CN113355266A - Recombinant rhamnolipid producing strain and application thereof - Google Patents
Recombinant rhamnolipid producing strain and application thereof Download PDFInfo
- Publication number
- CN113355266A CN113355266A CN202011470947.0A CN202011470947A CN113355266A CN 113355266 A CN113355266 A CN 113355266A CN 202011470947 A CN202011470947 A CN 202011470947A CN 113355266 A CN113355266 A CN 113355266A
- Authority
- CN
- China
- Prior art keywords
- recombinant
- rhamnolipid
- strain
- hemoglobin
- fermentation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- FCBUKWWQSZQDDI-UHFFFAOYSA-N rhamnolipid Chemical compound CCCCCCCC(CC(O)=O)OC(=O)CC(CCCCCCC)OC1OC(C)C(O)C(O)C1OC1C(O)C(O)C(O)C(C)O1 FCBUKWWQSZQDDI-UHFFFAOYSA-N 0.000 title claims abstract description 51
- 238000000855 fermentation Methods 0.000 claims abstract description 37
- 230000004151 fermentation Effects 0.000 claims abstract description 37
- 108010054147 Hemoglobins Proteins 0.000 claims abstract description 28
- 102000001554 Hemoglobins Human genes 0.000 claims abstract description 21
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 8
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 6
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 5
- 239000013604 expression vector Substances 0.000 claims abstract description 4
- 239000013612 plasmid Substances 0.000 claims description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 11
- 241000894006 Bacteria Species 0.000 claims description 10
- 241000863000 Vitreoscilla Species 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 229910002651 NO3 Inorganic materials 0.000 claims description 6
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 6
- 229910019142 PO4 Inorganic materials 0.000 claims description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
- 159000000003 magnesium salts Chemical class 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 6
- 239000010452 phosphate Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical group [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 5
- 159000000007 calcium salts Chemical class 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 5
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical group [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical group [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 4
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 claims description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 4
- 230000035939 shock Effects 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 229940099596 manganese sulfate Drugs 0.000 claims description 3
- 239000011702 manganese sulphate Substances 0.000 claims description 3
- 235000007079 manganese sulphate Nutrition 0.000 claims description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 230000001737 promoting effect Effects 0.000 claims description 3
- 235000010344 sodium nitrate Nutrition 0.000 claims description 3
- 239000004317 sodium nitrate Substances 0.000 claims description 3
- 235000012424 soybean oil Nutrition 0.000 claims description 3
- 239000003549 soybean oil Substances 0.000 claims description 3
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 3
- 229960001763 zinc sulfate Drugs 0.000 claims description 3
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 2
- 238000009630 liquid culture Methods 0.000 claims description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 2
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 2
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 2
- 235000010333 potassium nitrate Nutrition 0.000 claims description 2
- 239000004323 potassium nitrate Substances 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 239000013598 vector Substances 0.000 abstract description 3
- 230000012010 growth Effects 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 244000005700 microbiome Species 0.000 abstract description 2
- 241001052560 Thallis Species 0.000 abstract 1
- 238000003259 recombinant expression Methods 0.000 abstract 1
- 238000009423 ventilation Methods 0.000 abstract 1
- 238000005273 aeration Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000005265 energy consumption Methods 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 101150112266 vgb gene Proteins 0.000 description 3
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- 239000012880 LB liquid culture medium Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000003876 biosurfactant Substances 0.000 description 2
- 229960002518 gentamicin Drugs 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical class O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 1
- 241000607534 Aeromonas Species 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000005202 decontamination Methods 0.000 description 1
- 230000003588 decontaminative effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 238000005187 foaming Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/78—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Pseudomonas
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of applied microorganisms and genetic engineering, in particular to a recombinant rhamnolipid producing strain and application thereof in fermenting rhamnolipid produced by the strain. The method of the invention connects the hemoglobin gene vgb from Vitrotreoscilla faecalis (Vitroscilla sterncoraria) to an expression vector to construct a recombinant vector PBBR1MCS-5-vgb, and converts the recombinant expression vector into pseudomonas aeruginosa, the expression of hemoglobin promotes the growth of thalli and the synthesis of rhamnolipid, the ventilation and fermentation temperature required by fermentation are reduced, and a new technical scheme is provided for effectively reducing the fermentation production cost of rhamnolipid.
