title: Isolation and characterization of awamori yeast mutants with L-leucine accumulation that overproduce isoamyl alcohol.
abstract: Awamori shochu is a traditional distilled alcoholic beverage made from steamed rice in Okinawa, Japan. Although it has a unique aroma that is distinguishable from that of other types of shochu, no studies have been reported on the breeding of awamori yeasts. In yeast, isoamyl alcohol (i-AmOH), known as the key flavor of bread, is mainly produced from α-ketoisocaproate in the pathway of L-leucine biosynthesis, which is regulated by end-product inhibition of α-isopropylmalate synthase (IPMS). Here, we isolated mutants resistant to the L-leucine analog 5,5,5-trifluoro-DL-leucine (TFL) derived from diploid awamori yeast of Saccharomyces cerevisiae. Some of the mutants accumulated a greater amount of intracellular L-leucine, and among them, one mutant overproduced i-AmOH in awamori brewing. This mutant carried an allele of the LEU4 gene encoding the Ser542Phe/Ala551Val variant IPMS, which is less sensitive to feedback inhibition by L-leucine. Interestingly, we found that either of the constituent mutations (LEU4(S542F) and LEU4(A551V)) resulted in the TFL tolerance of yeast cells and desensitization to L-leucine feedback inhibition of IPMS, leading to intracellular L-leucine accumulation. Homology modeling also suggested that L-leucine binding was drastically inhibited in the Ser542Phe, Ala551Val, and Ser542Phe/Ala551Val variants due to steric hindrance in the cavity of IPMS. As we expected, awamori yeast cells expressing LEU4(S542F), LEU4(A551V), and LEU4(S542F/A551V) showed a prominent increase in extracellular i-AmOH production, compared with that of cells carrying the vector only. The approach described here could be a practical method for the breeding of novel awamori yeasts to expand the diversity of awamori taste and flavor.
authors: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. Electronic address: hiro@bs.naist.jp.
authors: Hiroshi
authors: H
authors: Takagi
authors: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
authors: Keisuke
authors: K
authors: Hashida
authors: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
authors: Daisuke
authors: D
authors: Watanabe
authors: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
authors: Ryo
authors: R
authors: Nasuno
authors: Nara Prefecture Institute of Industrial Development, 129-1 Kashiwagi, Nara, Nara 630-8031, Japan.
authors: Masataka
authors: M
authors: Ohashi
authors: BioJet Ltd., 315 Shioya, Uruma, Okinawa 904-2231, Japan.
authors: Tomoya
authors: T
authors: Iha
authors: BioJet Ltd., 315 Shioya, Uruma, Okinawa 904-2231, Japan.
authors: Maiko
authors: M
authors: Nezuo
authors: BioJet Ltd., 315 Shioya, Uruma, Okinawa 904-2231, Japan.
authors: Masatoshi
authors: M
authors: Tsukahara
mesh_terms: D015079:2-Isopropylmalate Synthase / Q000235:genetics / Q000378:metabolism; D000434:Alcoholic Beverages / Q000382:microbiology; D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004171:Diploidy; D005285:Fermentation; D005800:Genes, Fungal / Q000235:genetics*; D007564:Japan; D007651:Keto Acids / Q000378:metabolism; D007930:Leucine / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D000439:Pentanols / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D007651:Keto Acids; D000439:Pentanols; D029701:Saccharomyces cerevisiae Proteins; C013082:alpha-ketoisocaproic acid; C029683:isopentyl alcohol; D015079:2-Isopropylmalate Synthase; C080062:LEU4 protein, S cerevisiae; D007930:Leucine
keywords: 5,5,5-Trifluoro-dl-leucine resistance; Awamori; Isoamyl alcohol; Leucine; Saccharomyces cerevisiae; α-Isopropylmalate synthase