCTAGCTATCAAAGCGGATCAAGGTAGCGATCGCGATTTTCTCCAACTATCGCGTAAGCATGTGAT

CGATACCAATGAAGATAGCGCCCACATTAGTATTTTTGGCGGTAAATTAACCGACTGCTTGAACGTAGGTGATGAAA

TTGCAGAGGAAGTGGTTCGACTAGGCGTTAATCTCAGCGATATTAACTATCGCTGGTATGGTGAGCCGCCAGAACC

CGTTAAACAGCAGTTCATGGACCAGGCTAAACGGATGAACCTAGATGCGATGACCGCGCCAACATCCACCGAGCC

GCTCTCCTCGCGACTATGGCGTCGCTATGCAGAACAGGCCATGCAAATGCTAGAAAAAATCCGCCAGGATCCTGCA

GAAGCCGATATTTCATTGAAGGTACCGAGTATATTCGCTGTGAACTTGAGCATGCCCGAGATCACGAAATGATTAC

GCAGTTGGAGGACTTCCTGCGCCGAAGAGCCAAAGTCTCGCTGGTTGTTCACCATGAACAGCTGCGCCACTCTAA

GGGGCTGAAAGAAGCGTGTCGCGTTCTGTTCGGAGACCAAGCGGAAGAGCGTTTTGCCACCTACTTTGCTGAAAA

TCGTGATACTTCACAGCCTTATACTGCACCGGCAGAACCACAGCACACTCACTGATGATAAGAAATAATAAAAAAG

CCGGATTAATAATCTGGCTTTTTATATTCTCTTGACCCTTGGGGTTATCATTGGGCTTACTAGTGAGCTCCTCACCTTC

ATTTCCTTATGCTGAACACGGTAAGCTTGGCGATAACACCGCTACTGATGGCTAAAAATCAACGATGACTTCCCATT

GGATACTTGGCCCTGGGGCCATTGGTCGCCTGTTGGCGCATTCGCTTTCACCCATTACTGACATCACCTTGATTGGC

CGACGTGCATTGCCTGAACAGCAACGCCTGACCACCCTGAAGTGAAGAACGAACTCAGCGGCTAGCGAGCGT

TACGGTTGCGGAGCTAGCCTCTCACTCACTGCCTGTGCCTGGGTTTGTGCATATCACCACCAAAGCCATGGCCGCC

GAGGCGGCGCTTGCCAGCATTGCCGATGTGGTTGCGCCCACGACCCCGCTGGTGCTGTGGCAGAACGGTTTTTTG

GCACAGCCACGGCTGACTGATAGGTGGCCAGGGCCAGTGCTGTGTGCGACCACGACTCAAGGCGCTTACTTAACC

GGCAGTGATGGCGTGGTTCACGCCGGGCGCGGGCCGACGTTTATTGGTGATCTCAACAATCAACGCGCTGCGCTA

GCAAAAACACTGGCGCAGACGTTAGGTGAGGCAGGCTTTACCGCCACGCCGGTAGACGATATTCGCCAACGCCTG

TGGCAAAAGCTGGCGGTTAATGCGGCGATCAACCCATTGGTGGCGCTCAATGGCGTGCGCAATGGTGAGCTGCGC

GGTGATGCTTATGCAGTCCGTTTGGCAGCGGTGGTAAAGGAAGTCGCGGCGATTTTAAAGCAGGAAAACATTGCA

CCACCGAATGGTGGCGAAGGTGAAGACGCGTGGTTGGCGCTTGTGTGGCAGGTGGTGGAGAACACCGCTAACAA

TAAGGCCTCGATGTTGCAGGATGTTGAGGCCAGACGCACCACCGAGCGCGGGGCGATTTTAGGGCCGTTGATTGAT

AGCGCCGAGCGCCATGGGTTGCCGTGTGGGTTGTTGAAGGAGCTTGATAGCGAATTGGCTAAATTGGAGGCGAAG

TTTTAGGGAACA

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

The third aspect of the present invention provides a halophilic microorganism obtained by the preparation method described in the second aspect.

