Functional analysis of feedback inhibition-insensitive aspartate kinase identified in a threonine-accumulating mutant of Saccharomyces cerevisiae.

Humans and mammals need to ingest essential amino acids (EAAs) for protein synthesis. In addition to their importance as nutrients, EAAs are involved in brain homeostasis. However, elderly people are unable to efficiently consume EAAs from their daily diet due to reduced appetite and variations in the contents of EAAs in foods. On the other hand, strains of the yeast Saccharomyces cerevisiae that accumulate EAAs would enable elderly people to intakegest adequate amounts of EAAs and thus might slow down the neurodegenerative process, contributing to the extension of their healthy lifespan. In this study, we isolated a mutant (strain HNV-5) that accumulates threonine, an EAA, derived from a diploid laboratory yeast by conventional mutagenesis. Strain HNV-5 carries a novel mutation in the HOM3 gene encoding the Ala462Thr variant of aspartate kinase (AK). Enzymatic analysis revealed that the Ala462Thr substitution significantly decreased the sensitivity of AK activity to threonine feedback inhibition even in the presence of 50 mM threonine. Interestingly, Ala462Thr substitution did not affect the catalytic ability of Hom3, in contrast to previously reported amino acid substitutions that resulted in reduced sensitivity to threonine feedback inhibition. Furthermore, yeast cells expressing the Ala462Thr variant showed an approximately threefold increase in intracellular threonine content compared to that of the wild-type Hom3. These findings will be useful for the development of threonine-accumulating yeast strains that may improve the quality of life in elderly people.IMPORTANCEFor humans and mammals, essential amino acids (EAAs) play an important role in maintaining brain function. Therefore, increasing the intake of EAAs by using strains of the yeast Saccharomyces cerevisiae that accumulate EAAs may inhibit neurodegeneration in elderly people and thus contribute to extending healthy lifespan and improving their quality of life. Threonine, an EAA, is synthesized from aspartate. Aspartate kinase (AK) catalyzes the first step in threonine biosynthesis and is subject to allosteric regulation by threonine. Here, we isolated a threonine-accumulating mutant of S. cerevisiae by conventional mutagenesis and identified a mutant gene encoding a novel variant of AK. In contrast to previously isolated variants, the Hom3 variant exhibited AK activity that was insensitive to feedback inhibition by threonine but retained its catalytic ability. This resulted in increased production of threonine in yeast. These findings open up the possibility for the rational design of AK to increase threonine productivity in yeast.

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.

Functional Analysis of Feedback Inhibition-Insensitive Variants of N-Acetyl Glutamate Kinase Found in Sake Yeast Mutants with Ornithine Overproduction.

In the yeast Saccharomyces cerevisiae, N-acetyl glutamate kinase (NAGK), which catalyzes the phosphorylation of N-acetyl glutamate to form N-acetyl glutamyl-5-phosphate, is one of the rate-limiting enzymes in the ornithine and arginine biosynthetic pathways. NAGK activity is strictly regulated via feedback inhibition by the end product, arginine. We previously reported that the Thr340Ile variant of NAGK was insensitive to arginine feedback inhibition and that the interaction between Lys336 and Thr340 in NAGK may be important for arginine recognition. In the present study, we demonstrated that amino acid changes of Thr340 to Ala, Leu, Arg, Glu, Ile, and Asn removed arginine feedback inhibition, although the Thr340Ser variant was subject to the feedback inhibition. Therefore, these results indicate that the arginine-binding cavity formed via the interaction between the carbonyl group in the main chain of Lys336 and the hydroxyl group in the side chain of the residue at position 340 is critical for arginine recognition of NAGK. In addition, we newly identified two mutations in the ARG5,6 gene encoding the Cys119Tyr or Val267Ala variant of NAGK of sake yeast mutants with intracellular ornithine accumulation. Although it is unlikely that Cys119 and Val267 are directly involved in arginine recognition, we found here that two variants of NAGK were insensitive to arginine feedback inhibition and contributed to high-level production of ornithine. Structural analysis of NAGK suggests that these two amino acid substitutions influence the sensitivity to Arg feedback inhibition through alterations in local conformation around each residue. IMPORTANCE Ornithine has a number of physiological benefits in humans. Thus, an Orn-rich alcoholic beverage is expected to relieve feelings of fatigue after drinking. In the yeast Saccharomyces cerevisiae, N-acetyl glutamate kinase (NAGK) encoded by the ARG5,6 gene catalyzes the second step in ornithine and arginine biosynthesis, and its activity is subjected to feedback inhibition by arginine. Here, we revealed a role of key residues in the formation of the arginine-binding cavity which is critical for arginine recognition of NAGK. In addition, we analyzed novel arginine feedback inhibition-insensitive variants of NAGK in sake yeast mutants with ornithine overproduction and proposed that the amino acid substitutions in the NAGK variants destabilize the arginine-binding cavity, leading to the lower sensitivity to arginine feedback inhibition of NAGK activity. These findings provide new insight into the allosteric regulation of NAGK activity and will help to construct superior industrial yeast strains for high-level production of ornithine.

