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Method for heterologous production of linear triterpenes

A linear and triterpenoid technology, applied in the field of bioengineering, can solve the problems of immature genetic manipulation and research lag

Active Publication Date: 2022-08-09
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the immaturity of genetic manipulation, the functional identification of its CYP lags behind related studies in plants and prokaryotes (Hao Q, 2017)

Method used

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  • Method for heterologous production of linear triterpenes
  • Method for heterologous production of linear triterpenes
  • Method for heterologous production of linear triterpenes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Construction of recombinant yeast strains

[0030] Construction of yeast strains expressing CYP genes include:

[0031] Using the cDNA library of Ganoderma lucidum 5.616 as a template, the coding region sequence (CDS) fragment of cyp505d13 was obtained by PCR amplification with primer GL17184-F / R. The specific sequence of the primer is shown in Sequence Table 1;

[0032] The CDS fragments were then recombined into the expression vector pRS426 and verified by sequencing. The recombinant plasmid pRS426HF-cyp505d13 was obtained;

[0033] The recombinant plasmid was transformed into yeast strain YL-T3 to obtain yeast strain YL-T3-cyp505d13 expressing CYP gene.

[0034] Described recombinant plasmid pRS426HF-CYP17184 is obtained specifically in the following manner:

[0035] i) The pRS426 plasmid was linearized by SmaI followed by introduction of the yeast endogenous HXT7p promoter and FBA1t terminator at both ends.

[0036] Described HXT7p promoter and FBA1t terminator fr...

Embodiment 2

[0046] Product identification by fermentation of YL-T3-cyp505d13

[0047] YL-T3-cyp505d13 fermentation: 2.1) The constructed YL-T3-cyp505d13 yeast transformants were fermented in YPD medium (1% yeast powder, 2% beef peptone, 2% glucose), and the empty plasmid without CYP gene was used The strains were used as controls to compare the differences in metabolites between them. The specific operations were as follows:

[0048] 2.1.1) Fermentation culture of transformants. The positive transformants were inoculated into a 4mL SC-HLU seed culture test tube, cultured at 30°C and 220rpm for about 30h, and 1mL of the cultured seeds were aspirated, transferred to 50mL of YPD medium, and fermented at 30°C and 220rpm.

[0049] 2.1.2) On the 4th day of fermentation, take 20 mL of fermentation broth and add 20 mL of ethyl acetate and shake at 220 rpm for 30 min. Centrifuge at 5000rpm for 5min and absorb the upper organic phase, transfer to a rotary evaporator, spin dry ethyl acetate at 40°...

Embodiment 3

[0052] Fermentation product determination and dynamic accumulation process of yeast transformants

[0053] 3.1) High-performance liquid chromatography analysis method of fermentation product:

[0054] Instrument: Agilent 1200 analytical HPLC; Column: Agilent ZORBAX SB C18 reversed-phase column (5um, 4.6x250mm)

[0055] Column temperature: 30℃; flow rate: 1mL / min; injection volume: 20μL;

[0056] Phase A: methanol (containing 0.1% acetic acid), phase B: pure water;

[0057] Gradient elution program: initial phase A 80%, phase B 20%; 0-30 min, 80%-100% phase A; 30-40 min, 100% phase A. Detection wavelength 210nm.

[0058] 3.2) The transformed strain YL-T3-cyp505d13 containing CYP and the YL-T3 empty plasmid control bacteria were fermented in YPD medium, and the two strains grew (OD 600 ) and the dynamic process of product accumulation such as image 3 shown. YL-T3-cyp505d13 strain (solid line) and empty plasmid control strain YL-T3-Void plasmid (dashed line) grew similarly...

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Abstract

A method for discovering and heterologous production of linear triterpenes by means of synthetic biology, heterologous expression of cyp505d13 gene derived from Ganoderma lucidum in Saccharomyces cerevisiae cells, separation and purification, mass spectrometry and nuclear magnetic resonance analysis of fermentation products of yeast engineering strains, It was determined that linear triterpenoids 1, 2 and 3 were produced, of which compounds 1 and 2 had never been reported. The present invention includes a series of design and verification from the discovery of cyp505d13 gene, to product production identification, and finally obtaining heterologous biosynthetic linear triterpenoid substances, and also provides an example for heterologous production of other linear triterpenoid active substances.

Description

technical field [0001] The invention relates to a technology in the field of bioengineering, in particular to a method for biosynthesizing linear triterpenoids. Background technique [0002] Triterpenoids are the most widely distributed natural products, a class of C30 compounds with diverse structures and nearly 20,000 members (Sumit, 2016). According to their structural differences, triterpenoids can be divided into linear triterpenes (squalene type) and cyclic triterpenes (such as lanostane type, olean type, ursane type, rubutane type). Triterpenoids have many promising biological activities, including anticancer, antioxidant, anti-inflammatory, etc. (Malwina, 2015). Many of these linear triterpenes have strong antioxidant activities due to their multiple non-conjugated double bonds (Favink, 2007). It has a particularly important role in enhancing superoxide dismutase (SOD) activity in vivo, enhancing immunity, drug delivery and skin care (Bindubscm, 2015). Therefore, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/81C12P17/02C07D303/14C07D303/04
CPCC12N9/0071C12N15/81C12P17/02C07D303/14C07D303/04
Inventor 肖晗沈瑛宋欣
Owner SHANGHAI JIAO TONG UNIV
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