The fourth aspect of the present invention provides a use of the halophilic microorganism described in any of the above aspects, or the halophilic microorganism obtained by the preparation method described in the second aspect in producing metabolites.

Preferably, the halophilic microorganism utilizes crude glycerol to produce metabolites.

Preferably, the metabolite comprises PHA or its monomer.

Further preferably, the PHA monomer includes but is not limited to one, two or more of 2-hydroxypropionic acid, 3-hydroxybutyric acid, 4-hydroxybutyric acid, 3-hydroxyvaleric acid, 3-hydroxypropionic acid, 5-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 6-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid or 3-hydroxydodecanoic acid.

More preferably, the PHA includes, but is not limited to, one or more of P3HP, PHB, P(HB-LA), PHV, P34HB, PHBV, PHBHHx, PHBHHp, PHO, PHN, PHD, P3HB4HB3HV or P3HB4HB5HV.

In a fifth aspect of the present invention, there is provided a method for producing PHA or its monomers, the method comprising culturing the halophilic microorganism described in any one of the above aspects, or the halophilic microorganism obtained by the preparation method described in the second aspect;

The carbon source used in the culture includes crude glycerol, which is a byproduct of biodiesel.

Preferably, the biodiesel is obtained by esterifying vegetable oil, animal oil, waste oil or microbial oil with alcohol.

Further preferably, the crude glycerol is the substance remaining after extracting biodiesel after esterification. More preferably, the substance preferably includes fatty acid methyl ester or fatty acid ethyl ester.

Preferably, the extraction is extraction.

Preferably, in terms of mass ratio, the crude glycerol comprises glycerol, further preferably comprises any value of 30-80% glycerol, more preferably comprises any value of 70%-80% glycerol; for example, comprises 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80% glycerol.

Preferably, the crude glycerol further comprises one or more of water, methanol, soap, free fatty acids, methyl esters, catalysts, peptides, proteins or phospholipids.

Preferably, the carbon source also includes one or more of glucose, sucrose, glucosamine, glucosamine, 4-hydroxybutyric acid, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol, 1,12-dodecanediol, fructose, xylose, cellulose, lactose, lactic acid, lauric acid, acetic acid, caproic acid, propionic acid, valeric acid, caproic acid, butyric acid, palm oil or oleic acid.

In a specific embodiment of the present invention, the carbon source is crude glycerol, and the concentration of crude glycerol in the culture medium is greater than 1 g/L, preferably any value in the range of 10-300 g/L, and further preferably any value in the range of 10-50 g/L, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or 300 g/L.

Preferably, the culture medium further comprises an inorganic salt. Further preferably, the inorganic salt comprises one or more of potassium salt, zinc salt, magnesium salt, cobalt salt, sodium salt, calcium salt or phosphate, such as potassium dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, cobalt chloride, sodium chloride, potassium chloride, magnesium carbonate, calcium carbonate, sodium carbonate, etc.

Further preferably, the salt concentration in the culture medium is any value in the range of 2.5-200 g/L, further preferably 50-120 g/L, for example 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 64, 65, 66, 66.2, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 g/L.

Preferably, the pH value of the culture medium is any value between 6 and 11, for example, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5 or 11.

Preferably, the culture medium also includes a nitrogen source. Further preferably, the nitrogen source includes an organic nitrogen source and/or an inorganic nitrogen source, and more preferably, the organic nitrogen source includes but is not limited to one or more of peanut cake powder, soybean cake powder, cottonseed cake powder, corn steep liquor, yeast powder, fish meal, silkworm pupa powder, peptone, bran, waste mycelium or amino acids (also including amino acid salts, such as one or more of potassium salts, sodium salts, calcium salts, magnesium salts or zinc salts). More preferably, the inorganic nitrogen source includes but is not limited to one or more of ammonium salts (such as ammonium sulfate), nitrates or ammonia water.

In a specific embodiment of the present invention, the culture medium includes a carbon source, a nitrogen source, inorganic salts and trace elements.