Cryptic Oxidative Transamination of Hydroxynaphthoquinone in Natural Product Biosynthesis.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes are a group of versatile enzymes that catalyze various reactions, but only a small number of them react with O2. Here, we report an unprecedented PLP-dependent enzyme, NphE, that catalyzes both transamination and two-electron oxidation using O2 as an oxidant. Our intensive analysis reveals that NphE transfers the l-glutamate-derived amine to 1,3,6,8-tetrahydroxynaphthalene-derived mompain to form 8-amino-flaviolin (8-AF) via a highly conjugated quinonoid intermediate that is reactive with O2. During the NphE reaction, O2 is reduced to yield H2O2. An integrated technique involving NphE structure prediction by AlphaFold v2.0 and molecular dynamics simulation suggested the O2-accessible cavity. Our in vivo results demonstrated that 8-AF is a genuine biosynthetic intermediate for the 1,3,6,8-tetrahydroxynaphthalene-derived meroterpenoid naphterpin without an amino group, which was supported by site-directed mutagenesis. This study clearly establishes the NphE reaction product 8-AF as a common intermediate with a cryptic amino group for the biosynthesis of terpenoid-polyketide hybrid natural products.

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

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 to 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.

Isolation and analysis of a sake yeast mutant with phenylalanine accumulation.

Sake is a traditional Japanese alcoholic beverage brewed by the yeast Saccharomyces cerevisiae. Since the consumption and connoisseurship of sake has spread around the world, the development of new sake yeast strains to meet the demand for unique sakes has been promoted. Phenylalanine is an essential amino acid that is used to produce proteins and important signaling molecules involved in feelings of pleasure. In addition, phenylalanine is a precursor of 2-phenylethanol, a high-value aromatic alcohol with a rose-like flavor. As such, adjusting the quantitative balance between phenylalanine and 2-phenylethanol may introduce value-added qualities to sake. Here, we isolated a sake yeast mutant (strain K9-F39) with phenylalanine accumulation and found a missense mutation on the ARO80 gene encoding the His309Gln variant of the transcriptional activator Aro80p involved in the biosynthesis of 2-phenylethanol from phenylalanine. We speculated that mutation of ARO80 would decrease transcriptional activity and suppress the phenylalanine catabolism, resulting in an increase of intracellular phenylalanine. Indeed, sake brewed with strain K9-F39 contained 60% increase in phenylalanine, but only 10% less 2-phenylethanol than sake brewed with the parent strain. Use of the ARO80 mutant in sake brewing may be promising for the production of distinctive new sake varieties. ONE-SENTENCE SUMMARY: The ARO80 mutant is appropriate for controlling the content of phenylalanine and 2-phenylethanol.

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.

Enhancement of lysine biosynthesis confers high-temperature stress tolerance to Escherichia coli cells.

Lysine, a nutritionally important amino acid, is involved in adaptation and tolerance to environmental stresses in various organisms. Previous studies reported that lysine accumulation occurs in response to stress and that lysine supplementation enhances stress tolerance; however, the effect of lysine biosynthesis enhancement on stress tolerance has yet to be elucidated. In this study, we confirmed that lysine supplementation to the culture medium increased intracellular lysine content and improved cell growth of Escherichia coli at high temperature (42.5 °C). Lysine-overproducing strains were then isolated from the lysine analogue S-adenosylmethionine-resistant mutants by conventional mutagenesis and exhibited higher tolerance to high-temperature stress than the wild-type strain. We identified novel amino acid substitutions Gly474Asp and Cys554Tyr on ThrA, a bifunctional aspartate kinase/homoserine dehydrogenase (AK/HSDH), in the lysine-overproducing mutants. Interestingly, the Gly474Asp and Cys554Tyr variants of ThrA induced lysine accumulation and conferred high-temperature stress tolerance to E. coli cells. Enzymatic analysis revealed that the Gly474Asp substitution in ThrA reduced HSDH activity, suggesting that the intracellular level of aspartate semialdehyde, which is a substrate for HSDH and an intermediate for lysine biosynthesis, is elevated by the loss of HSDH activity and converted to lysine in E. coli. The present study demonstrated that both lysine supplementation and lysine biosynthesis enhancement improved the high-temperature stress tolerance of E. coli cells. Our findings suggest that lysine-overproducing strains have the potential as stress-tolerant microorganisms and can be applied to robust host cells for microbial production of useful compounds. KEY POINTS: • Lysine supplementation improved the growth of E. coli cells at high temperature. • The G474D and C554Y variant ThrA increased lysine productivity in E. coli cells. • The G474D substitution in ThrA reduced homoserine dehydrogenase activity. • E. coli cells that overproduce lysine exhibited high-temperature stress tolerance.

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.

Synthetic production of prenylated naringenins in yeast using promiscuous microbial prenyltransferases.

