Although “Harry Potter and the Deathly Hallows” marks the termination of J. K. Rowling’s popular novels, we recreate a fantastic world where patronus are produced by Saccharomyces cerevisiae to fight against dementors inside the cell. Ethanol fermented by yeast from lignocellulosic materials can be an environmentally friendly fuel. However, rapid and efficient fermentation of lignocellulosic hydrolysates is limited because of inhibitors generated during pretreatment in addition to monomeric sugars. Inhibitors strongly affect the normal physiology of yeast as well as its ethanol productivity, just like the dementors taking away people’s hope and happiness. Nevertheless, we reconstruct TOR protein, a central component of major signaling transduction network controlling cell growth, to increase the tolerance of yeast. A new TOR after directional mutation will play the role of patronus to defend the influence of inhibitors, keep the overall signaling networks in good order, and finally provide a prosperous world for ethanol production.

This year we are aimed at increasing the tolerance of Saccharomyces cerevisiae to composite inhibitors in lignocellulosic hydrolysates, such as furans, acetate and phenol (“FAP” for short) formed during pretreatment and hydrolysis. Lignocellulosic materials such as wood provide abundant and renewable energy sources. Lignocellulosics contain sugars polymerised to cellulose and hemicelluloses which can be liberated by hydrolysing the material using industrial waste acid, and subsequently fermented to ethanol by microorganisms, such as yeast. Lignocellulose-derived ethanol can be used as an environmentally friendly liquid fuel. However, rapid and efficient fermentation of the hydrolysates is limited because a range of toxic compounds are generated during steam pretreatment and hydrolysis of lignocellulosics in addition to monomeric sugars.

The inhibiting compounds are divided in three main groups based on origin: weak acids, furan derivatives, and phenolic compounds. Weak acids, especially acetate, which is widely known as a kind of food preservatives, could inhibit cell growth. The growth-inhibiting effect on microorganisms has been proposed to be due to the inflow of undissociated acid into the cytosol. Undissociated weak acids are liposoluble and can diffuse across the plasma membrane. In the cytosol, dissociation of the acid occurs due to the neutral intracellular pH, thus decreasing the cytosolic pH. With the obvious decrease of intracellular PH, acetate could cause severe amino acid starvation, repress the normal center carbon metabolism and thus damage the physiology of whole cell. Acetic acid has been shown to induce apoptosis in yeast, and TOR pathways (Tor1p) are involved in the signaling of acetic acid-induced apoptosis. Furan derivatives have been shown to reduce the specific growth rate, the cell-mass yield on ATP, the volumetric and specific ethanol productivities. They could give rise to production of oxidative stress, and consumption of enegy (as ATP) and reducing power (as NADPH, NADH). For example, HMF has been shown to cause accumulation of lipids and decrease the protein content in yeast cells. And furfural reduction to furfuryl alcohol by NADH dependent dehydrogenases has a higher priority than reduction of dihydroxyacetone phosphate to glycerol, and furfural causes inactivation of cell replication. Phenolic compounds partition into biological membranes may cause loss of integrity, thereby affecting their ability to serve as selective barriers and enzyme matrices. They have been suggested to exert a considerable inhibitory effect in the fermentation of lignocellulosic hydrolysates, the low molecular weight phenolic compounds being most toxic. However, the mechanism of the inhibiting effect has not been elucidated, largely due to a lack of accurate qualitative and quantitative analyses. What’s more, the three groups of inhibitors have been shown to interact synergistically or antagonistically when they coexist, making the inhibition mechanism more complicated.

With the treatment of composite inhibitors, the yeast will suffer from the raise in protein degradation, and ROS (Reactive Oxygen Species), unfolded protein response as well as excessive consumption of ATP, which may lead to the stasis of ethanol production and cell growth, and even autophagy.

Clarification of the inhibiting mechanism is indispensible for the manipulation and domestication of inhibitor-resistant yeast. However, traditional methods that only focus on the characterization of a few genes or proteins are too limited for elucidating the composite mechanism. Therefore, there is plainly a tendency that system biology, as well as synthetic biology in which varieties of “Omics” (proteomics, metabolomics, genomics, transcriptomics etc,.) is integrated, would become an advanced high-throughput technology to shed light on the key mechanism. We should regard the yeast cell as an entity and a system, and look into the overall comnination of the metabolite network, rather than several isolated genes and pathways. Following this discipline, we finally settled our target at TOR pathway.