Preferably, the trace elements include one or more of ammonium ferric citrate, calcium chloride, zinc sulfate heptahydrate, manganese chloride, boric acid, cobalt chloride hexahydrate, copper sulfate pentahydrate, nickel chloride hexahydrate or sodium molybdate dihydrate.

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

The "and/or" described in the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and/or B" includes "A", "A and B" and "B". For another example, "A, B and/or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

The terms “comprising” or “including” described in the present invention are open-ended terms. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

Beneficial effects obtained by this application:

A) Enhance the glycerol metabolic pathway in halophilic microorganisms so that they can use crude glycerol, a cheap carbon source, instead of glucose to produce PHA, greatly reducing the cost of PHA production.

B) It is suitable for the expansion and cultivation of strains and can be used to produce various products (such as various homo- or copolymeric PHAs).

C) The metabolic consumption of crude glycerol can alleviate the problem of excess crude glycerol, which is beneficial to economic sustainability, while avoiding the impact of crude glycerol on the environment, which is beneficial to environmental protection.

See Table 1 below for the Chinese and English translations involved in this application:

Table 1

DETAILED DESCRIPTION

The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

The bacterium used in the embodiment is Halomonas bluephagenesis WZY254, which is an outer membrane-defective strain. The Halomonas bluephagenesis WZY254 strain is a 4HB-CoA transferase encoding gene integrated into the recombinant Halomonas bluephagenesis TD01 genome, ΔphaP1, ΔlpxL, ΔlpxM, and then PMmp1-phaCAB is replaced with Pporin-phaCAB, wherein Δ represents knockout. The strain is disclosed in "Ziyu Wang et al., 2022. Hyperproduction of PHA copolymers containing high fractions of 4-hydroxybutyrate (4HB) by outer membrane-defected Halomonas bluephagenesis grown in bioreactors [J]. Microbial Biotechnology".

The Halomonas bluephagenesis TD01 strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 19, 2010, with the deposit registration number of CGMCC No.4353, and the classification name is Halomonas sp.TD01 (also known as Halomonas bluephagenesis TD01; it is recorded in the patent application publication number CN102120973A; the public can obtain the strain from Tsinghua University.

The competent Escherichia coli S17-1 used to construct the plasmid was purchased from Beijing Zhuangmeng International Bio-Gene Technology Co., Ltd.

The crude glycerol used in the examples was purchased from Hubei Carbon Source Water Treatment Co., Ltd., and the product name is Novel Sewage Treatment Crude Glycerol Composite Carbon Source.

Crude glycerin ingredients: glycerin, water, ash, dissolved sodium chloride, sodium fatty acid, fatty acid. The glycerin content is 70%-80%, and the pH is 7-8.

The culture medium formula in the embodiment is:

LB medium: contains 10 g/L NaCl, 10 g/L peptone, 5 g/L yeast extract, sterilized by high pressure steam at 121°C.

LB20 medium: contains 20 g/L NaCl, 10 g/L peptone, 5 g/L yeast extract, sterilized by high pressure steam at 121°C.

LB60 medium: contains 60 g/L NaCl, 10 g/L peptone, 5 g/L yeast extract, sterilized by high pressure steam at 121°C.

In the actual culture process, a certain concentration of antibiotics can be added to the above culture medium to maintain the stability of the plasmid. Commonly used antibiotics include: spectinomycin stock solution (100 mg/ml); chloramphenicol stock solution (25 mg/ml); kanamycin stock solution (25 mg/ml).

Fermentation medium: 60 g/L NaCl, 30 g/L glucose (or different concentrations of crude glycerol), 4 g/L urea, 0.2 g/L MgSO ₄ , 2.7 g/L Na ₂ HPO ₄ ·12H ₂ O, 3.3 g/L KH ₂ PO ₄ , 10 mL/L trace element solution I and 1 mL/L trace element solution II. The composition of trace element solution I is: 5 g/L ammonium ferric citrate, 2 g/L CaCl ₂ , prepared with 1M HCl. The composition of trace element solution II is: 100 mg/L ZnSO ₄ ·7H ₂ O, 30 mg/L MnCl ₂ ·4H ₂ O, 300 mg/L H ₃ BO ₃ , 200 mg/L CoCl ₂ ·6H ₂ O, 10 mg/L CuSO ₄ ·5H ₂ O, 20 mg/L NiCl ₂ ·6H ₂ O, 30 mg/L NaMoO ₄ ·2H ₂ O, prepared with 1M HCl. The above reagents were purchased from Sinopharm Chemical Reagent Company.