Reconstitution of prenylflavonoids using the flavonoid biosynthetic pathway and prenyltransferases (PTs) in microbes can be a promising attractive alternative to plant-based production or chemical synthesis. Here, we demonstrate that promiscuous microbial PTs can be a substitute for regiospecific but mostly unidentified botanical PTs. To test the prenylations of naringenin, we constructed a yeast strain capable of producing naringenin from l-phenylalanine by genomic integration of six exogenous genes encoding components of the naringenin biosynthetic pathway. Using this platform strain, various microbial PTs were tested for prenylnaringenin production. In vitro screening demonstrated that the fungal AnaPT (a member of the tryptophan dimethylallyltransferase family) specifically catalyzed C-3' prenylation of naringenin, whereas SfN8DT-1, a botanical PT, specifically catalyzed C-8 prenylation. In vivo, the naringenin-producing strain expressing the microbial AnaPT exhibited heterologous microbial production of 3'-prenylnaringenin (3'-PN), in contrast to the previously reported in vivo production of 8-prenylnaringenin (8-PN) using the botanical SfN8DT-1. These findings provide strategies towards expanding the production of a variety of prenylated compounds, including well-known prenylnaringenins and novel prenylflavonoids. These results also suggest the opportunity for substituting botanical PTs, both known and unidentified, that display relatively strict regiospecificity of the prenyl group transfer.

High-level production of ornithine by expression of the feedback inhibition-insensitive N-acetyl glutamate kinase in the sake yeast Saccharomyces cerevisiae.

We previously reported that intracellular proline (Pro) confers tolerance to ethanol on the yeast Saccharomyces cerevisiae. In this study, to improve the ethanol productivity of sake, a traditional Japanese alcoholic beverage, we successfully isolated several Pro-accumulating mutants derived from diploid sake yeast of S. cerevisiae by a conventional mutagenesis. Interestingly, one of them (strain A902-4) produced more than 10-fold greater amounts of ornithine (Orn) and Pro compared to the parent strain (K901). Orn is a non-proteinogenic amino acid and a precursor of both arginine (Arg) and Pro. It has some physiological functions, such as amelioration of negative states such as lassitude and improvement of sleep quality. We also identified a homo-allelic mutation in the ARG5,6 gene encoding the Thr340Ile variant N-acetylglutamate kinase (NAGK) in strain A902-4. The NAGK activity of the Thr340Ile variant was extremely insensitive to feedback inhibition by Arg, leading to intracellular Orn accumulation. This is the first report of the removal of feedback inhibition of NAGK activity in the industrial yeast, leading to high levels of intracellular Orn. Moreover, sake and sake cake brewed with strain A902-4 contained 4-5 times more Orn than those brewed with strain K901. The approach described here could be a practical method for the development of industrial yeast strains with overproduction of Orn.

Effects of a novel variant of the yeast γ-glutamyl kinase Pro1 on its enzymatic activity and sake brewing.

Sake is a traditional Japanese alcoholic beverage brewed with the yeast Saccharomyces cerevisiae. Sake taste is affected by sugars, organic acids, and amino acids. We previously isolated mutants resistant to the proline analogue azetidine-2-carboxylate derived from a diploid sake yeast strain. Some of the mutants produced a greater amount of proline in the brewed sake. One of them (strain K-9-AZC) carried a novel mutation in the PRO1 gene encoding the Gln79His variant of the γ-glutamyl kinase Pro1, a key enzyme in proline biosynthesis in S. cerevisiae. This mutation resulted in extreme desensitization to feedback inhibition by proline, leading to proline overproduction. Interestingly, sake brewed with K-9-AZC contained 3.7-fold more proline, but only 25% less succinate than sake brewed with the parent strain. Metabolome analysis suggests that the decrease in succinate was attributable to a lower level of 2-oxoglutarate, which is converted into glutamate. The approach here could be a practical method for breeding of yeast strains involved in the diversity of sake taste.

Identification of 8-amino-2,5,7-trihydroxynaphthalene-1,4-dione, a novel intermediate in the biosynthesis of Streptomyces meroterpenoids.

Furaquinocin is a natural polyketide-isoprenoid hybrid (meroterpenoid) produced by Streptomyces sp. strain KO-3988. All of the fur genes required for furaquinocin biosynthesis have been cloned, and the heterologous production of furaquinocin has been demonstrated in Streptomyces albus. Here, we report the identification of 8-amino-2,5,7-trihydroxynaphthalene-1,4-dione (8-amino-flaviolin) produced by the S. albus heterologous expression of the three contiguous genes encoding type III polyketide synthase (Fur1), monooxygenase (Fur2), and aminotransferase (Fur3) in the furaquinocin biosynthetic gene cluster. An S. albus transformant (S. albus/pWHM-Fur2_del3) harboring the fur gene cluster and lacking the fur3 gene did not produce furaquinocin, whereas furaquinocin production was restored when 8-amino-flaviolin was added to the culture medium of S. albus/pWHM-Fur2_del3. These results demonstrate that Fur3 aminotransferase is essential for furaquinocin biosynthesis and that 8-amino-flaviolin is an early-stage intermediate in furaquinocin biosynthesis. We propose that the biosynthetic pathway generating 8-amino-flaviolin is the common route for the biosynthesis of Streptomyces meroterpenoids.