Description
Technical Field
The invention relates to the technical field of applied microorganisms and genetic engineering, in particular to a recombinant rhamnolipid producing strain and application thereof in fermenting rhamnolipid produced by the strain.
Background
Rhamnolipids (rhamnolipids) is a microbially synthesized secondary metabolite belonging to the glycolipid class of biosurfactants. The rhamnolipid used as a biosurfactant has excellent surface/interface activity, foaming, emulsification, emulsion breaking, decontamination, solubilization, metal chelating and good stability under extreme environment, has low toxicity, is environment-friendly and easy to degrade compared with the prior chemical synthesis surfactants which are used in large quantity, and has wide application prospect in the fields of oil exploitation, pollution control, agriculture, industry, food and the like.
At present, the application problem of rhamnolipid is that the market price of rhamnolipid is far higher than that of cheap chemical synthetic surfactant due to lower yield and higher fermentation cost, thereby limiting the large-scale application of rhamnolipid. Therefore, the yield of the rhamnolipid is improved, the energy consumption in the fermentation process is reduced, the fermentation cost is reduced, and the marketization application of the rhamnolipid is facilitated.
The obligate aeromonas Vitreoscilla (Vitreoscilla) can survive in oxygen-deficient conditions because it expresses a hemoglobin (VHb) intracellularly, which increases the oxygen availability of the cell. VHb has a positive promoting effect on cell growth and product synthesis after expression in a variety of heterologous host cells, and therefore, the fermentation product yield can be increased and the required aeration during fermentation can be reduced by introducing hemoglobin, and in addition, the lower temperature conditions can amplify this positive effect of hemoglobin on strain growth and product synthesis. Therefore, the hemoglobin can be effective in reducing the fermentation cost of rhamnolipid to a certain extent.
Disclosure of Invention
The invention aims to provide a recombinant rhamnolipid producing strain, a construction method thereof and application of the strain in fermentation of rhamnolipid. Haemoglobin VHb of Vitreoscilla faecalis (Vitroscilla sterncoraria).
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a recombinant rhamnolipid producing strain contains recombinant plasmid of hemoglobin gene of Vitreoscilla coprophila.
The host strain is pseudomonas aeruginosa.
The recombinant plasmid of the hemoglobin gene of the Vitreoscilla faecalis is obtained by connecting the complete hemoglobin gene vgb of the Vitreoscilla faecalis to an expression vector pBBR1MCS-5 to construct a plasmid pBBR1MCS-5-vgb for expressing hemoglobin.
And (3) transforming the recombinant plasmid into pseudomonas aeruginosa by a calcium chloride heat shock method, and performing resistance screening to obtain the rhamnolipid-producing recombinant bacterium for expressing hemoglobin.
The application of a recombinant rhamnolipid producing strain in promoting the synthesis of rhamnolipid.
Culturing the recombinant strain in a seed solution at 30-37 ℃ and 150-180 rpm for 16-24 hours; then inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 3-5%, and culturing for 5-7 days at the temperature of 26-38 ℃ and the rpm of 100-200.
The seed liquid culture medium comprises, by weight, 0.5-1.2% of yeast powder, 1-2.4% of peptone, 1-2% of sodium chloride and the balance of water;
the fermentation medium comprises, by weight, 3-5% of glycerol, 1-1.5% of soybean oil, 0.3-0.4% of nitrate, 1.5-1.8% of phosphate, 0.1-0.15% of sodium chloride, 0.1-0.2% of potassium chloride, 0.07-0.09% of magnesium salt, 0.01-0.03% of calcium salt, 0.01-0.02% of manganese sulfate, 0.2-0.28% of zinc sulfate, and the balance of water, wherein the pH value is 6.7-7.0.
The nitrate is sodium nitrate and/or potassium nitrate; the magnesium salt is magnesium chloride and/or magnesium sulfate; the calcium salt is calcium carbonate and/or calcium sulfate; the phosphate is one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate and potassium dihydrogen phosphate.
The invention has the following beneficial effects:
the recombinant pseudomonas aeruginosa expression hemoglobin is obtained by using a genetic engineering technology, and the obtained recombinant strain is used for culturing to improve the oxygen utilization rate of the recombinant strain, so that the rhamnolipid yield is improved, the stirring amount and temperature required by fermentation are reduced, the fermentation production cost of the rhamnolipid is greatly reduced, and the large-scale and marketization application of the rhamnolipid is facilitated.
