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A strain of Escherichia coli engineering bacteria and its whole cell catalytic production method of steviol

A technology that catalyzes stevioside with Escherichia coli, which is applied in the field of bioengineering technology and the biosynthesis of natural compounds to achieve high conversion efficiency, save cumbersome steps, and high yield

Active Publication Date: 2021-08-17
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Application CN108707163A repeated the method of Zhang Zongying and others to verify the generation of isosteviol, and found that there were other by-products through hydrolysis of hydrochloric acid or sulfamic acid. In order to improve other by-products including isosteviol, acidolysis, silicon-based Steviol is prepared in 4 steps of oxidization, epoxidation and reduction. Although it can effectively solve the problem of by-products, the process involves a variety of organic reagents or other chemical reagents.

Method used

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  • A strain of Escherichia coli engineering bacteria and its whole cell catalytic production method of steviol

Examples

Experimental program
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Effect test

Embodiment 1

[0032] The β-glucosidase SPBGL1 (accession number WP_029622673.1) with stevioside hydrolysis activity was cloned from Sphingomonas elodea ATCC 3146. According to the report of patent ZL201210387271.8, the target gene sbgl (accession number KC986399) was cloned.

[0033] A strain of Escherichia coli engineering bacteria, its construction method operation steps are as follows:

[0034] (a) Clone the target gene sbgl, design the upstream primer sbgl-F (including NcoI restriction site) and the downstream primer sbgl-R (including EcoRI), and use pUC19-Ebgl1 as a template to amplify the target gene sbgl by PCR. After endonuclease NcoI and EcoRI target gene sbgl, it was ligated with the expression vector pSE380 that had also been digested by NcoI and EcoRI to obtain recombinant plasmid pSE-sbgl, which was transformed into Escherichia coli JM109;

[0035] sbgl-F: 5′-AACA CCATGG ATATGCAGGAAGCCGGTGCCCCGC-3′

[0036] sbgl-R:5′-ATT GAATTC TCAATCCTTCCAGCTACGCGCCGG-3′

[0037] (b) Clo...

Embodiment 2

[0041] Utilize embodiment 1 to construct the method for the co-expressed Escherichia coli engineering bacteria pSE-sbgl-spbgl1 catalyzed stevioside to produce steviol, the operation steps are as follows:

[0042] (1) 20°C, 0.5mM IPTG induced culture for 22 hours obtained in Example 1 co-expressed Escherichia coli engineered bacteria pSE-sbgl-spbgl1, after the fermentation was completed, the bacteria were collected and the wet weight of the bacteria was weighed;

[0043] (2) Wash the 0.1g wet weight thalli 3 times with 0.9% NaCl solution, 2 HPO 4 -resuspended bacteria in citric acid buffer to obtain 0.02g / ml co-expressed Escherichia coli engineering bacteria pSE-sbgl-spbgl1 whole cell catalytic solution; Na 2 HPO 4 - 0.2M Na in citrate buffer 2HPO 4 and 0.1M citric acid, pH 6.0;

[0044] (3) Adding stevioside at a concentration of 50 g / L to the whole-cell catalytic solution of the co-expressed Escherichia coli engineering bacteria pSE-sbgl-spbgl1 obtained in step (2), and ...

Embodiment 3

[0047] Utilize embodiment 1 to construct the method for the co-expressed Escherichia coli engineering bacteria pSE-sbgl-spbgl1 catalyzed stevioside to produce steviol, the operation steps are as follows:

[0048] (1) 20°C, 0.5mM IPTG induced culture for 22 hours obtained in Example 1 co-expressed Escherichia coli engineered bacteria pSE-sbgl-spbgl1, after the fermentation was completed, the bacteria were collected and the wet weight of the bacteria was weighed;

[0049] (2) Wash the 0.1g wet weight thalli 3 times with 0.9% NaCl solution, 2 HPO 4 -resuspended bacteria in citric acid buffer to obtain 0.02g / mL co-expressed Escherichia coli engineering bacteria pSE-sbgl-spbgl1 whole cell catalytic solution; Na 2 HPO 4 - 0.2M Na in citrate buffer 2 HPO 4 and 0.1M citric acid, pH 5.5;

[0050] (3) Adding stevioside at a concentration of 100 g / L to the whole-cell catalytic solution of the co-expressed Escherichia coli engineering bacteria pSE-sbgl-spbgl1 obtained in step (2), an...

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Abstract

The invention discloses a recombinant Escherichia coli engineering bacterium pSE‑sbgl‑spbgl1 and a method for catalyzing the production of steviol by using the whole cell of the strain. The whole-cell catalysis method uses the crude stevia rebaudiana extract as a substrate to catalyze the hydrolysis of stevioside and rubusoside to produce steviol. The present invention uses engineering bacteria pSE-sbgl-spbgl1 whole cells to catalyze stevioside to produce steviol, which has the advantages of high conversion rate, high yield, single product, low energy consumption, short time consumption, simple operation, high industrialization potential and the like.

Description

technical field [0001] The invention relates to the field of bioengineering technology and biosynthesis of natural compounds, in particular to a strain of Escherichia coli engineering bacteria and a method for producing steviol by catalyzing whole cells thereof. Background technique [0002] Stevia is a perennial herb native to Paraguay, South America (LEWIS W H. Early Uses of Stevia rebaudiana (Asteraceae) Leaves as a Sweetener in Paraguay [J]. Economic Botany, 1992, 46 (3): 336-7), It is also planted in Southwest my country, one of which is commonly known as sweet tea, which exists in large quantities in Guangxi (LIU Z, SCHWIMER J, LIU D, et al.Gallic acid is partially responsible for the antiangiogenic activities of Rubus leaf extract[J].Phytother Res , 2006, 20(9):806-13). Stevioside is a low-calorie sweetener extracted from stevia leaves. According to research, its sweetness is 200-350 times that of sucrose, but its calories are only 1 / 300 of that of sucrose (ORGANIZATIO...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P15/00C12R1/19
CPCC12N9/2445C12P15/00C12Y302/01021
Inventor 杜丽琴兰青文蔓蔓黄日波庞浩周洁韦宇拓
Owner GUANGXI UNIV
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