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.

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