Drawings
FIG. 1 is a diagram of the structure of pBBR1MCS-5-vgb according to the embodiment of the present invention.
FIG. 2 is a PCR-verified electrophoresis chart of the vector pBBR1MCS-5-vgb introduced into Pseudomonas aeruginosa according to the embodiment of the present invention.
Fig. 3 is a rhamnolipid standard curve provided by the embodiment of the present invention.
Detailed Description
The following examples are presented to further illustrate embodiments of the present invention, and it should be understood that the embodiments described herein are for purposes of illustration and explanation only and are not intended to limit the invention.
The materials used in the following examples are commercially available without specific reference.
Example 1: construction of hemoglobin-expressing plasmid pBBR1MCS-5-vgb
Using primers vgb-f: CGCGGATCCGGAAGACCCTCATGTTAGA, vgb-r: ATTATCTAGATTATTCAACCGCTTGAGCGTA, obtaining a complete vgb gene fragment from Vitreoscilla faecalis (ATCC 15218) by PCR, and connecting the fragment to a cloning vector pMD19-T to obtain a recombinant vector pMD 19-T-vgb.
The plasmid pBBR1MCS-5 and pMD19-T-vgb are subjected to double enzyme digestion by BamHI and SalI, a kit is used for recovering a large fragment after the enzyme digestion of the PBBR1MCS-5 after the enzyme digestion, pMD19-T-vgb is used for cutting glue to recover a vgb fragment, and the recovered two fragments are connected by T4 ligase to obtain the recombinant plasmid pBBR1MCS-5-vgb (see figure 1).
Example 2: construction of Pseudomonas aeruginosa engineering bacteria for expressing hemoglobin
The recombinant plasmid pBBR1MCS-5-vgb of the embodiment 1 is applied to a calcium chloride heat shock method to transform the pseudomonas aeruginosa competence, an LB solid plate containing 50 mu g/mL gentamicin is coated, the constant temperature culture is carried out at 37 ℃ until a transformant grows out, and the pseudomonas aeruginosa engineering bacteria for expressing VHb are obtained through PCR verification. The specific method comprises the following steps:
(1) the pseudomonas aeruginosa is inoculated into 5mL LB liquid culture medium for activated culture under the culture condition of 37 ℃ and 180 rpm. (2) The inoculating loop is dipped with the activated bacteria liquid and streaked on an LB plate, and cultured overnight at 37 ℃. (3) The single colony was inoculated into LB liquid medium and cultured at 37 ℃ and 180 rpm. (4) Transferring the obtained culture solution into a fresh LB liquid culture medium at a ratio of 1:100(v/v), and culturing for 2-3 h to OD600About 0.5. (5) The culture solution was transferred to a 50mL centrifuge tube and placed on ice for 20 min. (6) Then, the mixture was centrifuged at 4 ℃ and 3000r/min for 10min, and the supernatant was discarded. (7) 5mL of precooled 0.1mol/L calcium chloride is added into the collected precipitate, the bacterial liquid is gently washed, centrifuged for 10min, and then the supernatant is discarded. (8) 10mL of precooled calcium chloride is added again to resuspend the cells, the cells are placed on ice for 20min, centrifuged for 15min, and the supernatant is discarded. (9) The cells were resuspended in 50% glycerol and calcium chloride solution (3:7v/v) and placed on ice for 10 min. (10) About 10. mu.L of plasmid DNA obtained in the above example was added to the cell resuspension of the above step (9), mixed well, and then allowed to stand on ice for 30 min. (11) Quickly placing on ice for cooling for 5min after heat shock in 42 ℃ water bath for 90 seconds, finally adding sterile SOC culture medium to the total volume of 900 mu L, and carrying out shake culture at 37 ℃ and 100rpm for 1 h. (12) And (3) taking 100 mu L of the bacterial liquid, coating a gentamicin resistant plate, standing for 1-2 h with the front side upward, inverting, and culturing at constant temperature of 37 ℃ until a single colony grows out. (13) The single colony grown out was picked and verified with vgb-r/vgb-f primer, vgb-f: CGCGGATCCGGAAGACCCTCATGTTAGA, vgb-r: ATTATCTAGATTATTCAACCGCTTGAGCGTA are provided.
