Heat stress response module
Heat stress can cause growth and metabolic disturbances during fermentation process, including protein misfolding, electron spillover from the electronic respiratory chain, mitochondrial dysfunction and DNA damage [11-13]. To avoid inhibitions of growth and shutdown of cell metabolism inside microbial cells from stressors,HSF1 , MSN2/4 encoding TFs are the most common regulators in heat stress reaction (HSR), which induced the expression of stress response proteins and molecular chaperones [14-17] (Fig. 1).
Hsf1p is a TF known to be inhibited due to binding with Hsp70 under natural state and it can be phosphorylated and then translocated into the nucleus during heat stress, where they can rapidly recruit transcription-friendly mediators (such as Med2p, Med3p, Med15p) and RNA polymerase II to facilitate the transcription of the heat shock protein (HSPs) genes. HSPs genes can maintain the correct structure of proteins and promote the repairing or degradation of damaged proteins [18]. Furthermore, phosphorylation is a booster to Hsf1p activity. Under high temperature, the higher the phosphorylation level of Hsf1p is, the better the recruitment of the mediation complex will be. When Hsf1 is combined with Hsp70, its phosphorylation can also promote the transcription level of downstream stress-related genes [19]. In addition, Hsf1p also confers certain degree of resistance to toxic ribosomes through participating in the ribosomal pathway. Two independent TFs, Hsf1p and Ifh1pare involve in the ribosome assembly stress response (RASTR). They are induced by excessive production of orphan r-proteins and then enter into the nucleus to regulate the expression of ribosome synthetic genes and ribosome assembly genes (Fig. 1). In combination with correct operation of ribosome assembly, RASTR has led the generation of cells resistance to irreversible damage caused by incorrect ribosome assembly [20, 21].
In recent years, advanced characterization techniques such as Chromatin immunoprecipitation and deep sequencing (ChIP-seq), mRNA deep sequencing (RNA-seq) and proteomics technology have confirmed the function of another TFs Msn2/4p in high temperature. Msn2/4p mainly regulates yeast stress response via Ras-PKA signaling pathway. Under normal temperature, Ras protein is active, which results high cAMP level and promotes the interaction of cAMP and protein kinase A (PKA) to phosphorylate Msn2/4p, thus Msn2/4p activity are inhibition. Upon heat stress, this inhibition is removed, and, as a result, Msn2/4p can enter the nucleus and bind to the stress response elements (STREs) containing a CCCCT or AGGGG base sequences to activate the transcription of stress response genes [22-24]. Besides, Microarray-assisted, Sequential Window Acquisition of all Theoretical fragment ion spectra (SWATH) and Data independent acquisition (DIA) analyses combined with STRING or SGD database analysis have revealed multiple Msn2/4p-regulated genes and the interacting relationships between different TFs, such as Gcn4p directly interacts with Gal11p, Bas1p, Pho2p, which as a RNA pol II coactivator or several biosynthesis pathways regulator [25-27]. In this high temperature response module, the functions of TFs such as signal cascade transmission, phosphorylation and intracellular substance interactions help us understand the mechanism of high temperature at the transcription level and protein folding in yeast.
2.2Acetic acid stress response module
During the bioethanol producing, lignocellulose must go through pretreatment to facilitate downstream reactions [28, 29]. However, material hydrolysate products, such asacetic acid, furfural and furan undermines strains normal growth and fermentation during pretreatment of biomass [30, 31]. To cope with the effect, the major TF-mediated toxic molecular response systems include transporters, the adaptation of cell membrane components, Hog1p-depend reactions and retrograde regulation contribute to recovery from stress influence (Fig. 2).
