Improvement of valine and isobutanol production in sake yeast by Ala31Thr substitution in the regulatory subunit of acetohydroxy acid synthase.

The fruit-like aroma of two valine-derived volatiles, isobutanol and isobutyl acetate, has great impact on the flavour and taste of alcoholic beverages, including sake, a traditional Japanese alcoholic beverage. With the growing worldwide interest in sake, breeding of yeast strains with intracellular valine accumulation is a promising approach to meet a demand for sakes with a variety of flavour and taste by increasing the valine-derived aromas. We here isolated a valine-accumulating sake yeast mutant (K7-V7) and identified a novel amino acid substitution, Ala31Thr, on Ilv6, a regulatory subunit for acetohydroxy acid synthase. Expression of the Ala31Thr variant Ilv6 conferred valine accumulation on the laboratory yeast cells, leading to increased isobutanol production. Additionally, enzymatic analysis revealed that Ala31Thr substitution in Ilv6 decreased sensitivity to feedback inhibition by valine. This study demonstrated for the first time that an N-terminal arm conserved in the regulatory subunit of fungal acetohydroxy acid synthase is involved in the allosteric regulation by valine. Moreover, sake brewed with strain K7-V7 contained 1.5-fold higher levels of isobutanol and isobutyl acetate than sake brewed with the parental strain. Our findings will contribute to the brewing of distinctive sakes and the development of yeast strains with increased production of valine-derived compounds.

Characterization of a new Saccharomyces cerevisiae isolated from banana stems and its mutant with l-leucine accumulation for awamori brewing.

We isolated a new strain of the yeast Saccharomyces cerevisiae, 35a14, from banana stems in Okinawa. This strain did not belong to any industrial yeast groups in a phylogenetic tree and produced high levels of alcohol. Furthermore, awamori, an Okinawa's traditional distilled alcoholic beverage, brewed with an l-leucine overproducing mutant derived from 35a14 showed a high concentration of isoamyl acetate.

Improvement of Fusel Alcohol Production by Engineering of the Yeast Branched-Chain Amino Acid Aminotransaminase.

Branched-chain higher alcohols (BCHAs), or fusel alcohols, including isobutanol, isoamyl alcohol, and active amyl alcohol, are useful compounds in several industries. The yeast Saccharomyces cerevisiae can synthesize these compounds via the metabolic pathways of branched-chain amino acids (BCAAs). Branched-chain amino acid aminotransaminases (BCATs) are the key enzymes for BCHA production via the Ehrlich pathway of BCAAs. BCATs catalyze a bidirectional transamination reaction between branched-chain α-keto acids (BCKAs) and BCAAs. In S. cerevisiae, there are two BCAT isoforms, Bat1 and Bat2, which are encoded by the genes BAT1 and BAT2. Although many studies have shown the effects of deletion or overexpression of BAT1 and BAT2 on BCHA production, there have been no reports on the enhancement of BCHA production by functional variants of BCATs. Here, to improve BCHA productivity, we designed variants of Bat1 and Bat2 with altered enzyme activity by using in silico computational analysis: the Gly333Ser and Gly333Trp Bat1 and corresponding Gly316Ser and Gly316Trp Bat2 variants, respectively. When expressed in S. cerevisiae cells, most of these variants caused a growth defect in minimal medium. Interestingly, the Gly333Trp Bat1 and Gly316Ser Bat2 variants achieved 18.7-fold and 17.4-fold increases in isobutanol above that for the wild-type enzyme, respectively. The enzyme assay revealed that the catalytic activities of all four BCAT variants were lower than that of the wild-type enzyme. Our results indicate that the decreased BCAT activity enhanced BCHA production by reducing BCAA biosynthesis, which occurs via a pathway that directly competes with BCHA production. IMPORTANCE Recently, several studies have attempted to increase the production of branched-chain higher alcohols (BCHAs) in the yeast Saccharomyces cerevisiae. The key enzymes for BCHA biosynthesis in S. cerevisiae are the branched-chain amino acid aminotransaminases (BCATs) Bat1 and Bat2. Deletion or overexpression of the genes encoding BCATs has an impact on the production of BCHAs; however, amino acid substitution variants of Bat1 and Bat2 that could affect enzymatic properties-and ultimately BCHA productivity-have not been fully studied. By using in silico analysis, we designed variants of Bat1 and Bat2 and expressed them in yeast cells. We found that the engineered BCATs decreased catalytic activities and increased BCHA production. Our approach provides new insight into the functions of BCATs and will be useful in the future construction of enzymes optimized for high-level production of BCHAs.