The core of our project is the reconstruction of TOR (target of rapamycin) pathway, which is the core of yeast signaling transduction network. TOR protein is a central node of signaling network, and is involved in massive physiological processes, such as cell growth and nutrient uptake, etc. One of the most significant clues that relate TOR to the resistance of inhibitors is transcriptional profiling. Different “omics” analysis results revealed that expressions of TOR-relevant genes, RNAs and proteins differ greatly between tolerant strains to parental ones. The following heat map reveals that transcriptions of the Atg proteins in tolerant strains discriminate dramatically from parental ones in the presence of inhibitors.

Atg proteins are the key players involved in autophagy. While TORC1 is a generally-known negative regulator of autophagy whose activity controls the phosphorylation status of Atg13. When TORC1 is active (inhibitors absent), Atg13 is hyperphosphorylated, whereas rapamycin addition induces a rapid dephosphorylation of Atg1. The latter apparently stimulates the affinity of Atg13 for Atg1 and promotes Atg1–Atg13 complex formation which is a requirement for autophagy. It is possible that PP2A (a downstream substrate of TORC1) is involved in TORC1-dependent regulation of Atg13 phosphorylation, since it was recently shown that autophagy is negatively regulated by the Tap42-PP2A pathway (a major way for TORC1 to regulate nutrients uptake and amino acids synthesis).

Another strong evidence which links TOR to the allergy to FAP is the comparison between growth curves of deletion strains and parental ones. In the following image, several deletion strains lacking the gene of some transcriptional factor, such as msn2/4, rtg1/2, gln3, gcn2, crf1 and so on.

As shown above, the growth rate of original strain differs greatly from deletion strains, especially with the presence of FAP. However, some of the deletion effects are negative, and others are positive, indicating various physiological functions regulated by different transcriptional factors.

Seeing that TORC1-relevant regulators play crucial roles in inhibitors treatment, it should be useful to relieve or even switch the allergic symptoms of yeast through reconstructions of TOR pathway, especially modifications on the signaling transduction node “TORC1”, and regulations on corresponding downstream pathways.

With the summed up assumptions above, The core of our project is the directional modification of TOR protein(The target of rapamycin), a highly conserved Ser/Thr protein kinase which is the central component of a major signaling transduction network that controls cell growth in diverse eukaryotic organisms, ranging from yeast to human beings. Massive physiological processes, such as amino acid biosynthesis, transcription and translation machinery, carbohydrate metabolism, nucleotide biosynthesis, stress response, protein turnover and cell cycle are directly or indirectly linked with TOR pathway.

In contrast to most eukaryotes, yeast contains two TOR homologues, Tor1 and Tor2. In Saccharomyces cerevisiae, TOR1 and TOR2 genes encode two large (*280 kDa) and homologous (67% identical) proteins that belong to a family of phosphatidylinositol kinase-related kinases (PIKKs). Despite their resemblance to lipid kinases, they are thought to function solely as Ser/Thr protein kinases. Based on the homologous proteins Tor1 and Tor2, two functionally and structurally distinct TOR multiprotein complexes exist: TOR complex 1 (TORC1) and TOR complex 2 (TORC2). Both Tor1 as well as Tor2 can be found in the multiprotein TORC1 together with Lst8, Kog1 and Tco89. A separate pool of Tor2 also associates with Lst8, Avo1, Avo2, Avo3, Bit61 and Bit2 to form TORC2. However, only TORC1 is specifically inhibited by rapamycin.

The addition of rapamycin induces dramatic phenotypic changes such as cell cycle arrest and entry into G0, general downregulation of protein synthesis, accumulation of the reserve carbohydrate glycogen and the stress protectant trehalose, upregulation of stress response genes, autophagy and alterations in nitrogen and carbon metabolism, which became the research foundation of TOR pathway. It appears that TORC1 signalling controls the temporal aspects of cell growth in response to the quality of the available nitrogen and carbon sources. On the other hand, TORC2, which is insensitive to rapamycin and is less well characterized in comparison to TORC1, is thought to regulate the spatial aspects of growth, such as the control of actin polarization. Here, we will focus on TORC1, as only this complex modulates nutrient-induced signaling in response to starvations mainly of nitrogen sources and carbon sources triggered by FAP presence.

The TOR protein consists of several distinct domains (illustrated in the image above). The N-terminal half of TOR comprises 20 tandemly HEAT repeats. HEAT repeats mediate protein-protein interactions and are required for localization of TOR to the plasma membrane. The HEAT repeats are accompanied by a so-called FAT domain that possibly serves as a scaffold or protein interaction domain. Following the FAT domain are the FKBP-rapamycin binding domain, the kinase catalytic domain, and a C-terminal FATC domain. Because TOR contains a number of domains that may mediate protein-protein interactions, TOR has been proposed to exist in a multiprotein complex and multi pathways. The most well-characterized TORC1 mediates the rapamycin-sensitive signaling branch that positively regulates anabolic processes such as translation and ribosome biogenesis and negatively regulates catabolic processes such as RNA degradation, autophagy, and other degradative pathways.