Example 3: application of expression hemoglobin in improving rhamnolipid yield
Inoculating wild Pseudomonas aeruginosa and the hemoglobin-expressing strain obtained in example 2 into 250mL of flasks containing 100mL of seed medium, respectively, under 37 ℃ and 180 rpm; each culture experiment was performed in triplicate. The next day, inoculating the seed solution into 250mL of fermentation medium containing 150mL by using the inoculation amount of 3 wt%, culturing for 7 days at 37 ℃, wherein the rotation speed of a shaking table is 180rpm, the experiment is a three-time parallel experiment, and after the fermentation is finished, measuring the yield of wild pseudomonas aeruginosa and engineering bacteria rhamnolipid by using an oil-discharge ring method.
The fermentation medium comprises the following components in percentage by weight: 1.2% of yeast powder, 2.4% of peptone, 1% of sodium chloride and the balance of water; the fermentation medium comprises 2.5% of glycerol, 1% of soybean oil, 0.35% of nitrate, 1.7% of phosphate, 0.12% of sodium chloride, 0.15% of potassium chloride, 0.08% of magnesium salt, 0.02% of calcium salt, 0.012% of manganese sulfate, 0.25% of zinc sulfate and the balance of water, wherein the pH value is 6.7-7.0, the nitrate is sodium nitrate, the magnesium salt is magnesium sulfate, and the phosphate is potassium dihydrogen phosphate and dipotassium hydrogen phosphate.
The result shows that the rhamnolipid yield of the recombinant strain at the end of fermentation is 31.25 +/-0.063 g/L, the rhamnolipid yield of wild pseudomonas aeruginosa is 23.17 +/-0.046 g/L, and the rhamnolipid yield of the hemoglobin-expressing strain is improved by 30%.
Example 4: application of expression hemoglobin in reducing fermentation temperature
Wild-type P.aeruginosa and the hemoglobin-expressing strain obtained in example 2 were inoculated into 250mL of a flask containing 100mL of seed medium, respectively, under 37 ℃ and 180 rpm. The next day, inoculating the seed solution into 250mL of fermentation medium containing 150mL by using the inoculation amount of 3%, culturing for 7 days at 25-37 ℃ at different temperatures, setting the rotation speed of a shaking table to be 180rpm, setting three parallel fermentation temperatures, and measuring the yield of the original strain and the rhamnolipid of the engineering bacteria fermentation liquor under different temperature conditions by using an oil-discharge ring method after fermentation is finished.
The result shows that the recombinant bacterium can achieve the maximum rhamnolipid yield of 32.73 +/-0.078 g/L under the fermentation condition of 30 ℃, and is slightly higher than the maximum rhamnolipid yield (31.25 +/-0.063 g/L) of wild pseudomonas aeruginosa under the fermentation condition of 37 ℃, so that the optimal fermentation temperature of the rhamnolipid is reduced by 7 ℃ by adopting the expression of the recombinant hemoglobin, which is favorable for reducing the energy consumption in the fermentation process and the fermentation cost of the rhamnolipid.
Example 5: use of expressed hemoglobin for reducing upper agitation/aeration
Wild-type P.aeruginosa and the hemoglobin-expressing strain obtained in example 2 were inoculated into 250mL of a flask containing 100mL of seed medium, respectively, under 37 ℃ and 180 rpm. The next day, inoculating the seed solution into a 250mL triangular flask containing 150mL fermentation medium with the inoculation amount of 3%, culturing for 7 days at 30 ℃, performing at different table rotation speeds (50rpm,100rpm,150rpm,200rpm and 250rpm), setting three parallel rotation speed conditions, and measuring the yield of wild pseudomonas aeruginosa and engineering bacteria rhamnolipid under different rotation speeds/aeration conditions by an oil-discharge ring method after fermentation is finished.
The result shows that the recombinant strain can reach 31.75 +/-0.059 g/L of rhamnolipid yield under the condition of the rotation speed of 150rpm, while the wild pseudomonas aeruginosa can reach the maximum rhamnolipid yield (31.17 +/-0.033 g/L) under the rotation speed/aeration condition of 200rpm, so that the expression of hemoglobin effectively reduces the stirring amount/aeration amount required by fermentation, which is beneficial to reducing the foam generation and the fermentation energy consumption in the rhamnolipid fermentation process, and the fermentation cost of the rhamnolipid can be greatly reduced.