The method of Gibson assembly and construction of plasmid in the embodiment:

1) According to the principle of Gibson recombination, first design primers with 20-40bp homology arms. You can use NEBuilder to design, or you can use Snapgene to directly read the primer sequence containing the homology arm at the interface of the connecting fragment. The annealing temperature of each pair of primers cannot differ by more than 5°C.

2) Perform a conventional PCR reaction, and the vector can be selected for restriction digestion or PCR amplification, and then perform gel extraction and recovery according to the instructions of the gel extraction kit to obtain fragments with homology arms.

3) Use Nanodrop to determine the concentration of the recovered fragments, mix the fragments in equal molar numbers to ensure that each fragment is around 0.04 pmols, and the reaction system is 10 μl. Note that the pipette must be used to mix thoroughly. Do not use vortexing or centrifugation. The operation should be performed on ice.

4) After reacting at 50°C for 1 hour using a PCR instrument, chemically transform the competent cells. Because the recombination reaction efficiency is very high, for chemical transformation, the recombination product is first diluted 4 times (slowly pipetting to mix), and 2 μl is taken to transform the competent cells.

Conjugation transformation method in the embodiment:

1) The constructed plasmid was transformed into Escherichia coli S17-1 through chemical competence, the corresponding resistance plate was coated, and a single clone was picked and cultured in LB medium with shaking overnight;

2) Transfer the inoculum of 1% to a 100 ml small shake flask containing 20 ml LB medium, and transfer the inoculum of 1% of Halomonas to a 100 ml small shake flask containing 20 ml LB60 medium, and culture until OD600 = 0.6-0.8;

3) Take 1.5 ml of culture medium of Escherichia coli S17-1 and Halomonas, centrifuge at 2500 g for 2 minutes, discard the supernatant, add 1 mL of LB20 medium, gently wash the bacteria, centrifuge at 2500 g for 2 minutes, and discard the supernatant;

4) To ensure that E. coli and Halomonas can grow normally when mixed together, resuspend them in 50 μl of antibiotic-free LB20 medium respectively, then mix them in a 1:1 volume ratio, drop them onto LB20 antibiotic-free solid plates, and culture them for 6 hours. Scrape an appropriate amount of bacterial lawn and resuspend them in LB60 medium, apply the corresponding antibiotics onto LB60 solid medium, and culture them for 24-48 hours. Perform colony PCR identification when a single clone grows out.

The method for preparing the seed solution in the embodiment:

1) Bacteria activation

Take the glycerol tube of bacteria stored in a -80℃ refrigerator, streak it onto an LB60 medium plate, and culture it at 37℃ for 24h.

2) First-level seeds

A single bacterial colony was picked from the plate that completed step 1), inoculated into 20 mL of liquid LB60 medium, and cultured at 37°C and 200 rpm for 24 h.

3) Secondary Seed

Take the first-level seed solution obtained in step 2) and inoculate it into 20 mL of liquid LB60 culture medium at an inoculum size of 1%, and culture it at 37° C. and 200 rpm with shaking for 8-10 h.