High-Level Production of Lysine in the Yeast Saccharomyces cerevisiae by Rational Design of Homocitrate Synthase.

Homocitrate synthase (HCS) catalyzes the aldol condensation of 2-oxoglutarate (2-OG) and acetyl coenzyme A (AcCoA) to form homocitrate, which is the first enzyme of the lysine biosynthetic pathway in the yeast Saccharomyces cerevisiae. The HCS activity is tightly regulated via feedback inhibition by the end product lysine. Here, we designed a feedback inhibition-insensitive HCS of S. cerevisiae (ScLys20) for high-level production of lysine in yeast cells. In silico docking of the substrate 2-OG and the inhibitor lysine to ScLys20 predicted that the substitution of serine with glutamate at position 385 would be more suitable for desensitization of the lysine feedback inhibition than the substitution from serine to phenylalanine in the already known Ser385Phe variant. Enzymatic analysis revealed that the Ser385Glu variant is far more insensitive to feedback inhibition than the Ser385Phe variant. We also found that the lysine contents in yeast cells expressing the Ser385Glu variant were 4.62- and 1.47-fold higher than those of cells expressing the wild-type HCS and Ser385Phe variant, respectively, due to the extreme desensitization to feedback inhibition. In this study, we obtained highly feedback inhibition-insensitive HCS using in silico docking and enzymatic analysis. Our results indicate that the rational engineering of HCS for feedback inhibition desensitization by lysine could be useful for constructing new yeast strains with higher lysine productivity. IMPORTANCE A traditional method for screening toxic analogue-resistant mutants has been established for the breeding of microbes that produce high levels of amino acids, including lysine. However, another efficient strategy is required to further improve their productivity. Homocitrate synthase (HCS) catalyzes the first step of lysine biosynthesis in the yeast Saccharomyces cerevisiae, and its activity is subject to feedback inhibition by lysine. Here, in silico design of a key enzyme that regulates the biosynthesis of lysine was utilized to increase the productivity of lysine. We designed HCS for the high-level production of lysine in yeast cells by in silico docking simulation. The engineered HCS exhibited much less sensitivity to lysine and conferred higher production of lysine than the already known variant obtained by traditional breeding. The combination of in silico design and experimental analysis of a key enzyme will contribute to advances in metabolic engineering for the construction of industrial microorganisms.

An overview of branched-chain amino acid aminotransferases: functional differences between mitochondrial and cytosolic isozymes in yeast and human.

Branched-chain amino acid aminotransferase (BCAT) catalyzes bidirectional transamination in the cell between branched-chain amino acids (BCAAs; valine, leucine, and isoleucine) and branched-chain α-keto acids (BCKAs; α-ketoisovalerate, α-ketoisocaproate, and α-keto-ß-methylvalerate). Eukaryotic cells contain two types of paralogous BCATs: mitochondrial BCAT (BCATm) and cytosolic BCAT (BCATc). Both isozymes have identical enzymatic functions, so they have long been considered to perform similar physiological functions in the cells. However, many studies have gradually revealed the differences in physiological functions and regulatory mechanisms between them. In this article, we present overviews of BCATm and BCATc in both yeast and human. We also introduce BCAT variants found natively or constructed artificially, which could have significant implications for research into the relationship between the primary structures and protein functions of BCATs. KEY POINTS: • BCAT catalyzes bidirectional transamination in the cell between BCAAs and BCKAs. • BCATm and BCATc are different in the metabolic roles and regulatory mechanisms. • BCAT variants offer insight into a relationship between the structure and function.

Effect of the Ala234Asp replacement in mitochondrial branched-chain amino acid aminotransferase on the production of BCAAs and fusel alcohols in yeast.

In the yeast Saccharomyces cerevisiae, the mitochondrial branched-chain amino acid (BCAA) aminotransferase Bat1 plays an important role in the synthesis of BCAAs (valine, leucine, and isoleucine). Our upcoming study (Large et al. bioRχiv. 10.1101/2020.06.26.166157, Large et al. 2020) will show that the heterozygous tetraploid beer yeast strain, Wyeast 1056, which natively has a variant causing one amino acid substitution of Ala234Asp in Bat1 on one of the four chromosomes, produced higher levels of BCAA-derived fusel alcohols in the brewer's wort medium than a derived strain lacking this mutation. Here, we investigated the physiological role of the A234D variant Bat1 in S. cerevisiae. Both bat1∆ and bat1A234D cells exhibited the same phenotypes relative to the wild-type Bat1 strain-namely, a repressive growth rate in the logarithmic phase; decreases in intracellular valine and leucine content in the logarithmic and stationary growth phases, respectively; an increase in fusel alcohol content in culture medium; and a decrease in the carbon dioxide productivity. These results indicate that amino acid change from Ala to Asp at position 234 led to a functional impairment of Bat1, although homology modeling suggests that Asp234 in the variant Bat1 did not inhibit enzymatic activity directly. KEY POINTS: • Yeast cells expressing Bat1A234D exhibited a slower growth phenotype. • The Val and Leu levels were decreased in yeast cells expressing Bat1A234D. • The A234D substitution causes a loss-of-function in Bat1. • The A234D substitution in Bat1 increased fusel alcohol production in yeast cells.