Claims (8)
1. A recombinant rhamnolipid producing bacterium, which is characterized in that: the recombinant strain contains a recombinant plasmid of a hemoglobin gene of Vitreoscilla coprophila.
2. The recombinant rhamnolipid-producing strain according to claim 1, characterized in that: the host strain is pseudomonas aeruginosa.
3. The recombinant rhamnolipid-producing strain according to claim 1 or 2, characterized in that: the recombinant plasmid of the hemoglobin gene of the Vitreoscilla faecalis is obtained by connecting the complete hemoglobin gene vgb of the Vitreoscilla faecalis to an expression vector pBBR1MCS-5 to construct a plasmid pBBR1MCS-5-vgb for expressing hemoglobin.
4. The recombinant rhamnolipid-producing strain according to claim 3, characterized in that: and (3) transforming the recombinant plasmid into pseudomonas aeruginosa by a calcium chloride heat shock method, and performing resistance screening to obtain the rhamnolipid-producing recombinant bacterium for expressing hemoglobin.
5. Use of the recombinant rhamnolipid producer of claim 1, characterized in that: the recombinant rhamnolipid producing strain is applied to promoting the synthesis of rhamnolipid.
6. Use of the recombinant rhamnolipid producer according to claim 5, characterized in that: culturing the recombinant strain in a seed solution at 30-37 ℃ and 150-180 rpm for 16-24 hours; then inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 3-5%, and culturing for 5-7 days at the temperature of 26-38 ℃ and the rpm of 100-200.
7. Use of the recombinant rhamnolipid producer according to claim 5, characterized in that: the seed liquid culture medium comprises, by weight, 0.5-1.2% of yeast powder, 1-2.4% of peptone, 1-2% of sodium chloride and the balance of water;
the fermentation medium comprises, by weight, 3-5% of glycerol, 1-1.5% of soybean oil, 0.3-0.4% of nitrate, 1.5-1.8% of phosphate, 0.1-0.15% of sodium chloride, 0.1-0.2% of potassium chloride, 0.07-0.09% of magnesium salt, 0.01-0.03% of calcium salt, 0.01-0.02% of manganese sulfate, 0.2-0.28% of zinc sulfate, and the balance of water, wherein the pH value is 6.7-7.0.
8. Use of the recombinant rhamnolipid producer according to claim 7, characterized in that: the nitrate is sodium nitrate and/or potassium nitrate; the magnesium salt is magnesium chloride and/or magnesium sulfate; the calcium salt is calcium carbonate and/or calcium sulfate; the phosphate is one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate and potassium dihydrogen phosphate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011470947.0A CN113355266A (en) | 2020-12-14 | 2020-12-14 | Recombinant rhamnolipid producing strain and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011470947.0A CN113355266A (en) | 2020-12-14 | 2020-12-14 | Recombinant rhamnolipid producing strain and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113355266A true CN113355266A (en) | 2021-09-07 |
Family
ID=77524572
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011470947.0A Pending CN113355266A (en) | 2020-12-14 | 2020-12-14 | Recombinant rhamnolipid producing strain and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113355266A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030082611A1 (en) * | 1994-02-17 | 2003-05-01 | Affymax Technologies N.V. | Methods and compositions for cellular and metabolic engineering |
| CN101397548A (en) * | 2008-10-23 | 2009-04-01 | 江南大学 | Recombinant corynebacterium crematum by expression of vitreoscilla haemoglobin gene and use thereof |
| CN102041264A (en) * | 2009-10-10 | 2011-05-04 | 华东理工大学 | Genetic engineering bacterium of Gluconobacter oxydans (G.oxydans) and application thereof |
| CN104830889A (en) * | 2015-03-06 | 2015-08-12 | 西安海格生物技术研究所有限公司 | Genetic recombinant method of pseudomonas aeruginosa for high-yield producing rhamnolipid |
| CN105348384A (en) * | 2015-12-01 | 2016-02-24 | 山东隆科特酶制剂有限公司 | Vitreoscilla hemoglobin mutant and controllable expression of vitreoscilla hemoglobin mutant in genetically engineered bacteria |
