Yarrowia lipolytica strain for synthesizing isomaltooligosaccharide and its synthesis method

A technology of isomaltose oligosaccharide and Yarrowia lipolytica, which is applied in the field of Yarrowia lipolytica strain and its synthesis, and can solve the problem of high cost

Active Publication Date: 2021-02-12
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method does not need to add α-amylase, β-amylase, pullulanase and transglycosidase one by one like the classical method, but adds special amylase and branching enzyme at the same time, and then adds transglycosidase, which has Certain simplicity, but the disadvantage is that it also needs to prepare a variety of purified enzymes, which is very expensive

Method used

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  • Yarrowia lipolytica strain for synthesizing isomaltooligosaccharide and its synthesis method
  • Yarrowia lipolytica strain for synthesizing isomaltooligosaccharide and its synthesis method
  • Yarrowia lipolytica strain for synthesizing isomaltooligosaccharide and its synthesis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Embodiment 1, the Yarrowia lipolytica yeast strain that only contains β-amylase gene expression cassette transforms starch production Assay of finished product

[0102] Follow the steps below to implement:

[0103] (1) Construction of the Yarrowia lipolytica yeast strain containing only the β-amylase gene expression cassette: the β-amylase gene of Bacillus subtilis was fused with the anchor protein gene Pir1 of Yarrowia lipolytica BLC13 to form a fusion gene, and the 5' end of the fusion gene was fused with The hp4d promoter was connected, and its 3' end was connected with the terminator of the ura3 gene to form a β-amylase gene expression cassette. The upstream and downstream of the expression cassette are respectively connected with the homology arm integration sequence zeta to form an integrated gene expression cassette. The expression cassette was transformed into Yarrowia lipolytica BLC13 yeast, cultured in the basic medium containing starch as the only carbon...

Embodiment 2

[0109] Embodiment 2, Yarrowia lipolytica yeast containing α-amylase gene and β-amylase gene expression cassette Determination of products produced by strain transformed starch

[0110] Follow the steps below to implement:

[0111](1) Construction of Yarrowia lipolytica yeast strain containing α-amylase gene and β-amylase gene expression cassette: chemically synthesize and fuse the α-amylase gene and β-amylase gene of Bacillus subtilis (Note: α - The stop codon TAA of the amylase gene is removed and then fused with the β-amylase gene), and then fused with the anchor protein gene Pir1 to form a fusion gene, the 5' end of the fusion gene is connected with the hp4d promoter, and its 3' end is connected with the hp4d promoter The terminator of the ura3 gene was connected to form an α-amylase-β-amylase gene expression cassette. The upstream and downstream of the expression cassette are respectively connected with the homology arm integration sequence zeta to form an integrated ...

Embodiment 3

[0118] Embodiment 3, Yarrowia lipolytica ferment containing α-amylase gene and α-glucosidase gene expression cassette Determination of Products Produced by Transformed Starch by Mother Strain

[0119] Follow the steps below to implement:

[0120] (1) Construction of Yarrowia lipolytica yeast strain containing α-amylase gene and α-glucosidase gene expression cassette: the α-amylase gene of Bacillus subtilis and the α-glucosidase gene of Aspergillus niger were chemically synthesized and Fusion (note: the stop codon TAA of the α-amylase gene is removed and then fused with the α-glucosidase gene), and then fused with the anchor protein gene Pir1 to form a fusion gene, and the 5' end of the fusion gene is connected to the hp4d promoter , and its 3' end is connected with the terminator of ura3 gene to form an α-amylase-α-glucosidase gene expression cassette. The upstream and downstream of the expression cassette are respectively connected with the homology arm integration seque...

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Abstract

The invention discloses a Yarrowia lipolytica strain for synthesizing isomaltooligosaccharides and a synthesis method thereof; the invention uses the food-safe microorganism Yarrowia lipolytica as an enzyme expression host to reduce the synthesis of food-grade microorganisms Different genes of polyisomaltose were optimized and improved, and different gene expression cassettes were designed and constructed, and Yarrowia lipolytica was transformed to obtain engineering strains that can efficiently catalyze starch, dextrin or maltose to produce isomaltose oligosaccharides. The engineering yeast strain is cultivated to obtain cells, which are used as an enzyme source to realize the one-step synthesis of isomaltooligosaccharide from starch, dextrin or maltose, and the highest conversion rate reaches 75%. The food-grade bacterial strain provided by the invention and the method for synthesizing the isomaltooligosaccharide simplifies the production steps and improves the production efficiency, thereby significantly reducing the cost of large-scale production of the isomaltooligosaccharide.

Description

technical field [0001] The invention belongs to the field of food biotechnology, and relates to a Yarrowia lipolytica strain for synthesizing isomaltooligosaccharide and a synthesis method thereof. Background technique [0002] Isomalto-oligosaccharide (IMO) is an important functional oligosaccharide, generally connected by 2-10 glucose with glycosidic bonds, and these glycosidic bonds contain at least one α-1,6 glycosidic bond , may also contain very few α-1,3 glycosidic linkages. Isomaltooligosaccharides often contain multiple components, such as isomaltose (IG2), isomaltotriose (IG3), panose (P), isomaltotetraose (IG4), and the structural formula is as follows figure 1 , where A, B, and C represent isomaltose, panose, and isomaltotriose, respectively. [0003] The current national standard divides isomaltooligosaccharide into IMO-50 and IMO-90 according to the purity of the product. The content of IG2+P+IG3 in IMO-50 accounts for more than 35%, and the content of IG2+P+...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12P19/14C12P19/12C12P19/00C12R1/645
CPCC12N9/2402C12N9/2414C12N9/2425C12P19/00C12P19/12C12P19/14C12Y302/01001C12Y302/01002C12Y302/0102C12Y302/01106C12Y302/01133C12Y302/01135
Inventor 程海荣刘大文王思绮
Owner SHANGHAI JIAOTONG UNIV
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