Effects of Abolishing Whi2 on the Proteome and Nitrogen Catabolite Repression-Sensitive Protein Production

In yeast physiology, a commonly used reference condition for many experiments, including those involving Nitrogen Catabolite Repression (NCR), is growth in Synthetic Complete (SC) medium. Four SC formulations, SCCSH, 1990, SCCSH, 1994, SCCSH, 2005 and SCME, have been used interchangeably as the nitrogen-rich medium of choice (Cold Spring Harbor Yeast Course Manuals, SCCSH, and a formulation in The Methods in Enzymology SCME). It has been tacitly presumed that all of these formulations support equivalent responses. However, Chen et al. (2018) concluded that: (i) TorC1 activity is down regulated by the lower concentration of primarily leucine in SCME relative to SCCSH. (ii) The Whi2-Psr1/2 complex is responsible for this down regulation. TorC1 is a primary nitrogen-responsive regulator in yeast. Among its downstream targets is control of NCR-sensitive transcription activators Gln3 and Gat1. They in turn control production of catabolic transporters and enzymes needed to scavenge poor nitrogen sources (e.g., Proline) and activate autophagy (ATG14). One of the reporters used in Chen et al. was an NCR-sensitive DAL80-GFP promoter fusion. This intrigued us because we expected minimal if any DAL80 expression in SC medium. Therefore, we investigated the source of the Dal80-GFP production and the proteomes of wild type and whi2Δ cells cultured in SCCSH and SCME. We found a massive and equivalent reorientation of amino acid biosynthetic proteins in both wild type and whi2Δ cells even though both media contained high overall concentrations of amino acids. Gcn2 appears to play a significant regulatory role in this reorientation. NCR-sensitive DAL80 expression and overall NCR-sensitive protein production were only marginally affected by the whi2Δ. In contrast, the levels of 58 proteins changed by an absolute value of log2 between 3 and 8) when Whi2 was abolished relative to wild type. Surprisingly, with only two exceptions could those proteins be related in GO analyses, i.e., GO terms associated with carbohydrate metabolism and oxidative stress after shifting a whi2Δ from SCCSH to SCME for 6 hours. What was conspicuously missing were proteins related by TorC1- and NCR-associated GO terms.

Increased accumulation of squalene in engineered Yarrowia lipolytica through deletion of PEX10 and URE2

Squalene is a triterpenoid serving as an ingredient of various products in the food, cosmetic, pharmaceutical industries. The oleaginous yeast Yarrowia lipolytica offers enormous potential as a microbial chassis for the production of terpenoids, such as carotenoid, limonene, linalool, and farnesene as the yeast provides ample storage space for hydrophobic products. Here we present a metabolic design that allows the enhanced accumulation of squalene in Y. lipolytica . First, we improved squalene accumulation in Y. lipolytica by overexpressing the genes (ERG, HMG) coding for the mevalonate pathway enzymes. Second, we increased the production of lipid where squalene is accumulated by overexpressing DGA1 encoding for diacylglycerol acyltransferase and deleting PEX10 for peroxisomal membrane E3 ubiquitin ligase. Third, we deleted URE2 coding for a transcriptional regulator in charge of nitrogen catabolite repression (NCR) to induce lipid accumulation regardless of carbon to nitrogen ratio in culture media. The resulting engineered Y. lipolytica exhibited a 115-fold higher squalene content (22.0 mg/g DCW) than a parental strain. These results suggest that the biological function of Ure2p in Y. lipolytica is similar to that in S. cerevisiae , and its deletion can be utilized to enhance the production of hydrophobic target products in oleaginous yeast strains. IMPORTANCE This study demonstrated a novel strategy for increasing squalene production in Y. lipolytica . URE2, a bifunctional protein that is involved in both nitrogen catabolite repression and oxidative stress response, was identified and demonstrated correlation to squalene production. The data suggest that double deletion of PEX10 and URE2 can serve as a positive synergistic effect to help yeast cells on boosting squalene production. This discovery can be combined with other strategies to engineer cell factories to efficiently produce terpenoid in the future.

N- and C-terminal Gln3–Tor1 interaction sites: one acting negatively and the other positively to regulate nuclear Gln3 localization

Gln3 activates Nitrogen Catabolite Repression, NCR-sensitive expression of the genes required for Saccharomyces cerevisiae to scavenge poor nitrogen sources from its environment. The global TorC1 kinase complex negatively regulates nuclear Gln3 localization, interacting with an α-helix in the C-terminal region of Gln3, Gln3656–666. In nitrogen replete conditions, Gln3 is sequestered in the cytoplasm, whereas when TorC1 is down-regulated, in nitrogen restrictive conditions, Gln3 migrates into the nucleus. In this work, we show that the C-terminal Gln3–Tor1 interaction site is required for wild type, rapamycin-elicited, Sit4-dependent nuclear Gln3 localization, but not for its dephosphorylation. In fact, truncated Gln31-384 can enter the nucleus in the absence of Sit4 in both repressive and derepressive growth conditions. However, Gln31-384 can only enter the nucleus if a newly discovered second positively-acting Gln3–Tor1 interaction site remains intact. Importantly, the N- and C-terminal Gln3–Tor1 interaction sites function both autonomously and collaboratively. The N-terminal Gln3–Tor1 interaction site, previously designated Gln3URS contains a predicted α-helix situated within an unstructured coiled-coil region. Eight of the thirteen serine/threonine residues in the Gln3URS are dephosphorylated 3–15-fold with three of them by 10–15-fold. Substituting phosphomimetic aspartate for serine/threonine residues in the Gln3 URS abolishes the N-terminal Gln3–Tor1 interaction, rapamycin-elicited nuclear Gln3 localization, and ½ of the derepressed levels of nuclear Gln3 localization. Cytoplasmic Gln3 sequestration in repressive conditions, however, remains intact. These findings further deconvolve the mechanisms that achieve nitrogen-responsive transcription factor regulation downstream of TorC1.

