Yeast with high content of Abu, gamma-Glu-Abu, and/or gamma-Glu-Abu-Gly
A-glu-abu, high-content technology for yeast and yeast extracts, capable of solving any relationship unknown etc.
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Embodiment 1
[0160] Construction of M013Y strain, which has enhanced 2-aminobutyric acid biosynthesis system enzyme activity and weakened acetolactate synthase gene ( ILV2 )Express
[0161] A yeast strain (M013Y strain) was constructed by the following method, in which the enzyme activity of the 2-aminobutyric acid biosynthesis system was enhanced, and the gene encoding acetolactate synthase ( ILV2 ) is weakened for the purpose of enhancing the ability to produce 2-aminobutyric acid.
[0162] Saccharomyces cerevisiae AG1ura3-strain (Japanese Patent Publication (Kokai) No. 2012-213376) was used as a parent strain. AG1ura3-strain is the γ-glutamylcysteine synthetase gene of Saccharomyces cerevisiae Y006 strain (FERMBP-11299) ( GSH1 ) strains with enhanced expression. AG1ura3-strain deficiency URA3 gene, and show uracil auxotrophy.
[0163] According to the method of Sofyanovich et al. (Olga A.Sofyanovich et al., A New Method for Repeated "Self-Cloning" Promoter Replacement in Sac...
Embodiment 2
[0192] in M007Y strain and M013Y strain ILV2 Gene mRNA expression and Abu-related compound analysis
[0193] (1) Culture and sampling
[0194] The M007Y strain and M013Y strain constructed in Example 1 were inoculated into SD medium (50 mL) contained in a Sakaguchi flask with a volume of 500 mL in an amount of one loop, and cultured at 30° C. for 24 hours with shaking at 120 rpm.
[0195] The absorbance of each culture solution obtained was measured, and the culture solution was inoculated into SD medium (70 mL) contained in a Sakaguchi bottle with a volume of 500 mL at an OD600 of 0.01, and cultured at 30° C. for about 18 hours with shaking at 120 rpm ( main culture). Absorbance was measured by using a DU640 spectrophotometer (BECKMAN COULTER). When OD600 became 1.5, the culture solution was sampled.
[0196] (2) M007Y strain and M013Y strain ILV2 mRNA expression analysis
[0197] Cells contained in the culture solution (4 mL) obtained by the aforementioned method were...
Embodiment 3
[0213] has further weakened acetolactate synthase gene ( ILV2 ) Construction of the M014Y strain expressed
[0214] As mentioned above, it is estimated that the M007Y strain contains two or more copies of ILV2 Gene. Therefore, the additional copy in the M013Y strain as the parent strain was destroyed by the following method ILV2 gene , to prepare a strain (M014Y) in which the acetolactate synthase activity was further reduced.
[0215] (1) ILV2 Construction of Gene Disruption Strains
[0216] Amplified by PCR containing URA3 A DNA fragment of a gene using primer SEQ ID NO: 13 (5'-GTTACAATAGAAAGTATTTTACAAAATCTAAACCCTTTGAGCTAAGAGGAGATAAATACAACAGAATCAATTTTCAAAAGGGCAACGGTTCATCATC-3') comprising primers from ILV2 The 80-nucleotide upstream sequence of the initiation codon, primer SEQIDNO:14 (5'-TATAAGAAGCACGATTAAATAATAATAAAGTCTGCATTTTTACTGAAAATGCTTTTGAAATAAATGTTTTTTGAAATATTAGATATATACGCCAG-3'), which contains the ILV2 The 80-nucleotide downstream sequence of the stop codon...
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