The combination of NAD+-dependent deacetylase gene deletion and the interruption of gluconeogenesis causes increased glucose metabolism in budding yeast

RESEARCH ARTICLE The combination of NAD+-dependent deacetylase gene deletion and the interruption of gluconeogenesis causes increased glucose metabolism in budding yeast a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Masumoto H, Matsuyama S (2018) The combination of NAD+-dependent deacetylase gene deletion and the interruption of gluconeogenesis causes increased glucose metabolism in budding yeast. PLoS ONE 13(3): e0194942. https://doi.org/ 10.1371/journal.pone.0194942 Editor: Marie-Joelle Virolle, Universite Paris-Sud, FRANCE Received: October 6, 2017 Accepted: March 13, 2018 Published: March 26, 2018 Copyright: © 2018 Masumoto, Matsuyama. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by NIG Collaborative Research Program (2016-A1-2) and Special Coordination Funds for Promoting Science and Technology of the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government. Hiroshi Masumoto1¤*, Shigeru Matsuyama2 1 Transdisciplinary Research Integration Center, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka, Japan, 2 Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki, Japan ¤ Current address: Biomedical Research Support Center (BRSC), Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki, Nagasaki, Japan * Abstract Metabolic engineering focuses on rewriting the metabolism of cells to enhance native products or endow cells with the ability to produce new products. This engineering has the potential for wide-range application, including the production of fuels, chemicals, foods and pharmaceuticals. Glycolysis manages the levels of various secondary metabolites by controlling the supply of glycolytic metabolites. Metabolic reprogramming of glycolysis is expected to cause an increase in the secondary metabolites of interest. In this study, we constructed a budding yeast strain harboring the combination of triple sirtuin gene deletion (hst3Δ hst4Δ sir2Δ) and interruption of gluconeogenesis by the deletion of the FBP1 gene encoding fructose-1,6-bisphosphatase (fbp1Δ). hst3Δ hst4Δ sir2Δ fbp1Δ cells harbored active glycolysis with high glucose consumption and active ethanol productivity. Using capillary electrophoresis–time-of-flight mass spectrometry (CE–TOF/MS) analysis, hst3Δ hst4Δ sir2Δ fbp1Δ cells accumulated not only glycolytic metabolites but also secondary metabolites, including nucleotides that were synthesized throughout the pentose phosphate (PP) pathway, although various amino acids remained at low levels. Using the stable isotope labeling assay for metabolites, we confirmed that hst3Δ hst4Δ sir2Δ fbp1Δ cells directed the metabolic fluxes of glycolytic metabolites into the PP pathway. Thus, the deletion of three sirtuin genes (HST3, HST4 and SIR2) and the FBP1 gene can allow metabolic reprogramming to increase glycolytic metabolites and several secondary metabolites except for several amino acids. Competing interests: The authors have declared that no competing interests exist. PLOS ONE | https://doi.org/10.1371/journal.pone.0194942 March 26, 2018 1 / 13 Metabolic reprogramming of glycolysis Introduction Metabolic engineering is the science of rewriting the metabolism of cells to enhance native products or endow cells with the ability to produce new products [1]. This engineering has the potential for wide-range application, including the production of fuels, chemicals, foods and pharmaceuticals. The combination of bioinformatics and mathematical modeling methods, which enable quantitative analysis, has facilitated the development of metabolic engineering to generate genetic modifications that alter cellular metabolism to direct the fluxes toward the product of interest [1]. Glycolysis plays a pivotal role in central carbon metabolism and may become an important target of metabolic engineering. This biochemical reaction catabolizes glucose as a carbon source and produces pyruvate, adenosine triphosphate (ATP) and various glycolytic intermediates [2]. Glycolytic metabolites are employed in secondary metabolic reactions, such as lipid and amino acid metabolism, to produce considerable species of secondary metabolites [3]. For example, glycolysis shunts into the pentose phosphate (PP) pathway, producing muchneeded nucleotides for proliferation (Fig 1A). Increased glycolysis is utilized in cellular proliferation. The Warburg effect shifts from oxidative phosphorylation to aerobic glycolysis, characteristic of cancer cells [4–6]. Cancer cells drive glycolysis to generate ATP at a faster rate than oxidative phosphorylation, while producing less reactive oxygen species (ROS), nucleotides much needed throughout the PP pathway for rapid proliferation. Additionally, an increase in the fermentation of microbes contributes to human life. Budding yeast (Saccharomyces cerevisiae) harbors active glycolysis equipped with strong fermentation ability. This organism has been utilized to produce fermentative foods or beverages and has been recently utilized in the biofuel industry to produce biofuels such as ethanol and 1-butanol [7–9]. Metabolic engineering to activate glycolysis has the potential to achieve an increase in various secondary metabolic pathways. Gluconeogenesis, almost the reverse biochemical reaction of glycolysis, is activated to utilize a carbon source other than glucose [3]. In budding yeast, the main gluconeogenesis-specific enzymes are fructose-1,6-bisphosphatase (Fbp1), isocitrate lyase carboxykinase (Icl1), malate dehydrogenase (Mdh2), and phosphoenolpyruvate carboxykinase (Pck1) [3, 10]. An increase in both glycolysis and glucose storage is manifested in aged yeast cells, caused by an abnormal activation of gluconeogenesis [11, 12]. Some aging-related gene deletions exhibit a metabolic status that mimics that of aged yeast cells [11, 12]. NAD+-dependent deacetylases, which are also called sirtuins, are involved in multiple cellular functions, including gene silencing, genome maintenance, cellular metabolism and cellular aging [13, 14]. Among the five genes in the budding yeast sirtuin family (SIR2 and HST1/2/3/4), Sir2, Hst3 and Hst4 are involved in the regulation of cellular lifespan and cell metabolism [15, 16]. The increase in both glycolysis and glucose storage manifested in hst3Δ hst4Δ cells reflect enhanced gluconeogenesis [11, 12]. Additionally, sir2Δ cell exhibits enhanced gluconeogenesis by maintaining the acetylated form of Pck1 to prevent the conversion from phosphoenol pyruvate (PEP) to oxaloacetate [17]. Interestingly, the TDH2 gene encodes a glyceraldehyde 3-phosphate dehydrogena (...truncated)