The method of culturing in a 7L fermenter in the embodiment:

The secondary seed solution was prepared with reference to the "method for preparing seed solution in the embodiment", and inoculated into the fermentation medium according to the inoculation amount of 10%. The initial liquid of the 7.5L fermenter was 3L, and the fermentation system was directly fermented without sterilization. The temperature was controlled at 37°C, the initial dissolved oxygen was controlled at 30%-50%, and the dissolved oxygen was controlled by adjusting the rotation speed and ventilation. The starting rotation speed was 200rpm, the maximum rotation speed was 800rpm, and the maximum ventilation volume was 3vvm. After the rotation speed and ventilation reached the maximum, the dissolved oxygen was not controlled; during the fermentation process, the carbon source concentration was controlled between 10-15g/L by feeding, and the fermentation pH was controlled by 5M NaOH or ammonia water. The fermentation process includes a cell growth process and a product synthesis process: the feed solution of the cell growth process is 400ml750g/L sugar solution (or crude glycerol) with 15.2g of urea added; the product synthesis process begins after the feed solution is exhausted. The process needs to maintain nitrogen deficiency conditions to produce PHA, so 75% of the sugar solution (or crude glycerol) is continuously supplemented until the end of the fermentation.

During the fermentation period, the fermentation status needs to be monitored in real time to control parameters such as temperature, pH, dissolved oxygen, and carbon source concentration within the normal range. The sampling and analysis frequency is to take a 3-5 mL small sample every 2 hours and a 30 mL large sample every 4 hours; the small sample is used to determine the carbon source concentration and cell density to monitor the fermentation process and feeding rate, and the large sample is used for subsequent PHA content analysis and cell dry weight determination.

The freeze-drying method in the embodiment:

After fermentation, 35 mL of cell culture medium was taken and centrifuged at 8000 g for 15 min. The bacterial precipitate was collected, washed with water, and then freeze-dried (the centrifuge tube containing the bacterial precipitate was first placed at -80°C for 1 h, and then placed in a vacuum freeze dryer for 36 h) to obtain a freeze-dried product. Cell dry weight calculation method in the embodiment:

Measured as dry weight of cells per liter of fermentation system. The unit of dry weight of cells is g/L. Dry weight of cells (CDW) = (weight of centrifuge tube after freeze drying - weight of original empty centrifuge tube) ÷ 0.035; the weight of the centrifuge tube after freeze drying and the weight of the original empty centrifuge tube are both in g; 0.035 represents 0.035L.

Method for detecting the content of PHA in the bacteria and the content of each monomer in the embodiment:

The freeze-dried product was subjected to esterification and then the monomer content was determined by gas chromatography (GC);

Esterification reaction: take 60-70 mg of freeze-dried product into an esterification tube, add 2 mL of chloroform and 2 mL of esterification solution (containing 1 g/L benzoic acid and 3% concentrated sulfuric acid in methanol), mix well, cover tightly, and esterify in a 100°C metal bath for 4 hours; after cooling to room temperature, add 1 mL of distilled water, shake well and mix well, and stand to separate; after the chloroform phase and water are completely separated, take the chloroform phase for gas chromatography analysis;

10-100 mg of poly 3-hydroxybutyrate (P3HB) and γ-butyrolactone were taken for esterification reaction as standard samples;

Gas chromatography (GC) analysis parameters: HP-5 chromatographic column was used to separate the substances in Shimadzu GC-2014 gas chromatograph; the GC analysis temperature program was set, including injection port temperature (240°C), detector temperature (250°C), starting temperature and holding time (80°C, 1.5 min), first stage temperature rise (heating rate 30°C/min), second stage temperature rise (heating rate 40°C/min, maintained at 240°C for 2 min), and total program time 8 min;

The corresponding PHA monomer ratio was calculated by reading the internal standard peak area measured by gas chromatography, the PHA monomer methyl ester peak area of the standard sample, the internal standard peak area of the sample and the PHA monomer methyl ester peak area of the sample.

PHA content (wt%) = (mass of 3HB + mass of 4HB) ÷ mass of freeze-dried product × 100%;

Molar ratio of 3HB (mol%) = mole number of 3HB ÷ (mole number of 3HB + mole number of 4HB) × 100%;

The calculation method of the molar ratio of 4HB refers to the content of 3HB.