The precise function of these TOR interacting proteins is not known yet. They might play a role in the binding of the TOR complexes to their substrates, be the receivers of upstream signals and/or determine the localization of the complexes. The TOR complexes are likely dimeric built on a TOR–TOR dimer. Only TORC1 can bind FKBP12-rapamycin while in TORC2, the Tor2 FKBP12-rapamycin binding domain is probably not exposed for binding, explaining why only TORC1 signaling is sensitive to rapamycin treatment. Interestingly, the constitution of TORC1 appears to be unaffected by rapamycin, implying that rapamycin does not inhibit TORC1 signaling by interfering with TORC1 stability. Both TOR complexes are essential for viability, since deletion of TOR2 (inactivation of TORC2) or deletion of both TOR1 and TOR2, or rapamycin treatment (inactivation of TORC1) are lethal to yeast. Deletion of TOR1 alone, however, is not lethal, indicating that Tor1 and Tor2 have a redundant role in TORC1 signaling. Several studies investigated the localization of TORC1 and TORC2. Various different localization patterns were observed, which is possibly a reflection of the fact that TOR signaling controls a multitude of processes. TORC1 is mainly found at the vacuolar membrane, which is intriguing knowing that the vacuole is a reservoir of nutrients and that TORC1 signaling is believed to be regulated by nutrients. According to a recent study, TORC1 is also targeted to the nucleus where it induces 35S rRNA synthesis under favorable growth conditions.

In our project, increase the resistance of Saccharomyces cerevisiae to FAP equals removing the allergy of TORC1 to intracellular FAP. That is to say, to ignore the inhibiting signals from the extracellular environment to maintain normal physiological activity and keep a pretty high growth rate and ethanol productivity so that therefore it won’t trigger severe reactions like autophagy and apoptosis. Since the target has been identified on Tor protein, we should elucidate the corresponding mechanism.

Former transcriptional profiling data showed that inhibitors, especially acetate, would cause a series of phenotypic and intracellular changes induced by TORC1, including the entry of some transcription factors to the nucleus, some cell cycle arrest and entry into G0, general downregulation of protein synthesis, accumulation of the reserve carbohydrate glycogen, upregulation of stress response genes, autophagy and alterations in nitrogen and carbon metabolism. All the phenomena are originally controlled by an TOR initiated phosphorylation signaling network as the following image shows.

A major part of signaling transduction is regulated by the rapamycin-sensitive TORC1 complex either via the Tap42-Sit4/PPA2c or the recently identified Sch9 branches. In normal state (ample nutrients and no FAP), nutrients activate TORC1, resulting in the phosphorylation of Tap42-Sit4/PPA2 and Sch9, which would further transfer their phosphate groups to downstream transcription factors like Rtg1/3, Gln3, Maf1, Sfp1, Crf1, Msn2/4 and Rim15.

The precise mechanism how TORC1 regulates its downstream effectors are often not well-understood. As will be discussed in the section below, several processes appear to be regulated via the PP2A and the PP2A-related protein phosphatases. These phosphatases consist of heteromeric protein complexes. The PP2A holoenzyme contains one of the two redundant catalytic subunits (PP2Ac), Pph21, Pph22, a scaffolding subunit, Tpd3, and one of the two regulatory subunits, Cdc55 or Rts1. The PP2A-related phosphatase is mainly found as a complex between the catalytic subunit, Sit4, and one of the four regulatory subunits, Sap4, Sap155, Sap185 and Sap190. TORC1 controls the activity of these phosphatases via Tap42. When Tap42 is phosphorylated by TORC1, it will compete for binding the catalytic subunits of the phosphatases leading to the exclusion of other subunits of the phosphatase holoenzymes. Thereby, TORC1 stimulates the formation of a Tap42-associated phosphatase complex that further includes either one of the regulatory proteins Rrd1 or Rrd2, both of which are known to confer phosphotyrosyl phosphatase activity to the catalytic phosphatase subunits in vitro. Tap42 as well as Rrd1 and Rrd2 may redirect the substrate specificity of the catalytic phosphatase subunits, and as such, it is not surprising that the proteins have been attributed both inhibitory as well as activating roles, dependent on the substrate being studied. In actively growing cells, the Tap42-associated phosphatase complexes reside mainly at membranes where they associate with TORC1. Rapamycin treatment or nutrient starvation abrogates the TORC1 association and releases the Tap42-associated phosphatase complex into the cytosol. Once cytoplasmic, this complex then slowly dissociates, presumably concomitant with the dephosphorylation of Tap42. Several studies revealed an important role for yet another player in TORC1-dependent regulation of PP2Ac and Sit4, i.e. Tip41. This protein was initially identified as an inhibitor that could specifically interact with dephosphorylated Tap42. However, more recent data suggest that both Tip41 and Tap42 cooperate in determining the substrate specificity of PP2Ac and Sit4, and that both proteins may fulfill essentially a similar function in TORC1 signaling.

