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Recombinant vibrio natriticus for producing hydroxytyrosol and application of recombinant vibrio natriticus

A technology for producing hydroxytyrosol and sodium-requiring Vibrio, which is applied in the field of bioengineering, can solve the problem that the yield of biosynthetic hydroxytyrosol needs to be improved, etc., and achieves the effects of shortening the fermentation period, increasing the yield and increasing the growth speed.

Active Publication Date: 2022-08-09
SHENZHEN BLUEPHA BIOSCIENCES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the yield of biosynthetic hydroxytyrosol still needs to be improved

Method used

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  • Recombinant vibrio natriticus for producing hydroxytyrosol and application of recombinant vibrio natriticus
  • Recombinant vibrio natriticus for producing hydroxytyrosol and application of recombinant vibrio natriticus
  • Recombinant vibrio natriticus for producing hydroxytyrosol and application of recombinant vibrio natriticus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Construction of recombinant Vibrio sodium-requiring Vmax-HT and Escherichia coli E.coli-HT

[0050] 1. Download the sequences of the target genes HpaB, HpaC and FDH from the NCBI database and perform gene synthesis, wherein the genes HpaB (NCBI accession number: WP_000801472.1) and HpaC (NCBI accession number: WP_001175451.1) are derived from Escherichia coli, and the gene FDH (NCBI accession number: WP_009957650.1) is derived from Mycobacterium intracellulare.

[0051] 2. The synthetic gene was cloned and amplified, and the fragment was inserted between SacI and HindIII in the plasmid pETDuet using the Gibson assembly method to obtain the plasmid pETDuet-HpaB-HpaC-FDH.

[0052] 3. Clone and amplify the arabinose promoter sequence (SEQ ID NO. 1), clone and amplify the part of the plasmid pETDuet-HpaB-HpaC-FDH except the T7 promoter, and pass the amplified two pieces through Gibson assembly method was used for splicing to obtain plasmid pETDuet-pBAD-HpaB-HpaC-...

Embodiment 2

[0055] Example 2 Analysis of the growth of recombinant Vibrio natriureus Vmax-HT and recombinant Escherichia coli E.coli-HT

[0056] 1. The recombinant strains Vmax-HT and E.coli-HT constructed in Example 1 were taken out from the -80°C glycerol tube, streaked on an LB solid plate, and cultured at 37°C overnight. Then single clones were picked from the plate and then received 3ml LBv2 (peptone 10g / L, yeast extract 5g / L, sodium chloride 11.92g / L, potassium chloride 0.313g / L, magnesium chloride 2.203g / L) and LB (peptone 5g / L) 10g / L, yeast extract 5g / L, sodium chloride 10g / L) liquid medium, 37°C, 220rpm for activation.

[0057] 2. Connect the activated bacterial liquid of recombinant strains Vmax-HT and E.coli-HT to 20mL LBv2 and LB medium respectively, and control the initial OD 600 0.01, 37°C, 220rpm, culture in 150mL shake flask. Subsequent samples were taken at different time points to determine OD 600 value.

[0058] OD obtained at different time points 600 value to dra...

Embodiment 3

[0060] Example 3 Analysis of hydroxytyrosol degradation ability of recombinant Vibrio natriuresis Vmax-HT and recombinant Escherichia coli E.coli-HT

[0061] 1. The recombinant strains Vmax-HT and E.coli-HT were activated according to the method described in Example 2.

[0062] 2. Inoculate the activated bacterial liquid of recombinant strains Vmax-HT and E.coli-HT into 40 mL of LBv2 and LB medium respectively at 1%, cultivate at 37° C. and 220 rpm for 16 hours, and measure the OD. 600 value.

[0063] 3. According to the bacteria liquid OD 600 Take an appropriate amount of bacterial liquid and centrifuge at 4000 rpm for 10 min to collect the bacterial cells, so that the two bacterial strains have an equal amount of bacterial cells, and resuspend the bacterial cells with a 20 mL reaction system to make the final OD. 600 All equal to 4, and the cell-free reaction system was used as a control to incubate at 30°C and 220rpm. The above reaction system includes: disodium hydrogen...

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Abstract

The invention relates to the technical field of bioengineering, in particular to a recombinant vibrio natriureticus for producing hydroxytyrosol and application of the recombinant vibrio natriureticus. The invention provides a novel application of vibrio natriticus in production of hydroxytyrosol. The vibrio natriticus engineering bacterium obtained through transformation can be used for whole-cell catalytic synthesis of hydroxytyrosol, can efficiently promote accumulation of hydroxytyrosol, and can also be used as a chassis bacterium for constructing a hydroxytyrosol production strain. According to the method for producing the hydroxytyrosol based on the vibrio natriureticus, the production period of the hydroxytyrosol can be shortened while the yield of the hydroxytyrosol is guaranteed, the yield of the hydroxytyrosol is increased, and the removal difficulty of downstream endotoxin is reduced.

Description

technical field [0001] The invention relates to the technical field of biological engineering, in particular to a recombinant sodium-requiring vibrio for producing hydroxytyrosol and an application thereof. Background technique [0002] Hydroxytyrosol (HT) is a natural polyphenolic compound, its chemical name is 3,4-dihydroxyphenethyl alcohol, and its molecular formula is C 4 H 10 O 3 , the relative molecular mass is 154.16. [0003] Hydroxytyrosol is a light yellow oily liquid, which is an amphiphilic molecule with good water solubility and fat solubility. Epidemiological surveys have found that residents of Mediterranean countries who consume olive oil for a long time have lower morbidity and mortality rates of cardiovascular diseases, cancer, etc. This health effect is related to the polyphenolic compound hydroxytyrosol rich in olive oil. closely related. As a polyphenolic compound, hydroxytyrosol has good antioxidant properties, and studies have found that it has th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/53C12P7/22C12R1/63
CPCC12N9/0071C12N9/0008C12N15/74C12P7/22C12Y114/14009C12Y102/01002C12R2001/63Y02A50/30
Inventor 崔潇艺宗朕谈畅樊兵简依敏滕欣
Owner SHENZHEN BLUEPHA BIOSCIENCES CO LTD
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