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Method for synthesizing rebaudioside-A through high-density fermentation

A high-density fermentation and rebaudioside technology, applied in the field of bioengineering, can solve the problems of complex intracellular substances, unfavorable extraction and purification, and limited intracellular accumulation, and achieve high conversion rate, increased efficiency, and small cell damage Effect

Pending Publication Date: 2018-09-04
XINGHUA GL STEVIA CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, rebaudioside A, which is metabolized and synthesized by fermentation method, mainly exists in cells, and the intracellular accumulation is limited and the intracellular substances are complex, which is not conducive to extraction and purification (Lehmann M, Trueheart J, Zwartjens P, et al.DITERPENEPRODUCTION:WO, EP2806754[P].2014.)

Method used

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  • Method for synthesizing rebaudioside-A through high-density fermentation
  • Method for synthesizing rebaudioside-A through high-density fermentation
  • Method for synthesizing rebaudioside-A through high-density fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Knockout of a gene encoding a cell wall synthetase

[0060] Look up the sequences of genes FKS1, GSC2, OCH1, MNN9, DSE2, CHS3, and CRH1 in the NCBI database, and design primers with their gene homology arm sequences to amplify the above genes by PCR. The reaction system is:

[0061] ddH2O: 19 μL

[0062] P3: 2 μL

[0063] P4: 2 μL

[0064] Template: 2 μL

[0065] 2×Phanta Master Mix: 25μL

[0066] Total volume: 50 μL

[0067] Among them, denaturation, retreat temperature and corresponding time are calculated according to the primers.

[0068] Table 1 Primer sequences

[0069]

[0070]

Embodiment 2

[0071] Example 2: Construction of recombinant bacteria YLY01(△fks1), YLY01(△gsc2), YLY01(△och1), YLY01(△mnn9), YLY01(△dse2), YLY01(△chs2), YLY01(△crh1)

[0072] The NEO gene in Example 1 was separated and recovered by agarose gel, and the purified fragment was transformed into Saccharomyces cerevisiae competent YPH499 (Invitrogen) by chemical transformation method. Spread the yeast transformation solution on YPDA (containing a certain concentration of G418 resistance) medium plate (YPDA: yeast extract 10g / L, peptone 20g / L, glucose 20g / L, adenine 0.75g / L), and grow at 30°C Until a single colony grows, the positive clones are verified by colony PCR. The positive clones were named YLY01(△fks1), YLY01(△gsc2), YLY01(△och1), YLY01(△mnn9), YLY01(△dse2), YLY01(△chs2), YLY01(△crh1) and preserved kind.

Embodiment 3

[0073] Example 3 Effects of promoters on the expression levels of genes UGT76G1 and UGPase

[0074] Screen the promoter to increase the transcription level of the target gene. In this example, the following promoters are selected, TEF1, PGK1, HXT7, GPM1, CDC5, FBA1, ENO2, GAL10, and ALA1. The selected promoters were connected to the target gene by homologous recombination, and the enzyme activities of UGT76G1 and UGPase under the regulation of different promoters were compared.

[0075] The successfully constructed engineered bacteria were transferred to 100mL YPDA liquid medium and grown on a shaker at 30°C for 24h. The above-mentioned bacteria sludge was collected by centrifugation, and the bacteria sludge was washed three times with 100 mM, pH 8.0 phosphate buffer. The cells were disrupted with an ultrasonic disruptor for 20 min, the disrupted solution was centrifuged at 12000 rpm for 30 min, and the supernatant enzyme solution was collected.

[0076] The enzyme activity ...

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Abstract

The invention discloses a method for synthesizing rebaudioside-A through high-density fermentation. The method comprises the steps that firstly, genes encoded with a cell wall synthetase system is knocked out, and fermentation synthesis of the rebaudioside-A is achieved while the permeability of a cell wall is improved; secondly, thalluses obtained through fermentation are applied to a whole-cellcatalysis reaction, and the high recovery ratio of the rebaudioside-A is achieved; in the high-density fermentation process, when the thalluses grow to the amount of (10-25) g / L (DCW), a culture medium is subjected to fed-batch in a mode of constant dissolved oxygen index feeding, and when cells grow to the stable phase, the density OD<600> of the thalluses reaches to 100-150; the thalluses grow to the stable phase, the culture medium is subjected to fed-batch in a mode of constant speed feeding, and the content of the rebaudioside-A in fermentation liquid after fermentation is completed is 13.7+ / -1.6 g / L; and the thalluses collected in fermentation are subjected to buffer cleaning treatment by using potassium phosphate and then used for a 50 L whole-cell catalysis reaction, and the concentration of the rebaudioside-A in the system is 28.2+ / -4.9 g / L. According to the method, the problem that the conversion rate of stevioside in cell fermentation is low is solved well, operation is easy, production is easy to enlarge, and the good rebaudioside-A preparing prospects are achieved.

Description

technical field [0001] The invention relates to a method for high-density fermentation and synthesis of rebaudioside A, which belongs to the technical field of bioengineering. Background technique [0002] Rebaudioside A (Rebaudioside A, RA glycoside) is a natural sweetener extracted from Stevia rebaudiana Bert, and is one of the main components of stevioside. RA glycosides have good high temperature resistance, acid and alkali resistance, and can be stored stably for two years at room temperature and relative humidity of 60%. RA glycoside is an ideal natural sweetener, with zero calories, high sweetness, good taste, and no bad aftertaste. Its sweetness is about 350-450 times that of sucrose. However, the commercially available stevioside is mainly composed of stevioside (Stevioside, St glycoside), which has poor taste quality, slightly sweet and bitter taste and licorice off-flavor, which affects its application in the food industry. Therefore, increasing the ratio of RA ...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/81C12P19/56C12R1/865
CPCC12N9/1051C12N9/1241C12N15/81C12N15/905C12P19/56C12Y204/01017C12Y207/07009
Inventor 李艳周伯雅李阳阳贾红华张竹山于青海
Owner XINGHUA GL STEVIA CO LTD
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