Recombinant pichia pastoris engineering bacterium and application thereof to synthesis of RA (rebaudioside A)

A technology of Pichia pastoris and engineering bacteria, applied in the field of bioengineering, can solve the problems of high cost, poor effect, cumbersome process, etc., and achieve the effect of saving production cost, improving synthesis efficiency and raw material utilization rate

Active Publication Date: 2014-12-24
GUANGZHOU KANGLINNAI BIOLOGICAL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the methods for preparing high-purity rebaudioside A products include: cultivating high-yield rebaudioside A stevia varieties, resin adsorption or recrystallization

Method used

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  • Recombinant pichia pastoris engineering bacterium and application thereof to synthesis of RA (rebaudioside A)
  • Recombinant pichia pastoris engineering bacterium and application thereof to synthesis of RA (rebaudioside A)
  • Recombinant pichia pastoris engineering bacterium and application thereof to synthesis of RA (rebaudioside A)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Synthesis of Genes Sus1 and UGT76G1

[0040]Based on the amino acid sequence published by NCBI (http: / / www.ncbi.nlm.nih.gov / ), the sequence was optimized according to the codon preference of Pichia pastoris, and the codon-optimized Sus1 and UGT76G1 genes were obtained. The sequences are shown in Seq No.3 and Seq No.4 respectively. The whole gene synthesis was carried out by a biotechnology company (Nanjing GenScript Biotechnology Co., Ltd.), and the synthesized gene was cloned in the pUC57 plasmid (purchased from GenScript Biotechnology Co., Ltd. ), the plasmids pUC57-Sus1 and pUC57-UGT76G1 were obtained.

Embodiment 2

[0042] Construction of intracellular expression vector pPICZA-Sus1 and display expression vector pGCW61-UGT76G1

[0043] The forward primer SF (containing the EcoR I restriction site) and the reverse primer SR (containing the Not I restriction site) were designed according to the Sus1 gene sequence. PCR amplification was performed using the plasmid pUC57-Sus1 in Example 1 as a template and SF and SR as primers. The gel-recovered and purified PCR product was double-digested with restriction endonucleases EcoR I and Not I, and ligated with the plasmid pPICZA that had also been double-digested with EcoR I and Not I with T4 ligase overnight at 16°C, and the ligation product was chemically Transform E.coli Top10 (purchased from Invitrogen Yingjie Life Technology Co., Ltd., USA), and screen positive transformants by bleomycin resistance plate. Internal expression vector pPICZA-Sus1. The cloned strain E.coli Top10 / pPICZA-Sus1 containing the recombinant plasmid pPICZA-Sus1 was sto...

Embodiment 3

[0046] Construction of Recombinant Pichia Pastoris Engineering Strain GS115 / Sus1-UGT76G1 and Obtainment of Whole Cell Catalyst

[0047] The display expression vector pGCW61-UGT76G1 was linearized and purified by Kpn2 I single enzyme digestion, and then electrotransformed into Pichia pastoris GS115 (purchased from Invitrogen Yingjie Life Technology Co., Ltd., USA) competent cells, and positive transformants were screened on the MD plate; thus obtained Engineering bacteria GS115 / UGT76G1. The intracellular expression vector pPICZA-Sus1 was linearized and purified by Pme I, and then electrotransformed the above-mentioned GS115 / UGT76G1 competent cells, and positive transformants were screened on the bleomycin resistance plate; then recombinant Pichia pastoris was obtained Engineering bacteria GS115 / Sus1-UGT76G1.

[0048] Pick the above-mentioned recombinant Pichia pastoris GS115 / Sus1-UGT76G1 and inoculate it into BMGY medium, cultivate it at 30°C and 250rpm until the OD600 is...

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Abstract

The invention discloses a recombinant pichia pastoris engineering bacterium and a method for using the recombinant pichia pastoris engineering bacterium as a whole-cell catalyst for synthesizing RA (rebaudioside A). The recombinant pichia pastoris engineering bacterium is obtained through conversion of pichia pastoris after linearization of an expression vector containing exogenous DNA as follows: the DNA sequence for coding sucrose synthetase Sus1 is represented by SEQ ID NO.3, and the DNA sequence for coding UDP-glycosyl transferase UGT76G1 is represented by SEQ ID NO.4. The intracellular expression of the Sus1 and the surface expression of the UGT76G1 are realized by the aid of the recombinant pichia pastoris engineering bacterium, and the recombinant pichia pastoris engineering bacterium is used as the whole-cell catalyst and can be effectively used for synthesizing the RA, so that the synthetic efficiency of the RA is improved, the raw material utilization rate is increased, the production cost is reduced, and a new industrial way is opened for production and processing of the RA.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a recombinant Pichia pastoris engineering bacterium and its application in synthesizing rebaudioside A. Background technique [0002] Steviol glycoside (Steviol glycoside) is a high-sweet natural sweetener with many excellent properties, which has great application value in food, beverage, brewing, medicine, daily chemical industry and other fields. Stevioside has an aftertaste of bitterness and licorice odor, and the composition of the mixture is complex, so it is difficult to unify its quality specifications, which limits its application. Stevioside (Stevioside) and Rebaudioside A (Rebaudioside A) are the two most abundant components in stevioside, accounting for 5-10% and 2-4% of dry leaf weight, respectively. Rebaudioside A (RA) is a non-toxic, safe, low-calorie, high-sweet natural sweetener, its sweetness is 150-300 times that of sucrose, and its calorific value is o...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P19/56C12R1/84
Inventor 林影梁书利毛国红刘晓肖王蓓蓓
Owner GUANGZHOU KANGLINNAI BIOLOGICAL SCI & TECH CO LTD
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