4 Designed Artificial transcription factors
With
the development of gene editing, high-throughput testing,
bioinformatics, computational biology, researchers are no longer limited
to engineer native TFs to
improve
strains tolerance and regulate the biosynthesis of interest products,
they have attempted to developed
powerful TFs in
reconstruct
strains metabolism for meet industrial requirements and as a tool to use
in genetic biosensor.
ATF
is one type of powerful TFs originated from native TFs and its important
domains, such as DNA-binding domain (DBD) and Effector domain (ED) are
rational designed to suit various applications [88]. The type of DBD
are from native zinc fingers (ZFs), transcription activator-like
effectors (TALEs), catalytically inactive form of Cas9 (dCas9) [89],
while ED are divided into activator (VP16, VP64, CRP and Gal4) and
repressor (SID, KRAB, EAR, SRDX and Ume6) [90, 91]which
can co-interact with
transcription-related components in the cell to achieve transcription
control [92].
The
combination of function domains
selection and integration in the ATF construction is imperative to
give the transcriptional
performance and develop specialized bio-function devices. So, ATFs have
certain potentials as
transcription
remodeling by enhancing transcriptional activity, creating orthogonal
systems and adding bistable switch, even has been used to predict genes
associated with tolerance by the specificity of the structure binding to
DNA. In addition, while the biosensor devices constructed by the
conventional TFs system still bring new practical tools for synthetic
biology, ATFs also supply the
deficiencies of them and expands their application scope.
4.1 ATFs as
transcriptionalreconstruction partconstruct industrial
chassis cells
Constructing
chassis cells that can meet production needs is a research direction in
synthetic biology. Due to a serious
of pathway of growth, metabolism, cell cycle, or endoplasmic reticulum
are regulated by TFs, they are commonly used as part for genetic
modification to reconstruction of
gene transcription network. However, the known TFs sometimes fail to
satisfy more elaborate and complex regulation, and even their original
effects will be diluted by the complex metabolic network within the
cell.
Several specific ATFs have been
implicated in the reconstruction part to avoid these problems and to
improve strains tolerance and production capacity. For example,
Cys2-His2 zinc finger domain is
common DBD, which generally exists in the bZIP family [93]. And, TFs
in this family are usually associated with tolerance. Researchers
selected 40 human-derived ZFs and fused them with the CRP domain
(interact with RNA polymerase) of E. coli to construct
combinatorial libraries of ATFs. After transformed them into E.
coli and screening found that the combination of Z13, Z2 and Z25
resulted in the upregulation of genes (ompW , cpxP ,yliH and ybaT ) linked with heat-resistant. Similarly,
genes negatively related to heat tolerance (marR , marA andmarB ) were significantly down-regulated
[94].
Further study also found the
combination of truncated CRP and three ZFs (Z11, Z4, and Z23) could
enable the strain to survive in the environment of 1.5% (vol/vol)
butanol, and this hybrid TFs were
expedient in improving heat and butanol tolerance of E. coli[95]. Hence, this strategy of ATF with
hybridized
different domains also can favorably enhance yeast tolerance by regulate
metabolic networks related genes.Jin-Soo Kim et al .
respectively chosed 25 and 40 ZFs to create a pool of multiple ATFs
containing random combinations of three or four ZFs
coupled
to either an activation or inhibition domain. It had been noted that
different combinations leaded to the different acquisition of tolerance
to heat shock (52℃) and ketoconazole (50 µM) and swapping two different
ED showed a reversed phenotype. Besides, PDR5 and YLL053Cwere confirmed to participate in conferring ketoconazole resistance in
yeast. Subsequently, the acetic acid-tolerant strains were also obtained
by this ATFs library [96, 97]. It was not difficult to noted that
ATFs play a positive role in enhancing the stress tolerance and
discovering new regulated genes.
4.2 Gene Expression System withATFs switch
Despite
the intensive use of native elements in switches design, there is still
limitation of the quantity and quality, including the rarely response
parts and ability of regulation, orthogonality or
plasticity.
Variable structure and wider range
utilization of ATFs, lead to a great interest in development of
expression switch with novel
properties.
