3.3 Modified TFs
gTME based on changing genetic information by error-prone PCR offers a selective advantage to building tolerant mutants.  As a proof-of-strategy, researchers created a mutant library of the TF SPT15 encoding TATA-binding protein, followed by a screening to identify mutants showing tolerance improvement. Using this strategy, the authors were able to obtained a mutant (Phe177Ser, Tyr195His, Lys218Arg) that recovered growing normally under 6% ethanol and 120 g/L glucose and improved fermentation capacity. These results illustrated that change in Tyr195His amino acid and the synergistic interaction of the other two amino acids at the mutant site for the impact of inhibited effect of NC2 negative regulators on SPT15 and the interaction with the subunit SPT3-SAGA of the transcriptional regulatory complex, which leading to the genes transcription level and metabolic networks reconstructed [9]. ThePingkai Ouyang team also used mutate TF SPT15 adjust the pathways of glycolysis, respiration, gluconeogenesis and PPP in GX stage [79], and obtained high efficiency strains in the mixed medium of xylose and glucose. The utilization rates of xylose and glucose were up to 90.8% and 97.3%, respectively [80]. Furthermore, Transcription initiation complexes included TFs were operated by gTME. For instance, transcription factor D (TFIID) and Spt-Ada-Gcn5p-Acetyltransferase (SAGA) which jointly possess TATA-box binding protein (TBP) associated factors encoded bySPT3 and SPT8 are involved in the gene transcription process [81-84]. Spt3p and Spt8p as TFs can interact with TBP, and thereby involve nucleosome subunits deacetylated which is necessary for the initialization of transcription process. Although the SAGA complex binds to TBP via Spt3p, it is easier for Spt8p to bind to TFIIA and TBP than Spt3p. Mutated Spt8p changed the force, distance and quantity of hydrogen bonds that maintained the protein structure, leading to simultaneously alter its activity and charge transfer system. Finally, the ethanol tolerance and ethanol yield of the strains were increased [85]. Further, researchers used Random Insertional-deletional Strand Exchange (RAISE) technology to build Spt3p mutagenesis library and screened strain which could survive under the environment of 1.8 g/L lead. The degree of change in gene expression and the relationship between the tolerance phenotypes and gene genotypes analyses had identified that altered trehalose accumulation by mutated TF have contributed to enhanced yeast tolerance [86]. Interestingly, researchers mutated methylotrophic yeast (O.thermomethanolica ) TFHAC1by CRISPR-Cas9 technology to improve Endoplasmic reticulum (ER) resistant and desired product yield. Although strains had not been associated with any noticeable phenotypes in the resistant to ER stress, the mutation efficiency was 63%. It may be recommended that CRISPR-Cas9 has better mutation efficiency than gTME and proposed other option for mutant TF for future study [87].