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.

Characterization of a New Saccharomyces cerevisiae Isolated From Hibiscus Flower and Its Mutant With L-Leucine Accumulation for Awamori Brewing

“…The method for laboratory fermentation test was described previously (Takagi et al, 2015). In brief, 50 g of rice koji, 65 ml of water, and 0.1 ml of precultured yeast cells were mixed in a 200 ml Erlenmeyer flask.…”

“…Cell suspension was treated with a Multi-Beads Shocker (Yasui Kikai) at 4°C, for preparing the whole-cell extracts by disrupting with glass beads, and resultant supernatant was used for crude enzyme solution. IPMS activity was measured as described previously (Takagi et al, 2015). One unit of enzyme activity is defined as the amount of coenzyme A (CoA) liberated from acetyl-CoA, which produced by an enzymatic transacylation reaction from 2-ketoisovalerate to 2-isopropylmalate, at 37°C for 60 min.…”

“…Previously, we isolated a mutant of the diploid awamori yeast strain 101-18, which accumulated a higher amount of L -leucine selected from the L -leucine analog 5,5,5-trifluoro- DL -leucine (TFL)-resistant colonies. The mutant strain 101-T55 has mutations in the LEU4 gene, which confers overproduction of isoamyl alcohol and isoamyl acetate (Takagi et al, 2015). Such yeast breeding technology targeted to specific flavors is applicable to the improvement of the quality of awamori.…”

“…For awamori brewing, an awamori yeast strain of S. cerevisiae , Awamori 101, which is a diploid prototroph (supplied by National Research Institute of Brewing, Japan), has been dominantly used as a standard and conventional strain among awamori breweries. Strain 101-18 with high alcohol productivity has been isolated from Awamori 101 and is applicable for genetic analysis and awamori fermentation test (Takagi et al, 2015). Recently, several types of awamori have been manufactured and commercialized by awamori brewery, which brewed by use of yeast strains derived and isolated from nature in Okinawa, such as mango and brown sugar lump.…”

Yeast strains from coconut toddy in Sri Lanka show high tolerance to inhibitors derived from the hydrolysis of lignocellulosic materials

“…A widely used laboratory yeast strain, S288C (supplied by the Microbiology and fermentation Laboratory, University of the Ryukyus, Okinawa, Japan) and Awamori yeast 101-18 (supplied by the National Research Institute of Brewing, Hiroshima, Japan) strains were used as reference strains. Awamori yeast 101-18 strain is a kind of S. cerevisiae widely used in producing a local distilled alcoholic beverage, named as Awamori, from steamed rice in Okinawa, Japan [17]. Cell growth was monitored by measuring culture turbidity as McFarland units using a densitometer DEN-IB (Waken Btech Co. Ltd., Kyoto, Japan).…”