Truncated sophora flavescens isopentenyl transferase and application thereof

A technology of isopentenyl and transferase, applied in the field of bioengineering, can solve the problem of huge price difference

Active Publication Date: 2021-09-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently on the market, the price of 5mg of 8-PN with a purity of 98% is about 2,000 yuan, while the price of 5g of naringenin with a purity of 98% is about 100 yuan. The price difference between the two is huge.

Method used

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  • Truncated sophora flavescens isopentenyl transferase and application thereof
  • Truncated sophora flavescens isopentenyl transferase and application thereof
  • Truncated sophora flavescens isopentenyl transferase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Construction of embodiment 1 recombinant plasmid pY26-SfN8DT-1 and 7 truncated recombinant plasmids

[0031] (1) Construction of recombinant plasmid pY26-SfN8DT-1

[0032] The isopentenyltransferase SfN8DT-1 (Accession number: B1B3P3.1) derived from Sophora flavescens, whose nucleotide sequence is shown in SEQ ID NO.1, was synthesized by Suzhou Jinweizhi Company, constructed on pY26 plasmid, named For pY26-SfN8DT-1 ( figure 1 ).

[0033] (2) Construction of mutant plasmids

[0034] Using the recombinant plasmid pY26-SfN8DT-1 constructed in step (1) as a template, and the primer sequences used are shown in Table 1, 59, 60, 61, 62, and 81 N-terminal truncations of prenyltransferase were respectively constructed 101 or 110 amino acid mutant plasmids pY26-SfN8DT-1-t59, pY26-SfN8DT-1-t60, pY26-SfN8DT-1-t61, pY26-SfN8DT-1-t62, pY26-SfN8DT-1-t81 , pY26-SfN8DT-1-t101, pY26-SfN8DT-1-t110.

[0035] PCR reaction system: The total PCR system is 50 μL, including 25 μL of high-f...

Embodiment 2

[0041] Example 2 Recombinant truncated isopentenyl transferase expression Saccharomyces cerevisiae construction

[0042] The recombinant plasmid pY26-SfN8DT-1 and the mutant plasmids pY26-SfN8DT-1-t59, pY26-SfN8DT-1-t60, pY26-SfN8DT-1-t61, pY26-SfN8DT-1-t62, pY26-SfN8DT-1-t62, The pY26-SfN8DT-1-t81, pY26-SfN8DT-1-t101, pY26-SfN8DT-1-t110 plasmids were transferred into Saccharomyces cerevisiae PN01 by the Saccharomyces cerevisiae lithium acetate method, and spread on the YNB solid plate (adding 50mg / L Leu ), cultivated at 30°C for 2-3 days, picked positive clones, and constructed recombinant S. , PN01-pY26-ts81, PN01-pY26-ts101 and PN01-pY26-ts110.

Embodiment 3

[0043] Example 3 Recombinant Saccharomyces cerevisiae catalyzes the synthesis of 8-prenylnaringenin from naringenin

[0044] The recombinant Saccharomyces cerevisiae PN01-pY26-SfN8DT-1, PN01-pY26-ts59, PN01-pY26-ts60, PN01-pY26-ts61, PN01-pY26-ts62, PN01-pY26-ts81, PN01- pY26-ts101 and PN01-pY26-ts110 were respectively streaked, and single colonies were picked and transferred into YNB medium, cultured at 30°C and 220rpm for 16-18h to obtain seed liquid. The seed solution was transferred into fresh 25mL YPD medium with an addition of 1% by volume, and cultivated at 30°C and 220rpm. After 120 h of fermentation and cultivation, the fermentation broth was collected, and the 8-prenylnaringenin output was measured by HPLC.

[0045] The result is as figure 2 and image 3 As shown, the N-terminal truncation of prenyltransferase is 59, 60, 61, 62, 81, 101 or 110 amino acids, and the corresponding amino acid positions are H59, N60, L61, K62, E81 , R101, V110, the truncated recombin...

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Abstract

The invention discloses truncated sophora flavescens isopentenyl transferase and application thereof, and belongs to the field of bioengineering. On the basis of isopentenyl transferase SfN8DT-1 from sophora flavescens, truncation of different sites is respectively carried out on an N terminal, and a recombinant plasmid pY26-SfN8DT-1 and seven truncated isopentenyl transferase mutant plasmids are constructed. The recombinant plasmid is transferred into saccharomyces cerevisiae to obtain a recombinant saccharomyces cerevisiae strain for denovo synthesis of 8-isopentenyl naringenin. The recombinant saccharomyces cerevisiae can produce 9.33 mg / L of the 8-isopentenyl naringenin at most after being fermented in a YPD culture medium for 120 hours, the yield is improved by 290.10% compared with that of a control strain PN01-pY26-SfN8DT-1, and the yield of the 8-isopentenyl naringenin is the highest among currently reported yields of biosynthesized 8-isopentenyl naringenin. The strategy lays a foundation for producing the 8-isopentenyl naringenin by transforming the saccharomyces cerevisiae through metabolic engineering.

Description

technical field [0001] The invention relates to a truncated Sophora flavescens isopentenyl transferase and its application, belonging to the field of bioengineering. Background technique [0002] 8-PrenyInaringenin (8-PrenyInaringenin, 8-PN) is a prenylated flavonoid compound, mainly found in plants such as hops and Sophora flavescens, which can prevent cancer and relieve menopausal symptoms in women , prevention of osteoporosis and other effects. At present, the preparation methods of 8-prenyl naringenin are mainly plant extraction method and chemical synthesis method: 1) plant extraction method mainly extracts 8-PN from waste hops, but the content of 8-PN in hops is very low, There is only 0.025-0.060 g of 8-PN per kilogram of hop cones, and the existence of the isomer 6-prenylnaringenin in hops greatly increases the difficulty of the separation and purification of 8-PN; 2) The chemical synthesis method currently uses naringenin or isoxanthohumol as a substrate to synthe...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/81C12N1/19C12P17/06C12R1/865
CPCC12N9/1085C12N15/81C12P17/06
Inventor 周景文陈坚郭超杰高松曾伟主堵国成
Owner JIANGNAN UNIV
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