[1] I. Dilova, E. Easlon, S.-J. Lin. Calorie restriction and the nutrient sensing signaling pathways. Cell. Mol. Life Sci. 2007, 64: 752 - 767.
[2] H. Lempiäinen, A. Uotila, Jo. Urban et al. Sfp1 interaction with TORC1 and Mrs6 reveals feedback regulation on TOR signaling. Molecular Cell. 2009, 33: 704 - 716.
[3] Y.-X. Wang, N. L. Catlett, L. S. Weisman. Vac8p, a vacuolar protein with armadillo repeats, functions in both vacuole inheritance and protein targeting from the cytoplasm to vacuole. The Journal of Cell Biology. 1998, 140(5): 1064 - 1074.
[4] J. Urban,1 A. Soulard, A. Huber et al. Sch9 is a major target of TORC1 in Saccharomyces cerevisiae. Molecular Cell 2007, 26: 663 - 674.
[5] J. R. Rohde1, R. Bastidas, R. Puria et al. Nutritional control via TOR signaling in Saccharomyces cerevisiae. Current Opinion in Microbiology 2008, 11: 153 - 160.
[6] M. Wei, P. Fabrizio, F. Madia et al. Tor1/Sch9-regulated carbon source substitution is as effective as calorie restriction in life span extension. PLoS Genetics 2009, 5(5): 1 - 15.
[7] C. M. Alarcon, M. E. Cardenas, J. Heitman. Mammalian RAFT1 kinase domain provides rapamycin-sensitive TOR function in yeast. Genes Dev. 1996, 10: 279 - 288.
[8] I. Georis, J. J. Tate, A. Feller. Intranuclear function for protein phosphatase 2A: Pph21 and Pph22 are required for rapamycin-induced GATA factor binding to the DAL5 promoter in Yeast. Molecular and Cellular Biology, 2011, 31(1): 92 - 104.
[9] S. Wullschleger, R. Loewith, W. Oppliger et al. Molecular organization of target of Rapamycin Complex 2. The Journal of Biological Chemistry, 2005, 280(35): 30697 - 30704.
[10] E. Jacinto. What Controls TOR? Life, 2008, 60(8): 483 - 496.
[11] A. Adami, B. García-Álvarez, E. Arias-Palomo et al. Structure of TOR and its complex with KOG1. Molecular Cell 2007, 27: 509 - 516.
[12] L. Kuepfer, M. Peter, U. Sauer et al. Ensemble modeling for analysis of cell signaling dynamics. Nature Biotechnology 2007, 25(9): 1001 - 1006.
[13] B. Smets, R. Ghillebert, P. D. Snijder et al. Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae. Curr. Genet. 2010, 56: 1 - 32.
[14] M. A. Romanos, C. A. Scorer, J. J. Clare. Foreign gene expression in Yeast: a review. Yeast 1992, 8: 423 - 488.
[15] A. G. Hinnebusch, K. Natarajan. Gcn4p, a master regulator of gene expression, is controlled at multiple levels by diverse signals of starvation and stress. Eukaryotic Cell 2002, 1(1): 22 - 32.
[16] A. G. Hinnebusch. Translational regulation of Yeast GCN4. The Journal of Biological Chemistry 1997, 272(35): 21661 - 21664.
[17] B. Scherens, A. Feller, F. Vierendeels et al. Identification of direct and indirect targets of the Gln3 and Gat1activators by transcriptional profiling in response to nitrogen availability in the short and long term. FEMS Yeast Res 2006, 6: 777 - 791.
[18] J. L. Crespo, T. Powers, B. Fowler et al. The TOR-controlled transcription activators GLN3, RTG1, and RTG3 are regulated in response to intracellular levels of glutamine. PNAS 2002, 99(10): 6784 - 6789.
[19] S. M. Kingsman, D. Cousens, C. A. Stanway et al. High-efficiency Yeast expression vectors based on the promoter of the phosphoglycerate kinase gene. Methods in Enzymology 1990, 185(27): 329.
[20] M. Brunner, H. Bujard. Promoter recognition and promoter strength in the Escherichia coli system. The EMBO Journal 1987, 6(10): 3139 - 3144.
