High-Level Production of Isoleucine and Fusel Alcohol by Expression of the Feedback Inhibition-Insensitive Threonine Deaminase in Saccharomyces cerevisiae

Abstract

A variety of the yeast Saccharomyces cerevisiae with intracellular accumulation of isoleucine (Ile) would be a promising strain for developing a distinct kind of sake, a traditional Japanese alcoholic beverage, because Ile-derived volatile compounds have a great impact on the flavor and taste of fermented foods. In this study, we isolated an Ile-accumulating mutant (strain K9-I48) derived from a diploid sake yeast of S. cerevisiae by conventional mutagenesis. Strain K9-I48 carries a novel mutation in the ILV1 gene encoding the His480Tyr variant of threonine deaminase (TD). Interestingly, the TD activity of the His480Tyr variant was markedly insensitive to feedback inhibition by Ile, but was not upregulated by valine, leading to intracellular accumulation of Ile and extracellular overproduction of 2-methyl-1-butanol, a fusel alcohol derived from Ile, in yeast cells. The present study demonstrated for the first time that the conserved histidine residue located in a linker region between two regulatory domains is involved in allosteric regulation of TD. Moreover, sake brewed with strain K9-I48 contained 2-3 times more 2-methyl-1-butanol and 2-methylbutyl acetate than sake brewed with the parent strain. These findings are valuable for the engineering of TD to increase the productivity of Ile and its derived fusel alcohols. IMPORTANCE Fruit-like flavors of isoleucine-derived volatile compounds, 2-methyl-1-butanol (2MB) and its acetate ester, contribute to a variety of the flavors and tastes of alcoholic beverages. Besides its value as aroma components in foods and cosmetics, 2MB has attracted significant attention as second-generation biofuels. Threonine deaminase (TD) catalyzes the first step in isoleucine biosynthesis and its activity is subject to feedback inhibition by isoleucine. Here, we isolated an isoleucine-accumulating sake yeast mutant and identified a mutant gene encoding a novel variant of TD. The variant TD exhibited much less sensitivity to isoleucine, leading to higher production of 2MB as well as isoleucine than the wild-type TD. Furthermore, sake brewed with a mutant yeast expressing the variant TD contained more 2MB and its acetate ester than that brewed with the parent strain. These findings will contribute to the development of superior industrial yeast strains for high-level production of isoleucine and its related fusel alcohols.

Yap is a Nutrient Sensor Sensitive to the Amino Acid L-Isoleucine and Regulates Expression of Ctgf in Cardiomyocytes

“…Supplementation with threonine and L-isoleucine returned phosphorylated Yap signals to control levels in whole-cell lysate. We then focused on isoleucine as a rate-limiting amino acid, given there exists a feedback loop between threonine and isoleucine by threonine deaminase (the enzyme involved in catalyzing the first reaction which is inhibited by isoleucine allosteric binding to inhibit the reaction at high levels of isoleucine) [21]. As such, increased levels of threonine can be generated by conversion into isoleucine.…”

“…To further narrow down our focus we considered that threonine is catabolized to isoleucine and is regulated by an inhibitory feedback mechanism, where isoleucine binds allosterically to threonine deaminase (the first enzyme in the catabolism pathway) when isoleucine levels are high, therefore inhibiting the binding of threonine. In contrast, when isoleucine levels are low, threonine can bind and the catabolism to isoleucine can continue suggesting that isoleucine is a limiting factor [21]. Since the relationship between threonine and isoleucine relies on isoleucine as a limiting factor, all experiments moving forward were performed with L-isoleucine.…”

Improving the Catalytic Efficiency of a GH5 Processive Endoglucanase by a Combinatorial Strategy Using Consensus Mutagenesis and Loop Engineering

“…For its variants, because of the different enzyme activities of each mutant, the enzyme concentrations used were 0.25–0.5 μg/mL for soluble substrates and 0.5–1.0 μg/mL for insoluble substrates. Data recorded from continuous assays were analyzed via a nonlinear regression model using the Michaelis–Menten equation implemented in GraphPad Prism 9.0 (GraphPad Software, La Jolla, CA, USA) . All experiments were carried out in triplicate.…”

“…Data recorded from continuous assays were analyzed via a nonlinear regression model using the Michaelis−Menten equation implemented in GraphPad Prism 9.0 (GraphPad Software, La Jolla, CA, USA). 51 All experiments were carried out in triplicate.…”

Regulated use of alternative Transcription Start Sites controls the production of cytosolic or mitochondrial forms of branched-chain aminotransferase in Kluyveromyces marxianus

“…Some of the most significant aromas and flavours are derived from the degradation (catabolism) of amino acids in the Ehrlich pathway to produce a range of higher alcohols, acids and esters. These include the aromatic amino acids, phenylalanine and tyrosine, and the branched chain amino acids (BCAA), valine, isoleucine and leucine, and individual amino acids such as threonine and methionine (Hazelwood et al, 2008; Isogai et al, 2022). In each case, the first catabolic step is a transamination to form an α-ketoacid by distinct aromatic amino acid (AAT) or branched chain amino acid aminotransferases (BCAT).…”