Example 1 Overexpression of endogenous glycerol kinase and glycerol-3-phosphate dehydrogenase genes to construct recombinant strain GLY01

Halomonas bluephagenesis WZY254 has a glycerol metabolic pathway, and glycerol kinase and glycerol-3-phosphate dehydrogenase play an important role. Using CRISPR/Cas9 genome editing technology (see CRISPR/Cas9 editing genome of extremophile Halomonas spp (Qin et al, Metabolic Engineering, 2018)), endogenous glycerol kinase gene (gyk gene) and glycerol-3-phosphate dehydrogenase gene (gpdh gene) were inserted into the G4 site of the strain genome (see CRISPR/Cas9 editing genome of extremophile Halomonas spp (Qin et al, Metabolic Engineering, 2018)) to construct a recombinant strain GLY01, which improves the strain's ability to utilize crude glycerol.

1. Construction of pDK01 plasmid using Gibson assembly method

The backbone of the pDK01 plasmid is pSEVA341 (for plasmid, see the literature: Silva-Rocha, Rafael, et al. “The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes.” Nucleic Acids Research 41. D1 (2013): D666-D675.). The sequence of the inserted plasmid is SEQ ID NO: 13, in which from 5' to 3' end are sgRNA expression module (nucleotides 1-152), upstream homology arm (nucleotides 153-1152), gyk gene promoter module (nucleotides 1153-1249), gyk gene (nucleotides 1250-2800), terminator module (nucleotides 2801-2932), gpdh gene promoter module (nucleotides 2933-3089), gpdh gene (nucleotides 3090-4745), terminator module (nucleotides 4746-4836), and downstream homology arm (nucleotides 4837-5836).

2. Construction of recombinant strain GLY01

The Cas9 protein expression plasmid used in the embodiment is pQ08 plasmid (see Qin Q, Ling C, Zhao Y, et al. CRISPR / Cas9 editing genome of extremophile Halomonas spp [J]. Metabolic engineering, 2018, 47: 219-229.). The pQ08 plasmid and the pDK01 plasmid are sequentially transferred into Halomonas bluephagenesis WZY254 by conjugation transformation, and the gyk and gpdh genes are successfully integrated into the Halomonas bluephagenesis WZY254 genome by PCR and sequencing verification to obtain the recombinant strain GLY01. Then, the strain after successful editing is continuously and repeatedly passaged in non-resistant LB60 liquid culture medium, and streaked and cultured on spectinomycin resistance, chloramphenicol resistance and non-resistant plates, respectively, to identify stable strains with plasmid loss.

3. Recombinant strain GLY01 produces PHB using crude glycerol

The secondary seed liquid of the recombinant strain GLY01 and Halomonas bluephagenesisWZY254 were inoculated into 50 ml fermentation medium at an inoculum of 1%, and the carbon source was 30 g/L glucose or 30 g/L crude glycerol. The pH was adjusted to 8.5 with 5M NaOH solution, 200 rpm, 37°C, and cultured for 48 hours. After 48 hours, the bacteria were collected and the cell dry weight and PHB content were detected. Three parallel samples were set for each group of experiments, and the results were averaged. The results are shown in Table 2.

The results showed that Halomonas bluephagenesis WZY254 was able to utilize glucose to grow and produce PHB well, but its ability to utilize crude glycerol was poor, and its cell dry weight and PHB content were low. The recombinant strain GLY01 had greatly improved its ability to utilize crude glycerol for cell growth and production of PHB, with a cell dry weight of 8.52 g/L and PHB accounting for 54.89% of the cell dry weight.

Table 2 Production of PHB by strain GLY01 using crude glycerol

Example 2: Using a strong promoter to express glycerol kinase and glycerol-3-phosphate dehydrogenase genes to construct a recombinant strain GLY02

Although the ability of the recombinant strain GLY01 to grow and metabolize using crude glycerol was enhanced, the cell dry weight and PHB content were still not as good as those when the strain used glucose. In order to further improve the ability of the strain to use glycerol, the promoters of the gyk gene and gpdh gene were replaced with the strong promoter P _porin140 and inserted into the G4 site of the genome of the WZY254 strain to construct the recombinant strain GLY02.

