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 2-3 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.

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Expression, Enzymatic Characterization, and High-Level Production of Glucose Isomerase from Actinoplanes missouriensis CICIM B0118(A) in Escherichia coli

Zeitschrift für Naturforschung C ◽

10.1515/znc-2011-11-1210 ◽

2011 ◽

Vol 66 (11-12) ◽

pp. 605-613 ◽

Cited By ~ 2

Author(s):

He Wang ◽

Ruijin Yang ◽

Xiao Hua ◽

Zhong Zhang ◽

Wei Zhao ◽

...

Keyword(s):

Ph Value ◽

Glucose Isomerase ◽

Medium Composition ◽

Gene Encoding ◽

E Coli ◽

Optimum Ph ◽

One Step ◽

Ph Range ◽

High Level ◽

Level Production

High-level production of recombinant glucose isomerase (rGI) is desirable for lactulose synthesis. In this study, the xylA gene encoding glucose isomerase from Actinoplanes missouriensis CICIM B0118(A) was cloned and expressed in E. coli BL21(DE3), and high-level production was performed by optimization of the medium composition. rGI was purified from a recombinant E. coli BL21(DE3) and characterized. The optimum pH value of the purified enzyme was 8.0 and it was relatively stable within the pH range of 7.0 - 9.0. Its optimum temperature was around 85 °C, and it exhibited good thermostability when the temperature was lower than 90 °C. The maximum enzyme activity required the presence of both Co2+ and Mg2+, at the concentrations of 200 μM and 8 mM, respectively. With high-level expression and the simple one-step chromatographic purification of the His-tagged recombinant enzyme, this GI could be used in industrial production of lactulose as a potential economic tool

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Engineering Betalain Biosynthesis in Tomato for High Level Betanin Production in Fruits

Frontiers in Plant Science ◽

10.3389/fpls.2021.682443 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ramona Grützner ◽

Ramona Schubert ◽

Claudia Horn ◽

Changqing Yang ◽

Thomas Vogt ◽

...

Keyword(s):

Cytochrome P450 ◽

Biosynthetic Pathway ◽

Feedback Inhibition ◽

Red Pigment ◽

Tomato Plants ◽

Dehydrogenase Gene ◽

Yellow Orange ◽

Arogenate Dehydrogenase ◽

High Level ◽

Level Production

Betalains are pigments found in plants of the Caryophyllales order, and include the red-purple betacyanins and the yellow-orange betaxanthins. The red pigment from red beets, betanin, is made from tyrosine by a biosynthetic pathway that consists of a cytochrome P450, a L-DOPA dioxygenase, and a glucosyltransferase. The entire pathway was recently reconstituted in plants that do not make betalains naturally including potato and tomato plants. The amount of betanin produced in these plants was however not as high as in red beets. It was recently shown that a plastidic arogenate dehydrogenase gene involved in biosynthesis of tyrosine in plants is duplicated in Beta vulgaris and other betalain-producing plants, and that one of the two encoded enzymes, BvADHα, has relaxed feedback inhibition by tyrosine, contributing to the high amount of betanin found in red beets. We have reconstituted the complete betanin biosynthetic pathway in tomato plants with or without a BvADHα gene, and with all genes expressed under control of a fruit-specific promoter. The plants obtained with a construct containing BvADHα produced betanin at a higher level than plants obtained with a construct lacking this gene. These results show that use of BvADHα can be useful for high level production of betalains in heterologous hosts. Unlike red beets that produce both betacyanins and betaxanthins, the transformed tomatoes produced betacyanins only, conferring a bright purple-fuschia color to the tomato juice.

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High-level production of valine by expression of the feedback inhibition-insensitive acetohydroxyacid synthase in Saccharomyces cerevisiae

Metabolic Engineering ◽

10.1016/j.ymben.2018.02.011 ◽

2018 ◽

Vol 46 ◽

pp. 60-67 ◽

Cited By ~ 10

Author(s):

Natthaporn Takpho ◽

Daisuke Watanabe ◽

Hiroshi Takagi

Keyword(s):

Saccharomyces Cerevisiae ◽

Feedback Inhibition ◽

Acetohydroxyacid Synthase ◽

High Level ◽

Level Production

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

Applied and Environmental Microbiology ◽

10.1128/aem.00600-21 ◽

2021 ◽

Author(s):

Shota Isogai ◽

Tomonori Matsushita ◽

Hiroyuki Imanishi ◽

Jirasin Koonthongkaew ◽

Yoichi Toyokawa ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

In Silico ◽

Feedback Inhibition ◽

Yeast Cells ◽

Enzymatic Analysis ◽

In Silico Docking ◽

Key Enzyme ◽

Homocitrate Synthase ◽

High Level ◽

Level Production

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 to 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 variant Ser385Phe. Enzymatic analysis revealed that the Ser385Glu variant is far more insensitive to feedback inhibition than the Ser385Phe variant. We also found that the lysine content in yeast cells expressing the Ser385Glu variant was 4.62-fold and 1.47-fold higher than that 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 and 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.

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

Metabolic Engineering ◽

10.1016/j.ymben.2020.08.005 ◽

2020 ◽

Vol 62 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Masataka Ohashi ◽

Ryo Nasuno ◽

Shota Isogai ◽

Hiroshi Takagi

Keyword(s):

Saccharomyces Cerevisiae ◽

Feedback Inhibition ◽

Yeast Saccharomyces Cerevisiae ◽

High Level ◽

Level Production

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Effect of taste receptor protein T1R3 on the development of islet tissue of the murine pancreas

Доклады Академии наук ◽

10.31857/s0869-56524841117-120 ◽

2019 ◽

Vol 484 (1) ◽

pp. 117-120

Author(s):

V. O. Murovets ◽

E. A. Sozontov ◽

T. G. Zachepilo

Keyword(s):

Parent Strain ◽

Gene Knockout ◽

Taste Receptor ◽

Morphological Characteristics ◽

Sweet Taste ◽

Pancreatic Tissue ◽

Receptor Protein ◽

Gene Encoding ◽

Islet Tissue ◽

Knockout Animals

Protein T1R3, the main subunit of sweet, as well as amino acid, taste receptor, is expressed in the epithelium of the tongue and gastro intestinal tract, in β–cells of the pancreas, hypothalamus, and numerous other organs. Recently, convincing witnesses of T1R3 involvement in control of carbohydrate and lipid metabolism, and control of production of incretines and insulin, have been determined. In the study on Tas1r3-gene knockout mouse strain and parent strain C57Bl/6J as control, priority data concerning the effect of T1R3 on the morphological characteristics of Langerhans islets in the pancreas, are obtained. In Tas1r3 knockout animals, it is found that the size of the islets and their density in pancreatic tissue are reduced, as compared to the parent strain. Additionally, a decrease of expression of active caspase-3 in islets of gene-knockouts is demonstrated. The obtained data show that the lack of a functional, gene encoding sweet-taste receptor protein causes a dystrophy of the islet tissue and associates to the development of pathological changes in the pancreas specific to type-2 diabetes and obesity in humans.

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