-
2020
- 2020-12-14 CN CN202011470947.0A patent/CN113355266A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030082611A1 (en) * | 1994-02-17 | 2003-05-01 | Affymax Technologies N.V. | Methods and compositions for cellular and metabolic engineering |
| CN101397548A (en) * | 2008-10-23 | 2009-04-01 | 江南大学 | Recombinant corynebacterium crematum by expression of vitreoscilla haemoglobin gene and use thereof |
| CN102041264A (en) * | 2009-10-10 | 2011-05-04 | 华东理工大学 | Genetic engineering bacterium of Gluconobacter oxydans (G.oxydans) and application thereof |
| CN104830889A (en) * | 2015-03-06 | 2015-08-12 | 西安海格生物技术研究所有限公司 | Genetic recombinant method of pseudomonas aeruginosa for high-yield producing rhamnolipid |
| CN105348384A (en) * | 2015-12-01 | 2016-02-24 | 山东隆科特酶制剂有限公司 | Vitreoscilla hemoglobin mutant and controllable expression of vitreoscilla hemoglobin mutant in genetically engineered bacteria |
Non-Patent Citations (2)
| Title |
|---|
| H. KAHRAMAN等: "Rhamnolipid Production by Pseudomonas aeruginosa Engineered with the Vitreoscilla Hemoglobin Gene", 《APPLIED BIOCHEMISTRY AND MICROBIOLOGY》 * |
| 冯蕾等: "含血红蛋白基因（VHb）的铜绿假单胞菌重组菌的构建及鼠李糖脂表达条件的研究", 《工业微生物》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108102995B (en) | D-psicose 3-epimerase production strain and immobilization method thereof | |
| KR102030776B1 (en) | A Method of Producing Lactobionic Acid Using Pseudomonas taetrolens By Optimization of Culture Condition | |
| US20130089901A1 (en) | Novel thraustochytrid-based microalgae, and method for preparing bio-oil by using same | |
| CN105331572B (en) | A method of fermentation high yield DHA | |
| CN110117550B (en) | Process for producing phloretin based on saccharomyces cerevisiae fermentation and saccharomyces cerevisiae | |
| CN106434510A (en) | Genetically engineered bacterium for producing L-aspartic acid through fermentation | |
| CN112941119B (en) | Method for increasing yield of fatty acid ethyl ester of saccharomyces cerevisiae engineering bacteria | |
| CN107227286B (en) | Genetically engineered bacterium capable of producing succinic acid at high yield, and construction method and application thereof | |
| CN113355266A (en) | Recombinant rhamnolipid producing strain and application thereof | |
| CN105555947A (en) | High expression zymomonas promoters | |
| JP2023534879A (en) | N-Acetylglucosamine-producing strain and method for construction and use thereof | |
| CN102676408A (en) | Method for producing rhodotorula benthica by subsurface fermentation of high-density liquid | |
| JPS6018397B2 (en) | How to convert D-glucose to D-fructose | |
| CN104293817B (en) | Construction methods and uses of recombinant plasmid for producing succinic acid, and genetic engineering bacterium | |
| CN114107356B (en) | Method for transforming pseudomonas putida to assimilate D-galactose | |
| CN112852668B (en) | Acid-resistant streptomyces albidoflavus and application thereof in epsilon-polylysine fermentation | |
| CN110885765B (en) | Rhodotorula and culture method thereof and application of rhodotorula in production of biological grease | |
| CN110452862B (en) | Pseudomonas fluorescens strain and application thereof | |
| CN109161570B (en) | Method for improving fermentation production of N-acetylneuraminic acid and fermentation liquor | |
| CN101892250A (en) | Method for constructing cryptococcus albidus engineering bacterial strain for producing oleic acid in high yield | |
| CN110982705A (en) | Fermentation process of schizochytrium limacinum | |
| CN113293119B (en) | Bacterial high-density liquid fermentation medium and fermentation method thereof | |
| Dyrset et al. | Development of a fermentation process for production of an alginate G-lyase from Klebsiella pneumoniae | |
| CN106754838B (en) | Method for improving protease producing capability of aspergillus niger | |
| CA2173793C (en) | Pentose fermentation by recombinant zymomonas |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210907 |
|
| RJ01 | Rejection of invention patent application after publication |