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.