Role of Gln79 in Feedback Inhibition of the Yeast γ-Glutamyl Kinase by Proline.

Awamori, the traditional distilled alcoholic beverage of Okinawa, Japan, is brewed with the yeast Saccharomyces cerevisiae. During the distillation process after the fermentation, enormous quantities of distillation residues containing yeast cells must be disposed of, and this has recently been recognized as a major problem both environmentally and economically. Proline, a multifunctional amino acid, has the highest water retention capacity among amino acids. Therefore, distillation residues with large amounts of proline could be useful in cosmetics. Here, we isolated a yeast mutant with high levels of intracellular proline and found a missense mutation (Gln79His) on the PRO1 gene encoding the γ-glutamyl kinase Pro1, a limiting enzyme in proline biosynthesis. The amino acid change of Gln79 to His in Pro1 resulted in desensitization to the proline-mediated feedback inhibition of GK activity, leading to the accumulation of proline in cells. Biochemical and in silico analyses showed that the amino acid residue at position 79 is involved in the stabilization of the proline binding pocket in Pro1 via a hydrogen-bonding network, which plays an important role in feedback inhibition. Our current study, therefore, proposed a possible mechanism underlying the feedback inhibition of γ-glutamyl kinase activity. This mechanism can be applied to construct proline-accumulating yeast strains to effectively utilize distillation residues.

Purification and characterization of a halotolerant serine proteinase from thermotolerant Bacillus licheniformis RKK-04 isolated from Thai fish sauce.

A gram-positive thermotolerant bacterium, designated strain RKK-04, was isolated from a fermented Thai fish sauce broth as it demonstrated high proteolytic activity. A phylogenetic analysis based on comparisons of 16S rRNA gene sequences showed that strain RKK-04 is Bacillus licheniformis. The proteolytic enzyme, which was purified 80-fold with 18% yield, has a molecular mass of 31 kDa and an isoelectric point higher than 9.3. The optimum pH and temperature of the enzyme activity were found to be 10.0 and 50 degrees C, respectively. The addition of diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride completely inhibited enzymatic activity. These results showed that the enzyme is a subtilisin-like alkaline serine proteinase. On the other hand, the enzyme exhibited unique cleavage sites in oxidized insulin B-chain that differed from those of other subtilisin-like proteases. High enzymatic activity was also retained under high salt conditions (30% NaCl). The myosin heavy chain of fish protein was completely digested by reaction with this enzyme. Thus the halotolerant proteinase from B. licheniformis RKK-04 is a key enzyme for fish sauce fermentation.

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

Since flavors of alcoholic beverages produced in fermentation process are affected mainly by yeast metabolism, the isolation and breeding of yeasts have contributed to the alcoholic beverage industry. To produce awamori, a traditional spirit (distilled alcoholic beverage) with unique flavors made from steamed rice in Okinawa, Japan, it is necessary to optimize yeast strains for a diversity of tastes and flavors with established qualities. Two categories of flavors are characteristic of awamori; initial scented fruity flavors and sweet flavors that arise with aging. Here we isolated a novel strain of Saccharomyces cerevisiae from hibiscus flowers in Okinawa, HC02-5-2, that produces high levels of alcohol. The whole-genome information revealed that strain HC02-5-2 is contiguous to wine yeast strains in a phylogenic tree. This strain also exhibited a high productivity of 4-vinyl guaiacol (4-VG), which is a precursor of vanillin known as a key flavor of aged awamori. Although conventional awamori yeast strain 101-18, which possesses the FDC1 pseudogene does not produce 4-VG, strain HC02-5-2, which has the intact PAD1 and FDC1 genes, has an advantage for use in a novel kind of awamori. To increase the contents of initial scented fruity flavors, such as isoamyl alcohol and isoamyl acetate, we attempted to breed strain HC02-5-2 targeting the L-leucine synthetic pathway by conventional mutagenesis. In mutant strain T25 with L-leucine accumulation, we found a hetero allelic mutation in the LEU4 gene encoding the Gly516Ser variant α-isopropylmalate synthase (IPMS). IPMS activity of the Gly516Ser variant was less sensitive to feedback inhibition by L-leucine, leading to intracellular L-leucine accumulation. In a laboratory-scale test, awamori brewed with strain T25 showed higher concentrations of isoamyl alcohol and isoamyl acetate than that brewed with strain HC02-5-2. Such a combinatorial approach to yeast isolation, with whole-genome analysis and metabolism-focused breeding, has the potentials to vary the quality of alcoholic beverages.

