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Method for heterologous production of linear triterpenes

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

Active Publication Date: 2020-07-03
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 includes:

[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 sequences of the primers are shown in Sequence Table 1;

[0032] Subsequently, the CDS fragment was 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, thereby obtaining yeast strain YL-T3-cyp505d13 expressing CYP gene.

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

[0035] i) The pRS426 plasmid was linearized by SmaI, and then the yeast endogenous HXT7p promoter and FBA1t terminator were introduced at both ends thereof.

[0036] The HXT7p promoter and FBA1t...

Embodiment 2

[0046] Product Identification of Fermentation YL-T3-cyp505d13

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

[0048] 2.1.1) Fermentation culture of transformants. Inoculate positive transformants into 4mL SC-HLU seed culture test tubes, culture at 30°C, 220rpm for about 30h, absorb 1mL of cultured seeds, transfer to 50mL of YPD medium, and ferment at 30°C, 220rpm.

[0049] 2.1.2) On the fourth day of fermentation culture, take 20 mL of fermentation broth and add 20 mL of ethyl acetate to 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°C at -0.09Mpa on a rotary evaporator, add...

Embodiment 3

[0052] Determination and Dynamic Accumulation Process of Fermentation Products of Yeast Transformants

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

[0054] Instrument: Agilent 1200 Analytical HPLC; Chromatographic column: Agilent ZORBAX SB C18 reverse-phase chromatographic column (5um, 4.6x250mm)

[0055] Column temperature: 30°C; 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%; 0-30min, 80%-100% phase A; 30-40min, 100% phase A. The detection wavelength is 210nm.

[0058] 3.2) The transformed bacterial strain YL-T3-cyp505d13 comprising CYP and the YL-T3 empty plasmid control bacteria were fermented in the YPD medium, and the growth of the two strains (OD 600 ) and the dynamic process of product accumulation such as image 3 shown. The growth conditions of the YL-T3-cyp505d13 strain ...

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Abstract

The present invention discloses a method for heterogeneous production of linear triterpenes by using a synthetic biology means. Ganoderma lucidum-derived cyp505d13 gene in saccharomyces cerevisiae cells is heterologously expressed, separation, purification, mass spectrometry and nuclear magnetic resonance and other analysis are conducted for fermentation products of yeast engineering strains, andlinear triterpenoid compounds 1, 2, and 3 are determined and produced, wherein the compounds 1 and 2 are never reported. The method comprises a series of designs and verifications from discovery of the cyp505d13 gene to identification of product production and finally to obtaining of the heterologous biosynthetic linear triterpenoids, etc., and also provides an example for the 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 lanosterane type, oleane 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 activity because they contain multiple non-conjugated double bonds (Farvink, 2007). It plays a particularly important role in improving the activity of superoxide dismutase (SOD) in the body, enhancing the body's immunity, drug delivery and skin ca...

Claims

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Application Information

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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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