1. Construction of pDK02 plasmid using Gibson assembly method

The backbone of the pDK02 plasmid is pSEVA341. The sequence inserted into the plasmid is SEQ ID NO: 14, and from 5' to 3' in SEQ ID NO: 14, there are sgRNA expression module (nucleotides 1-152), upstream homology arm (nucleotides 153-1152), P _porin140 promoter module (nucleotides 1153-1347), gyk gene (nucleotides 1348-2898), terminator module (nucleotides 2899-3030), P _porin140 promoter module (nucleotides 3031-3273), gpdh gene (nucleotides 3274-4929), terminator module (nucleotides 4930-5020), and downstream homology arm (nucleotides 5021-6020).

2. Construction of recombinant strain GLY02

The Cas9 protein expression plasmid used in this embodiment is a pQ08 plasmid. The pQ08 plasmid and the pDK02 plasmid were sequentially transferred into Halomonas bluephagenesis WZY254 by conjugation transformation, and the P _porin140 -gyk and P _porin140 -gpdh genes were successfully integrated into the Halomonas bluephagenesis WZY254 genome by PCR and sequencing to obtain the recombinant strain GLY02. Then, the GLY02 strain after successful editing was continuously and repeatedly passaged in a non-resistant LB60 liquid culture medium, and streaked on spectinomycin resistance, chloramphenicol resistance and non-resistant plates, respectively, to identify stable strains with plasmid loss.

3. Production of PHB by recombinant strain GLY02 using crude glycerol

The secondary seed liquid of the recombinant strains GLY01, GLY02 and Halomonas bluephagenesisWZY254 were inoculated into 50 ml fermentation medium at an inoculum of 1%, and the carbon source was 30 g/L glucose or 30 g/L crude glycerol. The pH was adjusted to 8.5 with 5M NaOH solution, 200 rpm, 37°C, and cultured for 48 hours. After 48 hours, the cells were collected and the cell dry weight and PHB content were detected. Three parallel samples were set for each group of experiments, and the results were averaged. The results are shown in Table 3.

The results showed that compared with GLY01, the ability of recombinant strain GLY02 to use crude glycerol for cell growth and production of PHB was further improved, with a cell dry weight of 14.57 g/L and PHB accounting for 82.78% of the cell dry weight. The effect was comparable to the cell dry weight and PHB content of GLY02 using glucose, indicating that the recombinant strain GLY02 can efficiently use crude glycerol to produce PHA.

Table 3 Production of PHB by GLY02 strain using crude glycerol

Example 3: Production of PHB by recombinant strain GLY02 in a 7L fermenter

In order to explore the efficiency of the recombinant strain GLY02 in utilizing crude glycerol during the expansion culture, the recombinant strain GLY02 was subjected to batch fed-batch fermentation to produce PHB in a 7L fermenter, and the carbon source in the cell growth process and product synthesis process was replaced with crude glycerol. The fermentation experiment with glucose as the carbon source was used as a control, and three parallel samples were set up for each group of experiments. After the fermentation, the cell dry weight and PHB content in the fermentation broth were detected, and the results are shown in Table 4.

The results showed that in a 7L fermenter, the recombinant strain GLY02 could still efficiently utilize crude glycerol for cell growth and production of PHB. The cell dry weight reached 102.09 g/L, which was not much different from that when glucose was used; at the same time, the proportion of PHB in the cell dry weight was 89.78%, which was even slightly higher than the PHB content when glucose was used. This result shows that the recombinant strain GLY02 is suitable for expanding the cultivation of crude glycerol to produce PHA. Since crude glycerol is cheap, it can greatly reduce the raw material cost of PHA production.

Table 4 PHB production by 7L fermentation tank experiment of GLY02 strain

Example 4: Production of P3HB4HB by recombinant strain GLY02 in a 7L fermenter

The WZY254 strain can not only produce high PHB, but also produce P3HB4HB containing a higher proportion of 4HB under the condition of adding butyrolactone during the fermentation process. In order to explore the efficiency of the recombinant strain GLY02 in producing P3HB4HB using crude glycerol, the recombinant strain GLY02 was used as a fermentation strain in a 7L fermenter under the conditions of Example 3 to produce P3HB4HB containing 10% 4HB by adding butyrolactone. The fermentation experiment with glucose as the carbon source was used as a control, and three parallel samples were set up for each group of experiments. After the fermentation, the cell dry weight, P3HB4HB content and 4HB molar ratio in the fermentation broth were detected, and the results are shown in Table 5.