Crucial roles of reactive chemical species in modification of respiratory syncytial virus by nitrogen gas plasma.

The exact mechanisms by which nanoparticles, especially those composed of soft materials, are modified by gas plasma remain unclear. Here, we used respiratory syncytial virus (RSV), which has a diameter of 80-350nm, as a model system to identify important factors for gas plasma modification of nanoparticles composed of soft materials. Nitrogen gas plasma, generated by applying a short high-voltage pulse using a static induction (SI) thyristor power supply produced reactive chemical species (RCS) and caused virus inactivation. The plasma treatment altered the viral genomic RNA, while treatment with a relatively low concentration of hydrogen peroxide, which is a neutral chemical species among RCS, effectively inactivated the virus. Furthermore, a zero dimensional kinetic global model of the reaction scheme during gas plasma generation identified the production of various RCS, including neutral chemical species. Our findings suggest the nitrogen gas plasma generates RCS, including neutral species that damage the viral genomic RNA, leading to virus inactivation. Thus, RCS generated by gas plasma appears to be crucial for virus inactivation, suggesting this may constitute an important factor in terms of the efficient modification of nanoparticles composed of soft materials.

Nitrogen gas plasma treatment of bacterial spores induces oxidative stress that damages the genomic DNA.

Gas plasma, produced by a short high­voltage pulse generated from a static induction thyristor power supply [1.5 kilo pulse/sec (kpps)], was demonstrated to inactivate Geobacillus stearothermophilus spores (decimal reduction time at 15 min, 2.48 min). Quantitative polymerase chain reaction and enzyme­linked immunosorbent assays further indicated that nitrogen gas plasma treatment for 15 min decreased the level of intact genomic DNA and increased the level of 8-hydroxy-2'-deoxyguanosine, a major product of DNA oxidation. Three potential inactivation factors were generated during operation of the gas plasma instrument: Heat, longwave ultraviolet-A and oxidative stress (production of hydrogen peroxide, nitrite and nitrate). Treatment of the spores with hydrogen peroxide (3x2­4%) effectively inactivated the bacteria, whereas heat treatment (100˚C), exposure to UV-A (75­142 mJ/cm2) and 4.92 mM peroxynitrite (•ONOO­), which is decomposed into nitrite and nitrate, did not. The results of the present study suggest the gas plasma treatment inactivates bacterial spores primarily by generating hydrogen peroxide, which contributes to the oxidation of the host genomic DNA.

Nitrogen Gas Plasma Generated by a Static Induction Thyristor as a Pulsed Power Supply Inactivates Adenovirus.

Adenovirus is one of the most important causative agents of iatrogenic infections derived from contaminated medical devices or finger contact. In this study, we investigated whether nitrogen gas plasma, generated by applying a short high-voltage pulse to nitrogen using a static induction thyristor power supply (1.5 kilo pulse per second), exhibited a virucidal effect against adenoviruses. Viral titer was reduced by one log within 0.94 min. Results from detection of viral capsid proteins, hexon and penton, by Western blotting and immunochromatography were unaffected by the plasma treatment. In contrast, analysis using the polymerase chain reaction suggested that plasma treatment damages the viral genomic DNA. Reactive chemical products (hydrogen peroxide, nitrate, and nitrite), ultraviolet light (UV-A) and slight temperature elevations were observed during the operation of the gas plasma device. Viral titer versus intensity of each potential virucidal factor were used to identify the primary mechanism of disinfection of adenovirus. Although exposure to equivalent levels of UV-A or heat treatment did not inactivate adenovirus, treatment with a relatively low concentration of hydrogen peroxide efficiently inactivated the virus. Our results suggest the nitrogen gas plasma generates reactive chemical products that inactivate adenovirus by damaging the viral genomic DNA.