Acetic acid is the most abundant byproduct of biomass pretreatment, and when it goes into yeast cell dissociating into two different states of ions, the intracellular pH drops and the ATP synthesis is disrupted [32]. There are numbers transporters specifically transporting anions and cations on cell membrane to decrease the acetic acid effects in yeast.FPS1 coded aquaglyceroporin channel is generally required for permeating acetate molecules into the cell. In response to acetic acid, active osmoregulation TF Hog1p is phosphorylated and then activates the regulatory factor Rgc2p, which in turn divorces Fps1p and closes aquaglyceroporin channel to prevent acetic acid permeating the yeast cell and to decrease its toxic effects [33, 34]. When the acidity becomes more serious, Hog1p directly interacts with Fps1p and thus causes Fps1p phosphorylated and then degraded by endocytosis [35]. In addition to Fps1p, proton channel protein Pma1p and anion ABC transporters (Pdr10p, Pdr12p and Pdr18p) also assist in resistance to acetic acid [36-38]. Recently, the TFs-involved retrograde regulation has been discovered as a system to against acetic acid destruction. It is formed by complex comprising subunits (Rtg2p, Rtg1p, Rtg3p and Mks1p) and occurs to alleviate the metabolic disorders caused by the disturbance of mitochondria [39]. Rtg3p-Rtg1p complex activity is induced by the activity of Mks1p-Rtg2p complex, which serves as an activator and be repressed by the Mks1-Bmh1/2p complex. Active Rtg3p-Rtg1p complex smoothly enters the nucleus to stimulate the expression of acetic acid stress-responsive genes, including IDH1/2 (NAD+-dependent isocitrate dehydrogenase), DLD3 (D-lactate dehydrogenase) and PYC1 (pyruvate carboxylase). So, retrograde regulation system confers the ability to harness metabolic networks plastic to generate enough glutamic acid for synthesizing other metabolites when mitochondrial activity was impaired [40-46]. Another research reveals that Hog1p as a TF can be induced by overexpressing OLE1 and then sequentially phosphorylate, entering the nucleus to control the expression of a stretch of regulons (CTT1 ,HSP12 , and STL1 ) to reconstruct metabolic networks and enhance acetic acid tolerance in yeast [47, 48]. In addition, OLE1 encoding the sole Δ-9 desaturase catalyzes saturated fatty acids (SFA) into unsaturated fatty acid (UFA), which is a process known to change of the membrane fluidity in yeast [49]. The cell membranes status such as fluidity and lipid composition can change in order to cope with acetic acid stress. So, the acetic acid module presents the key roles of different types of stress responsive TFs in pathway, including ion channel regulation, retrograde regulation and cell membrane state changes.
2.3 Oxidative stress response module
Oxidative Stress is a type of damage in which the original balance of intracellular pro-oxidant/antioxidant is disturbed and metabolic disorders. Sometimes, it due to the accumulation of reactive oxygen species (ROS) which mainly comes from the peroxisomes, endoplasmic reticulum (ER) or mitochondria [50, 51], or it causes by electrophilic compounds, such as cobalt, mercury, furfural and ferulic acid [52-54]. Innately, overcoming oxidative stress depend on different TFs-related signaling which reconstruct metabolic networks in yeast [55]. In addition, some enzymatic or nonenzymatic molecules defense system such as thioredoxin and glutathione also play an important role in ROS detoxification (Fig. 3).
In most ROS-induced regulatory pathways, Yap1p is a major TF that controls the key metabolic networks of oxidative stress resistant systems, including glutathione system, multidrug resistance MDR, glycolysis pathway and cellular detoxification [56]. Upon exposure to inhibitory concentration of ROS, it connecting with scaffold protein Ybp1p forms intramolecular-disulfide bonds between N-terminal and C-terminal the nuclear export signal (NES) is hided, thus inhibit the expression of antioxidant genes. In addition, some electrophilic compounds can also direct adduct to C-terminal Cys residue of Yap1, which is activated in a manner independent of Gpx3 [57, 58]. For instance, Hyr1p encoding thiol peroxidase catalytic catalyzes the formation of intramolecular disulfide bonds in Yap1p, which inhibits Yap1p nuclear outputting and accumulating in the nucleus. And, to cope with oxidized proteins by ROS,Ire1-Hac1pathway of unfolded protein response (UPR) maintains endoplasmic reticulum homeostasis and correct protein folding in yeast. Ire1p is an endonuclease located on endoplasmic reticulum. When senses the unfolded protein, it activates Hac1p by splicing introns. Hac1p as a TF regulating the UPR to cope with ROS, and it transcriptionally induces a number of genes regulating growth and mitochondria homeostasis of yeast [59, 60]. Besides, the thioredoxin and glutaredoxin systems involve in maintain redox state in strains. They utilize proteins with reductive amino acid residues to reduce oxidizing molecules. At the same time, various reductases use NADPH as an electron donor to transform into an oxidized state to achieve recycling. The mainly three oxidative responsive TFs (Yap1p, Ire1p and Hac1p) forms part of the classical oxidative stress response module and occupies an important position in maintaining redox state and reconstruction metabolic pathways in yeast. And some reductive molecules as another branch of the oxidation response system help to eliminate oxidizing molecules to maintain strains homeostasis.
3 Conventional TFs engineering implicated in meetingindustrial requirements
A clear correlation between the strains industrial value and TFs engineering reveals that proper reconstruction of gene transcription network can change organelle structure and pathway flux into the direction of enhancing strains tolerance and improving productivity, which has shown to be more important during strain modification. In combination with genetic modification or increasing the diversity of genetic information by heterologous expression and global transcription machinery engineering (gTME), conventional TFs engineering will markedly enhance the generation of cells resistance to various stress and arise with the desired phenotype.
