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.

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