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Escherichia coli engineering bacterium and method for whole-cell catalytic production of steviol

A technology of Escherichia coli that catalyzes stevioside, applied in the field of bioengineering technology and biosynthesis of natural compounds, to achieve clear physical and chemical properties, save cumbersome steps, and simple operation

Active Publication Date: 2019-12-13
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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  • Escherichia coli engineering bacterium and method for whole-cell catalytic production of steviol

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Experimental program
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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 whole-cell catalytic production of steviol by the recombinant escherichia coli engineeringbacterium pSE-sbgl-spbgl1. The strain co-expresses double beta-glucosidase SPBGL1 and SBGL in a polycistron form, and with a stevia rebaudiana crude extract as a substrate, stevioside and rubusosidein the stevia rebaudiana crude extract are catalyzed to be hydrolyzed to produce steviol by using the whole-cell catalysis method. The engineering bacterium pSE-sbgl-spbgl1 is used for catalyzing stevioside to produce steviol in a whole-cell mode, and the method has the advantages of being high in conversion rate, high in yield, single in product, low in energy consumption, short in consumed time,easy to operate, high in 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 Applications(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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