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Strains of s.cerevisiae, capable of growing in media with melibiose, stachyose and raffinose

A yeast and genetic technology, applied in the field of Saccharomyces cerevisiae strains, can solve problems such as changing DNA, low growth rate, interfering with replication and transcription process growth and respiration

Inactive Publication Date: 2012-05-09
UNIV DA CORUNA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A disadvantage of strains obtained by treatment with UV-induced mutations is that this treatment fundamentally alters the DNA due to the configuration of the pyrimidine primer causing local distortions of the double helix structure, which interferes with normal complementary base pairing; this in turn Interfering with replication and transcription processes and subsequent growth and respiration
Therefore, in addition to being able to have other unwanted mutations, thus mutated strains also often have low growth rates

Method used

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  • Strains of s.cerevisiae, capable of growing in media with melibiose, stachyose and raffinose
  • Strains of s.cerevisiae, capable of growing in media with melibiose, stachyose and raffinose
  • Strains of s.cerevisiae, capable of growing in media with melibiose, stachyose and raffinose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Obtaining a modified yeast strain for the production of α-galactosidase

[0091] Materials and methods

[0092] 1. Cloning of the nucleotide sequence from the gene encoding α-galactosidase

[0093]Two pairs of primers were designed to amplify the MEL1 gene encoding Saccharomyces cerevisiae α-galactosidase by PCR (Polymerase Chain Reaction, polymerase chain reaction). Two fragments are amplified, one of which corresponds to the complete gene of α-galactosidase (SEQ ID NO: 4), using the following primers with SEQ ID NO: 5 and SEQ ID NO: 6 for amplification; the other fragment corresponds to the removal of the coding secretion The 54-nucleotide α-galactosidase gene of the signal (SEQ ID NO: 7) was amplified by the following primers having SEQ ID NO: 8 and SEQ ID NO: 9. Insert the complete gene of α-galactosidase into the vector YEpFLAG1 (Eastman Kodak classification number IB13400) for ADH2 (Alcohol Dehydrogenase, alcohol dehydrogenase 2) promoter (sequence number: 1...

Embodiment 2

[0107] Strains [A] with MEL1 under the ADH2 promoter and strains with MEL1 under the ADH1 promoter Comparison of α-galactosidase activity of strain [B]

[0108] The details of cloning and the determination of α-galactosidase activity are described in Example 1. The recombinant strain [A] was cultured on a synthetic medium containing 1% glucose.

[0109] figure 2 Shows the comparison of the data obtained from the strain [B] described in US Patent No. 5,055,401 and the recombinant strain [A] of the present invention. For purposes, data were extracted from Figures 7A and 7B in US 5055401 and they have been compared. It can be observed that the total activity of the α-galactosidase of the strain described in US 5055401 reaches 8000E.U. / ml in 36-54 hours, while the total activity of the strain [A] of the present invention reaches the total The activity was 10,000 E.U. / ml to 20,000 E.U. / ml and, as discussed previously, reached about 32,000 E.U. / ml at a later stage of the cul...

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Abstract

The invention relates to strains of S. cerevisiae capable of secreting Alpha-galactosidase into culture medium, as well as to methods for obtaining Alpha-galactosidase using said strains and methods for producing biomass and bioethanol by means of culturing said strains in media rich in galactose. In another aspect, the invention provides methods for expressing recombinant proteins using a composition rich in galactose as a culture medium for the microorganisms producing said protein.

Description

technical field [0001] The present invention relates to S. cerevisiae strains capable of producing alpha-galactosidase. For its better secretion, a signal sequence was fused to the gene of α-galactosidase. Said bacterial strain is used in a method for producing α-galactosidase, biomass and ethanol in a medium rich in raffinose, melibiose and / or stachytetraose, and if the bacterial strain contains The secondary construct of the functional protein can then be used to produce the therapeutic protein. Background technique [0002] α-Galactosidase (EC 3.2.1.22) catalyzes the hydrolysis of galactose residues, which combine with galactooligosaccharides and galactomannose polymers (mannose polymers with galactose branches) via α (1,6) Glycosidic bonding; also hydrolyzes oligosaccharides such as stachyose, raffinose and melibiose present in beans, soybeans and other legumes. [0003] These enzymes are widely distributed in animals, plants and microorganisms. But most monogastric ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/40C12N9/42C12N15/81
CPCC12N15/81C12N9/2465C12Y302/01022C12P7/08
Inventor M·贝塞拉 费尔南德斯M·E·塞尔当 维拉纽瓦R·费尔南德斯 莱罗M·I·冈萨雷斯 斯索A·佩雷拉 罗德里格兹
Owner UNIV DA CORUNA
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