The results showed that compared with glucose, under the condition of crude glycerol as the carbon source, the cell dry weight and P3HB4HB yield of the recombinant strain GLY02 were 87.05% and 67.31% respectively, and the molar ratio of 4HB in P3HB4HB could reach 9.89%, which was comparable to the effect when glucose was used as the carbon source.

Table 5 GLY02 strain 7L fermenter experimental production of P3HB4HB

Therefore, the recombinant strain GLY02 can efficiently produce various PHAs such as PHB and P3HB4HB using crude glycerol and can greatly reduce the production cost. At the same time, the consumption and metabolism of crude glycerol is also beneficial to environmental protection.

The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims (10)

1. An engineered halophilic microorganism for the production of a metabolite using crude glycerol, wherein said halophilic microorganism overexpresses glycerol kinase and/or glycerol-3-phosphate dehydrogenase;

The crude glycerol is a byproduct of biodiesel;

The halophilic microorganisms are moderately halophilic microorganisms.

2. The halophilic microorganism according to claim 1, wherein the moderately halophilic microorganism comprises a genus chromobacillus (Chromohalobacter), a genus Halomonas (Halomonas), a genus Vibrio (Halovibrio), a genus Vibrio (Vibrio), a genus cobicillium (Cobetia) or a genus kesteriella (Kushneria);

Preferably, the Halomonas (Halomonas) comprises Halomonas bluephagenesis、Halomonas campaniensis、Halomonas desiderata、Halomonas cupida、Halomonas smymensis、Halomonas levan、Halomonas lutescens、Halomonas elongata、Halomonas venusta or Halomonas alkaliantarctica.

3. A halophilic microorganism according to claim 1 or 2, wherein the glycerol kinase and/or glycerol-3-phosphate dehydrogenase is controlled by a self promoter or by a constitutive promoter or by an inducible promoter;

Preferably, the glycerol kinase and/or glycerol-3-phosphate dehydrogenase is expressed on a plasmid or on a chromosome.

4. A halophilic microorganism according to claim 3, wherein the constitutive promoter comprises a P _porin promoter or a mutant thereof or a P _Sp6 promoter or a variant thereof;

Preferably, the mutant of the P _porin promoter comprises P _porin203、P_porin221、P_porin194、P_porin278、P_porin68、P_porin58、P_porin42 or P _porin140.

5. The halophilic microorganism according to any one of claims 1-4, wherein the metabolite comprises PHA.

6. A method for producing a halophilic microorganism according to any one of claims 1 to 5, wherein the method comprises introducing a nucleotide sequence encoding a glycerol kinase and/or a nucleotide sequence encoding a glycerol-3-phosphate dehydrogenase into the halophilic microorganism.

7. The method of claim 6, wherein the nucleotide sequence encoding glycerol kinase comprises SEQ ID NO:5, and a nucleotide sequence shown in SEQ ID NO.

8. The method of claim 6 or 7, wherein the nucleotide sequence encoding glycerol-3-phosphate dehydrogenase comprises the nucleotide sequence of SEQ ID NO:6, and a nucleotide sequence shown in FIG. 6.

9. Use of a halophilic microorganism according to any one of claims 1 to 5, or obtained by a method according to any one of claims 6 to 8, for the production of a metabolite;

Preferably, the halophilic microorganisms utilize crude glycerol to produce metabolites;

Preferably, the metabolite comprises PHA.

10. A process for producing PHA, characterized in that it comprises culturing a halophilic microorganism according to any one of claims 1 to 5, or a halophilic microorganism obtained by the production process according to any one of claims 6 to 8;

The carbon source used for the culture comprises crude glycerol;

The crude glycerol is a byproduct of biodiesel;

preferably, the biodiesel is obtained by esterifying vegetable oil, animal oil, waste grease or microbial grease with alcohols.