The fine modulation of gene
expression by ATF-related switch
will be of benefit to promote the subsequently multilayered metabolic
pathways run accurately such as decoupled the two states of the growth
and production. Additionally, universal ATF switch breaks species
limitations for metabolic network reconstruction.
A
study revealed that two ATFs with opposite effect could establish for a
bistable
switch. The repressors consisted of
DNA
binding domain
(SrpR,
PhlF, TetR, Bm3R1, and LacI) and the SV40 nuclear localization signal
(NLS),
and the activators had the same NLS, threshold of the more types of DNA
binding domain (TarA, Orf2, LexA, and TetR) and additional C-terminal
VP16 activation domain. So, paired with the corresponding inducible
promoters (GAL1 and THI4 prompter), the bistable switch could thus be
well suited even for repressing or activating the expression of
regulated genes, and switched between two states. For instance, this
ATFs-based bistable switch was used to fine-tune the expression of VioC
and VioD which were the parts of heterologous violacein pathway, the
metabolism pathway successfully reconstructed and developed towards the
synthesis of proviolacein (green pigment), deoxyviolacein (red pigment)
and violacein (violet pigment) [98].
Besides, a period motif or the
whole of TFs from the Arabidopsis coupled to heterologous AD and
their cognate binding sites were constructed an orthorhombic gene
expression switch together in yeast. In this system, some ATFs with high
output combinations (e.g., NLS-GAL4AD-JUB1and NLS-GAL4AD-ATAF1)
which exceeded the strong constitutive yeast TDH3 promoter were
identified by detecting the strength of yEGFP, and a link between the
copy numbers of DNA-binding sites and plant-derived ATFs transcriptional
output were suggested [99]. Employing the mentioned above ATFs
library, researches also
created COMPASS which was a
high-throughput cloning method. And this artificial constructed
orthogonal transcription system balanced the multiple genes expression
and decoupled the two states of the
growth and production, achieving an increase in the target product yield
in Saccharomyces cerevisiae[100].
Moreover,
dCas9-based or TALEs-based ATFs were also used in
orthogonal
systems. TALEs is DBD derived fromXanthomonas spp and no
endonuclease activity dCas9 is a transcription activator be guided to a
20 bp DNA target by a small single RNA derived from Streptococcus
pyogenes.After
the two were respectively fused with AD (VP4 and VP64), they bound to
the matched synthetic promoter
(PSyn) to regulate
the genes expression and satisfied a wide range of inducible expression
strengths. ATF-related switch was indispensable for the design of
orthogonally regulated signaling and metabolic
network.
And,
among
various causes of ATF-related switch plasticity, the number and
direction of binding sites (BS) were considered as the main influencing
factors, which associated with the spectrum of transcriptional outputs
owing to the number of transcription initiation complexes recruited by
BS. Further, a steric hindrance effect caused by the oriented BSs and
spacers also affected the
interaction between AD and the basal transcription machinery or TFs. If
people want to fine-tune the ATFs regulation intensity, it can be
achieved via introducing 1-2 single nucleotide mismatches into the BSs
[101].
Although
the above expression system can meet orthogonality, the application is
still limited by creating matching promoter which increases the
difficulty of large-scale application to engineered strains. Therefore,
a flexible and highly adaptable ATFs system is needed to satisfy
industrial needs.
A
suggested
that the synthetic expression system (SES) constituted with core
promoters (CPs) which from S. cerevisiae, Aspergillus niger andTrichoderma reesei , ATFs (LexA-VP16 or Bm3R1-NLS-VP16) as well as
BSs achieved working in different host such as Saccharomyces
cerevisiae ; Yarrowia lipolytica ; Aspergillus niger ;Pichia pastoris; Trichoderma reesei and Pichia
kudriavzevii .
And, four strategies, involving
changing the number or type of CP, optimizing codons for ATFs act
respective to switching intensity [102]. Overall, ATFs have expanded
the switch type for modifying metabolic pathways.
4.3 ATFs-dependent device applicated inquantitatively evaluate
Upon
ATFs development drove by the design concept of synthetic biology,
modular synthetic devices were invented. In general, they are used to
evaluate metabolites of interest and optimized by adjusting the
adaptability and diversity of functional elements.