[21] T. Kodadek, D. Sikder, K. Nalley. Keeping transcriptional activators under control. Cell 2006, 127: 261 - 264.
[22] K. Natarajan, M. R. Meyer, B. M. Jackson et al. Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in Yeast. Molecular and Cellular Biology 2001, 21(13): 4347 - 4368.
[23] A. G. Hinnebusch. Translational regulation of GCN4 and the general amino acid control of Yeast. Annual Review of Microbiology 2005, 59: 407 - 450.
[24] E. Nevoigt, J. Kohnke, C. R. Fischer et al. Engineering of promoter replacement cassettes for fine-tuning of gene expression in Saccharomyces cerevisiae. Applied and Encironmental Microbiology 2006, 72(8): 5266 - 5273.
[25] K.EJ Tyo, K. Kocharin, J. Nielsen. Toward design-based engineering of industrial microbes. Current Opinion in Microbiology 2010, 13: 255–262.
[26] Y. Ohne, T. Takahara, R. Hatakeyama et al. Isolation of hyperactive mutants of mammalian target of rapamycin. The Journal of Biological Chemistry 2008, 283(46): 31861 - 31870.
[27] J. Urano, T. Sato, T. Matsuo et al. Point mutations in TOR confer Rheb-independent growth in fission yeast and nutrient-independent mammalian TOR signaling in mammalian cells. PNAS 2007, 104(9): 3514 - 3519.
[28] T. W. Sturgill, M. N. Hall. Activating mutations in TOR are in similar structures as oncogenic mutations in PI3KCα. ACS Chemical Biology 2009, 4(12): 999 - 1015.
[29] M. Hardt, N. Chantaravisoot, F. Tamanoi. Activating mutations of TOR (target of rapamycin). Genes to Cells 2011, 16: 141–151.
[30] E. Palmqvist, B. Hahn-Hägerdal. Fermentation of lignocellulosic hydrolysates. I: inhibition and detoxification. Bioresource Technology 2000, 74: 17 - 24.
[31] E. Palmqvist, B. Hahn-Hägerdal. Fermentation of lignocellulosic hydrolysates. II: inhibition and detoxification. Bioresource Technology 2000, 74: 25 - 33.
[32] S. I. Mussatto, I. C. Roberto. Alternatives for detoxification of diluted-acid lignocellulosic hydrolyzates for use in fermentative processes: a review. Bioresource Technology 2004, 93: 1 - 10.
[33] Z. L. Liu. Molecular mechanisms of yeast tolerance and in situ detoxification of lignocellulose hydrolysates. Appl. Microbiol. Biotechnol. 2011, 90: 809 - 825.
[34] A. Breitkreutz, H.Choi, J. R. Sharom et al. A global protein kinase and phosphatase interaction network in Yeast. Science 2010, 328: 1043 - 1046.
[35] Z. D. Sharp. Aging and TOR: interwoven in the fabric of life. Cell. Mol. Life Sci. 2011, 68: 587 - 597.
[36] D. Carmona-Gutierrez, T. Eisenberg, S Büttner et al. Apoptosis in yeast: triggers, pathways, subroutines. Cell Death and Differentiation 2010, 17: 763 - 773.
[37] P. Fabrizio, V. D. Longo. Chronological aging-induced apoptosis in yeast. Biochem. Biophys. Acta. 2008, 1783(7): 1280 - 1285.
[38] D. S. Evansa, P. Kapahic, W. - C. Hsueha et al. TOR signaling never gets old: aging, longevity and TORC1 activity. Ageing Research Reviews 2011, 10: 225 - 237.
[39] B. Almeida, S. Ohlmeier, A. J. Almeida et al. Yeast protein expression profile during acetic acid-induced apoptosis indicates causal involvement of the TOR pathway. Proteomics 2009, 9: 720 - 732.
[40] C. J. Bashor, N. C. Helman, S. Yan et al. Using engineered scaffold interactions to reshape MAP kinase pathway signaling dynamics. Science 2008, 319: 1539 - 1543.
[41] E. Nevoigt. Progress in metabolic engineering of Saccharomyces cerevisiae. Microbiology and Molecular Biology Reviews 2008, 72(3): 379 - 412.