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

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.

Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae for high-level production of isoprenoids

2007 ◽  
Vol 9 (2) ◽  
pp. 160-168 ◽  
Author(s):  
Yoichiro Shiba ◽  
Eric M. Paradise ◽  
James Kirby ◽  
Dae-Kyun Ro ◽  
Jay D. Keasling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pyruvate Dehydrogenase ◽  
High Level ◽  
Level Production

scholarly journals Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via β-alanine

2015 ◽  
Vol 27 ◽  
pp. 57-64 ◽  
Author(s):  
Irina Borodina ◽  
Kanchana R. Kildegaard ◽  
Niels B. Jensen ◽  
Thomas H. Blicher ◽  
Jérôme Maury ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Synthetic Pathway ◽  
High Level ◽  
Hydroxypropionic Acid ◽  
Level Production

scholarly journals Combined in silico and 19F NMR analysis of 5-fluorouracil metabolism in yeast at low ATP conditions

2019 ◽  
Vol 39 (12) ◽  
Author(s):  
Piotr H. Pawłowski ◽  
Paweł Szczęsny ◽  
Bożenna Rempoła ◽  
Anna Poznańska ◽  
Jarosław Poznański
Keyword(s):  
Oxidative Stress ◽  
Saccharomyces Cerevisiae ◽  
In Silico ◽  
31P Nmr ◽  
Yeast Cells ◽  
Atp Concentration ◽  
In Silico Modeling ◽  
Intracellular Metabolism ◽  
Hydrolysis Of ◽  
Careful Design

Abstract The cytotoxic effect of 5-fluorouracil (5-FU) on yeast cells is thought to be mainly via a misincorporation of fluoropyrimidines into both RNA and DNA, not only DNA damage via inhibition of thymidylate synthase (TYMS) by fluorodeoxyuridine monophosphate (FdUMP). However, some studies on Saccharomyces cerevisiae show a drastic decrease in ATP concentration under oxidative stress, together with a decrease in concentration of other tri- and diphosphates. This raises a question if hydrolysis of 5-fluoro-2-deoxyuridine diphosphate (FdUDP) under oxidative stress could not lead to the presence of FdUMP and the activation of so-called ‘thymine-less death’ route. We attempted to answer this question with in silico modeling of 5-FU metabolic pathways, based on new experimental results, where the stages of intracellular metabolism of 5-FU in Saccharomyces cerevisiae were tracked by a combination of 19F and 31P NMR spectroscopic study. We have identified 5-FU, its nucleosides and nucleotides, and subsequent di- and/or triphosphates. Additionally, another wide 19F signal, assigned to fluorinated unstructured short RNA, has been also identified in the spectra. The concentration of individual metabolites was found to vary substantially within hours, however, the initial steady-state was preserved only for an hour, until the ATP concentration dropped by a half, which was monitored independently via 31P NMR spectra. After that, the catabolic process leading from triphosphates through monophosphates and nucleosides back to 5-FU was observed. These results imply careful design and interpretation of studies in 5-FU metabolism in yeast.

Multiple-yapsin-deficient mutant strains for high-level production of intact recombinant proteins in Saccharomyces cerevisiae

2010 ◽  
Vol 149 (1-2) ◽  
pp. 1-7 ◽  
Author(s):  
Eun Young Cho ◽  
Seon Ah Cheon ◽  
Hyunah Kim ◽  
Jinho Choo ◽  
Dong-Jik Lee ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Recombinant Proteins ◽  
Deficient Mutant ◽  
Mutant Strains ◽  
High Level ◽  
Level Production

scholarly journals Desensitization of Feedback Inhibition of the Saccharomyces cerevisiae γ-Glutamyl Kinase Enhances Proline Accumulation and Freezing Tolerance

2007 ◽  
Vol 73 (12) ◽  
pp. 4011-4019 ◽  
Author(s):  
Tomoko Sekine ◽  
Akari Kawaguchi ◽  
Yoshimitsu Hamano ◽  
Hiroshi Takagi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Feedback Inhibition ◽  
Random Mutagenesis ◽  
Proline Accumulation ◽  
Molecular Surface ◽  
Yeast Cells ◽  
Freezing Stress ◽  
Ethanol Stress ◽  
Plasmid Library ◽  
Glycerol Synthesis

ABSTRACT In response to osmotic stress, proline is accumulated in many bacterial and plant cells as an osmoprotectant. The yeast Saccharomyces cerevisiae induces trehalose or glycerol synthesis but does not increase intracellular proline levels during various stresses. Using a proline-accumulating mutant, we previously found that proline protects yeast cells from damage by freezing, oxidative, or ethanol stress. This mutant was recently shown to carry an allele of PRO1 which encodes the Asp154Asn mutant γ-glutamyl kinase (GK), the first enzyme of the proline biosynthetic pathway. Here, enzymatic analysis of recombinant proteins revealed that the GK activity of S. cerevisiae is subject to feedback inhibition by proline. The Asp154Asn mutant was less sensitive to feedback inhibition than wild-type GK, leading to proline accumulation. To improve the enzymatic properties of GK, PCR random mutagenesis in PRO1 was employed. The mutagenized plasmid library was introduced into an S. cerevisiae non-proline-utilizing strain, and proline-overproducing mutants were selected on minimal medium containing the toxic proline analogue azetidine-2-carboxylic acid. We successfully isolated several mutant GKs that, due to extreme desensitization to inhibition, enhanced the ability to synthesize proline better than the Asp154Asn mutant. The amino acid changes were localized at the region between positions 142 and 154, probably on the molecular surface, suggesting that this region is involved in allosteric regulation. Furthermore, we found that yeast cells expressing Ile150Thr and Asn142Asp/Ile166Val mutant GKs were more tolerant to freezing stress than cells expressing the Asp154Asn mutant.

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