Sometimes,
TF-based
biosensors
have huge potential in high-throughput screening. For example, the
developed 3-hydroxypropionic acid (3-HP) biosensors consisted with
LysR-type transcriptional regulator
(LTTR), MmsR TF protein, PmmsA promoter and GFP reporter
gene were involved in the response of the concentration of 3-HP inPseudomonas denitrificans which covered a wide dynamic range
(0-100mM) [103]. To take
advantage of the TFs sensitivity and avoiding narrow detection ranges of
native TFs, ATFs
biosensors
system was introduced in the strain to monitor and respond metabolites
in vivo.
Studies
have shown that a orthogonal
ATF-based cAMP detection device to mammalian cells
consisted of cAMP receptor protein
CRP from E.coli and VP16 transactivation domain from Herpes
simplex virus. And, this tool was
constructed into two plasmids, the one contained ATF which combined with
intracellular cAMP and another as a reporter part contained a single CRP
specific operator site OCRP that could be bind to ATF and a chimeric
promoter. Compared with the traditional CRE reporter
device, the new detection device
avoided the interference of calcium signaling, MAPK signaling or Rho
signaling, and achieved accurate monitoring of cAMP signals in mammalian
cell populations [104].
Although traditional TF-based
biosensors are easy to application in screening small molecules such as
Itaconic acid, flavonoids, mevalonate [105], ATFs biosensors lack of
creation in Eukaryote such as yeast.
5
Conclusions
As
a regulator of the expression of genes, TF disturbed at the
transcriptional level acted like a domino effect, affecting the whole
metabolic network in yeast. With cascade multiple signaling pathways of
TFs in stress have been identified from research, conventional TFs
engineering can contribute to modified strains in combination with
theoretical basis, which may give strains new phenotypes and avoid the
limitations of some engineering methods. At the same time, he emergence
of ATFs originated from native TF has promoted the development of
conventional TFs engineering towards the diversification of functions.
ATFs can not only meet the intervention in enhancing transcriptional
activity and reconstructing metabolism, but also be designed as
standardized components to expand the number of synthetic biological
tools, such as ATFs switch or ATFs biosensors, which have been shown to
be more effective in controlling gene expression or quantitatively
evaluating interest products compared with native TFs. And, the
development of ATFs-based biosensors provides prospects for screening
valuable engineering strains in the future. TFs engineering harnesses
metabolic networks becoming increasingly active various directions in
yeast.
There are several challenges
underlying the application of TFs engineering. Transcriptomic,
proteomics, GC-MS, and QTL (Quantitative trait locus) analysis of yeast
response to stress revealed determine changes in metabolic fluxes and
multiple genes expression levels. These can find more meaningful key
TFs, and then improve strains tolerance by adjusting or combinatorial
regulating them. However, sometimes the effect is not very effective
[65, 106]. It due to the intricacies in the regulation of TFs as
well as determined by specific circumstances, it has become a difficulty
to objectively characterize the strain under the stress. Besides, the
functional redundancy of TFs negatively affects the study of gene
function. Truly showing the characteristics of binding to target genes
and might be necessary for future research [107].
Although there has been a robust
engineered strain by traditional stress-resistant system based on
understanding of the growth and metabolism mechanism, the chassis for
high productivity is still limited by less novel defense
means. So,
researchers
not
only need to pay attention to existing regulatory network, but also
devoted to taped
new
TFs or transform known TFs to build a ”unique” defense system, which
causes a high-quality chassis cell factory achieve efficient production
of target products [108, 109].
Even,
actively modifying strains phenotype would be energetically a less
costly and convenient if creating a universal ATFs which would not
restrict by species.
In
conclusion, TFs engineering harnessed
metabolic
networks has power and ability to avoid the limitations of methods
including laboratory evolution, random mutation, and many others and
achieves reconstruction metabolic networks under stress as advanced
genetic tools in yeast. With these continuous efforts, it will soon be a
mainstream strategy for improving strains tolerance and the biosynthesis
of industrial products in engineered
chassis.
Similarly,
ATF originated from native TFs improved capacity in metabolic networks
design promotes the research of TFs engineering advancement.