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.