3.1G enetic modification of native TFs
With many omics and genome sequencing datasets providing valuable understanding of tolerance traits, environmental stress responsive native TFs functioning as activators or inhibitors have been repeatedly overexpressed or knocked-out in harnessing metabolic networks in yeast[61, 62]. For example, HAA1 is a TF which not only directly controls stress-induced genes expression such as sphingolipid and ceramide metabolism (YPC1 ), carbohydrate metabolism (TOS3 ), and cell wall related secretory glycoprotein (YGP1 ), but also indirectly regulates Msn4p, Nrg1p, Fkh2p, Stp4p, Com2p and other TFs in response to acid stress [63]. The above regulated genes have played important roles in prevent stress-induced cellular dysfunctions. After overexpressing HAA 1 treatment, modified xylose strains tolerated an initial acidity level of 50mM acetic acid, 50 g/L xylose environment and increased the ethanol yield from 0.26 g/g to 0.32 g/g. In addition, engineered strains could also grow in non-detoxified hardwood hydrolysate which contain glucose, xylose, acetic acid, furfural and other inhibitors, and the utilization rate of glucose and xylose had been improved in yeast [64, 65]. Similarly, overexpressing Whi2p interacted with Prs1p leaded multiple genes upregulated in vivo, such as Catalase (CTA1 ), glutathione antioxidant system (GSH1 ), reducing furan aldehyde enzymes (ADH7 ), global TFs (Msn2/4 ) [66]. Further, asides increasing strains tolerance, TFs engineering can promote the synthesis of natural products such as squalene, glycyrrhizic acid, and glycyrrhetinic acid and changes the xylose conversion rate. For instance, overexpressingINO2 had improved squalene production in engineered yeast. Ino2p and Ino4p are TFs that regulate lipid synthesis, while Opi1p acts as a negative regulator to inhibit the activity of Ino2p and Ino4p. By overexpressing INO2 , the area of the endoplasmic reticulum had been expanded to provide a high-quality microenvironment for the enzymes (ERG9 , HMG1and PPDS ) in the MVA pathway, resulting in increasing glycolysis flux and acetyl-CoA content and enhancing protein synthesis capacity squalene production(634 mg/L) [67].
Besides overexpressing is used to conversion of sensitive phenotype to valuable microorganisms, yeast has the potential to convert lignocellulose into ethanol only by introducing an effective xylose metabolizing pathway[68]. Heterologous xylose system can effect native metabolic networks, and the transcription level of some important genes were down-regulation, such as in carbon metabolism (FBA1, GPM1 and TDH2), DNA damage stimulus respond and repair (HTA2), structural constituent of ribosome (RPL22A, RPL22B and RPL7A) during the GX stage (glucose-xylose co-fermentation). And, cell cycle-related or vitamin metabolic-related TFs Nrm1p, Yhp1p, and Thi2p had also slightly down-regulation. Thi2p is a thiamine biosynthetic TF that interacts with Thi3p and Pdc2p and regulates the transcription of a number of target genes. Knocking out THI2(encoding Thi2p) resulted in the upregulation of genes involved in ribosome and transcription initiation factor complex subunits synthesis and energy metabolism, which had positive effects on the utilization of xylose in the GX stage (increase 67.7%). Even though, an efficient xylose utilization in the THI2 knocked-out yeast strain is limited by carbon source conditions, in which glucose responding system (Snf3p/Rgt2p, Snf1p/Mig1p and cAMP/PKA) still strongly controlled the initiation of signaling processes in fermentative metabolism. Therefore, knocking out ofTHI2 didn’t increase the utilization of xylose in the X stage(xylose fermentation)) [69, 70].
Another study investigating ethanol production displays critical role played by TFs of chromatin remodeling (such as Swi3p ) in knock-out strains [71]. SWI/SNF (BAF) Chromatin Remodeling Complex utilizes the energy released by ATP hydrolysis to drive the movement of nucleosomes, which facilitates chromatin structure remodeling and promotes genes expression of ethanol dehydrogenase (ADH1/2 ) and galactokinase (GAL1 andSUC2 ). So, reducing ADH2 expression might be one of the major contributions to ethanol production by knocking out SWI3 which encodes a subunit of SWI/SNF chromatin remodeling complex [72-76].
3. 2 Heterologous expression of native TFs
Introducing heterogeneous stress-related TFs genes realizes the expression of dominant genes from different sources. It breaks through the genetic information communication barrier between different species to meet the purpose of acquiring new genetic material quickly. TFs engineering using heterologous expression had also been proven to effectively modify the phenotypes in plants (Table 1), while this method had been reported in yeast. Transforming the Kluyveromyces marxianus TF Msn2p or Hsf1p inSaccharomyces cerevisiae favorably led the bioethanol production up to 27.6 g/L and 27.2 g/L respectively in the presence of 104.8 g/L glucose, 43°C, and the best one made a 46% higher than control. One reason was due to DNA binding domain was different from primitive structure [77, 78]. And, transcriptomic analysis revealed that the reconstruction of lipid metabolism, glucose transport and glycolysis or gluconeogenesis process display critical role played by TFs of heterologous expression in enhancing ethanol tolerance and ethanol production. So, TFs engineering can harness metabolic networks to obtain industrial strains and apply for different organisms.