Regulatory Networks Governing Methionine Catabolism into Volatile Organic Sulfur-Containing Compounds in Clonostachys rosea

ABSTRACT Adaptation to environmental perturbations requires living systems to coordinately regulate signaling pathways, gene expression, and metabolism. To better understand the mechanisms underlying adaptation, the regulatory nodes within networks must be elucidated. Here, ARO8-2 (which encodes an aminotransferase), PDC (which encodes a decarboxylase), and STR3 (which encodes a demethiolase) were identified as key genes involved in the catabolism of methionine in the mycoparasitic fungus Clonostachys rosea, isolated from Tuber melanosporum ascocarps. Exogenous Met induced the transcription of ARO8-2 and PDC but repressed the transcription of STR3, which is controlled by the putative MSN2 and GLN3 binding sites responding to nitrogen catabolite repression. Met and its structural derivatives function as glutamine synthetase inhibitors, resulting in the downregulation of STR3 expression. The putative GLN3 binding site was necessary for STR3 downregulation. In Saccharomyces cerevisiae, Met and its structural derivatives also triggered downregulation of demethiolase gene expression. Altogether, the results indicated that exogenous Met triggered nitrogen catabolite repression, which stimulated the Ehrlich pathway and negatively regulated the demethiolation pathway via the methionine sulfoximine-responsive regulatory pathway. This finding revealed the regulatory nodes within the networks controlling the catabolism of Met into volatile organic sulfur-containing compounds, thereby enhancing our understanding of adaptation. IMPORTANCE Methionine shuttles organic nitrogen and plays a central role in nitrogen metabolism. Exogenous Met strongly induces the expression of ARO8-2 and PDC, represses the expression of STR3, and generates volatile organic sulfur-containing compounds via the Ehrlich and demethiolation pathways. In this study, we used genetic, bioinformatic, and metabolite-based analyses to confirm that transcriptional control of the aminotransferase gene ARO8-2, the decarboxylase gene PDC, and the demethiolase gene STR3 modulates Met catabolism into volatile organic sulfur-containing compounds. Importantly, we found that, in addition to the Ehrlich pathway, the demethiolation pathway was regulated by a nitrogen catabolite repression-sensitive regulatory pathway that controlled the transcription of genes required to catabolize poor nitrogen sources. This work significantly advances our understanding of nitrogen catabolite repression-sensitive transcriptional regulation of sulfur-containing amino acid catabolism and provides a basis for engineering Met catabolism pathways for the production of fuel and valuable flavor alcohols.

Regulation of Nitrogen Metabolism by GATA Zinc Finger Transcription Factors in Yarrowia lipolytica

ABSTRACT Nitrogen source is commonly used to control lipid production in industrial fungi. Here we identified regulators of nitrogen catabolite repression in the oleaginous yeast Y. lipolytica to determine how the nitrogen source regulates lipid metabolism. We show that disruption of both activators and repressors of nitrogen catabolite repression leads to increased lipid accumulation via activation of carbon catabolite repression through an as yet uncharacterized method. Fungi accumulate lipids in a manner dependent on the quantity and quality of the nitrogen source on which they are growing. In the oleaginous yeast Yarrowia lipolytica, growth on a complex source of nitrogen enables rapid growth and limited accumulation of neutral lipids, while growth on a simple nitrogen source promotes lipid accumulation in large lipid droplets. Here we examined the roles of nitrogen catabolite repression and its regulation by GATA zinc finger transcription factors on lipid metabolism in Y. lipolytica. Deletion of the GATA transcription factor genes gzf3 and gzf2 resulted in nitrogen source-specific growth defects and greater accumulation of lipids when the cells were growing on a simple nitrogen source. Deletion of gzf1, which is most similar to activators of genes repressed by nitrogen catabolite repression in filamentous ascomycetes, did not affect growth on the nitrogen sources tested. We examined gene expression of wild-type and GATA transcription factor mutants on simple and complex nitrogen sources and found that expression of enzymes involved in malate metabolism, beta-oxidation, and ammonia utilization are strongly upregulated on a simple nitrogen source. Deletion of gzf3 results in overexpression of genes with GATAA sites in their promoters, suggesting that it acts as a repressor, while gzf2 is required for expression of ammonia utilization genes but does not grossly affect the transcription level of genes predicted to be controlled by nitrogen catabolite repression. Both GATA transcription factor mutants exhibit decreased expression of genes controlled by carbon catabolite repression via the repressor mig1, including genes for beta-oxidation, highlighting the complex interplay between regulation of carbon, nitrogen, and lipid metabolism. IMPORTANCE Nitrogen source is commonly used to control lipid production in industrial fungi. Here we identified regulators of nitrogen catabolite repression in the oleaginous yeast Y. lipolytica to determine how the nitrogen source regulates lipid metabolism. We show that disruption of both activators and repressors of nitrogen catabolite repression leads to increased lipid accumulation via activation of carbon catabolite repression through an as yet uncharacterized method.