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].