Stabilized proline transporter

A transporter, proline technology, applied in the field of stabilized proline transporter, can solve the problem of no amino acid residues, and achieve the effects of promoting assimilation, increasing productivity, and saving resources

Inactive Publication Date: 2007-05-30
SUNTORY HLDG LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, there is no report on the amino acid residue involved in the stabilization of Put4, which is involved in the uptake of proline, one of the main amino acids contained in alcoholic beverage raw materials such as wort.

Method used

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  • Stabilized proline transporter
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1. Isolation and mutagenesis of the PUT4 gene

[0061]Because the PUT4 gene of yeast Saccharomyces cerevisiae has been cloned and its base sequence has been reported, based on this information, the PUT4 gene was isolated by PCR amplification. The template for PCR was used from the laboratory yeast X2180-1A [Rose, M.D., Winston, F. and Hieter, P. (1990): Methods in Yeast Genetics: A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY] Chromosomal DNA prepared. At this time, for the purpose of testing the stability of the Put4p protein expressed later, the amino acid sequence 10 residues from influenza virus hemagglutinin (HA) was used as a tag and added upstream of the ATG initiation codon of the PUT4 ORF to prepare PUT4 ORF (Chen, et al., Proc. Natl. Acad. Sci. USA, 90, 6508, 1993). PCR primers for amplifying the ORF of PUT4 used 5'-GAGCTCATGTACCCATACGA TGTTCCGGAT TACGCTAGCG TAAATACTGCCCTTCCAC AAGA-3' (SEQ ID NO: 1) and 5'-GG...

Embodiment 2

[0063] Example 2. Expression of Mutant PUT4 Gene in Saccharomyces cerevisiae

[0064] A part of the 408bp SacI-KpnI fragment encoding the ORF of the three mutant PUT4 genes, and a 1508bp KpnI-BamHI fragment encoding the remaining part of the ORF prepared separately from pCR-PUT4 were simultaneously inserted into the SacI / BglII site of the expression vector pUP3GLP, Plasmids were prepared for expressing mutant mPut4#10, #18, and #20 in S. cerevisiae. As a control, the natural-type PUT4 ORF with HA tag was prepared as a 1916bp SacI-BamHI fragment, and inserted into the SacI / BglII site of the expression vector pUP3GLP to make a natural-type Put4 expression plasmid. Using the obtained plasmid for expression, the lithium acetate method [Rose, M.D., Winston, F. and Hieter, P. (1990): Methods in Yeast Genetics: A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY] Transformation of brewer's yeast Saccharomycescerevisiae VLB Rasse J strain. Since...

Embodiment 3

[0066] Example 3. In vivo accumulation evaluation of mutant Put4

[0067] From the culture fluid of 10ml, reclaim the bacterial strain of expression mutant type proline transporter mPut4#10, mPut4#18, mPut4#20 and natural type transporter Put4 (hereinafter referred to as " natural type Put4 ") of logarithmic growth phase, In the dissolution buffer [8M urea, 5% (w / v) SDS, 40mM Tris-HCl (pH6.8), 0.1mM EDTA, 1% β-mercaptoethanol], the cells were destroyed by stirring with glass beads to obtain cell extracts. A sample of 60 μg of total protein was separated by SDS-gel electrophoresis, transferred to a nitrocellulose membrane, and subjected to Western blot analysis using a rabbit polyclonal anti-HA antibody (manufactured by Santa Cruz Biotechnology). The detection of HA-tagged native Put4 and mutant Put4 was performed by chemiluminescence using an ECL kit from Amersham Biosciences. Compared with the natural type Put4, the mutant proline transporter mPut4#20 has inhibited intrace...

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Abstract

The present invention relates to a stabilized proline transporter Put4 obtained by gene mutation and a gene encoding the same, as well as a Saccharomyces yeast strain which is obtained by yeast transformation with the gene and is capable of efficiently using proline in a source material such as wort. The stabilized mutated proline transporter Put4 can be used to achieve efficient use of nitrogen sources such as poorly assimilable proline contained in source materials (e.g., wort) for alcohol beverages. Fermentation using yeast capable of taking up a wide variety and large amounts of nitrogen sources facilitates carbon source assimilation and allows improvement of productivity for alcohol beverages. Moreover, the use of poorly assimilable nitrogen sources leads to resource savings and enables environmentally friendly production of alcohol beverages.

Description

technical field [0001] The invention relates to a stabilized proline transporter, a gene encoding the protein, and a yeast of the genus Saccharomyces obtained by transforming yeast with the gene and capable of efficiently utilizing proline in raw materials such as wort. Background technique [0002] Proline is one of the main amino acids contained in raw materials for alcoholic beverages such as wort. However, proline cannot be actively absorbed and assimilated by yeast when it coexists with nitrogen sources such as other amino acids and ammonia that yeast is more suitable for. This is because in the presence of other amino acids, ammonia, etc., the transcription of the proline transporter PUT4 gene (hereinafter referred to as "PUT4 gene") responsible for proline uptake is inhibited and the proline transporter Put4 (hereinafter referred to as "Put4 gene") is inhibited. ”) due to the extremely low stability. [0003] Therefore, attempts have been made to improve the utiliza...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K14/395C12C11/00C12G1/02C12N1/19C12C12/00
CPCC07K14/395C12C12/006C12C12/004
Inventor 大村文彦福井宣之近藤平人
Owner SUNTORY HLDG LTD
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