High-level production of lysine in the yeast saccharomyces cerevisiae by rational design of homocitrate synthase

Shota Isogai, Tomonori Matsushita, Hiroyuki Imanishi, Jirasin Koonthongkaew, Yoichi Toyokawa, Akira Nishimura, Xiao Yi, Romas Kazlauskas, Hiroshi Takagi

Research output: Contribution to journalArticlepeer-review

7 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)1-12
Number of pages12
JournalApplied and environmental microbiology
Issue number15
StatePublished - Jul 2021

Bibliographical note

Funding Information:
This research was partially supported by the Matching Planner Program (MP27215668049) from the Japan Science and Technology Agency (JST) to H.T. We declare no conflicts of interest.

Publisher Copyright:
© 2021 Isogai et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license


  • feedback inhibition
  • homocitrate synthase
  • in silico docking
  • lysine
  • Saccharomyces cerevisiae
  • yeast
  • Metabolic Engineering
  • Lysine/metabolism
  • Oxo-Acid-Lyases/chemistry
  • Saccharomyces cerevisiae/genetics
  • Feedback, Physiological
  • Fungal Proteins/chemistry
  • Molecular Docking Simulation
  • Amino Acid Substitution

PubMed: MeSH publication types

  • Research Support, Non-U.S. Gov't
  • Journal Article


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