Abstract
BackgroundBesides being a major regulator of the response to acetic acid in Saccharomyces cerevisiae, the transcription factor Haa1 is an important determinant of the tolerance to this acid. The engineering of Haa1 either by overexpression or mutagenesis has therefore been considered to be a promising avenue towards the construction of more robust strains with improved acetic acid tolerance.ResultsBy applying the concept of global transcription machinery engineering to the regulon-specific transcription factor Haa1, a mutant allele containing two point mutations could be selected that resulted in a significantly higher acetic acid tolerance as compared to the wild-type allele. The level of improvement obtained was comparable to the level obtained by overexpression of HAA1, which was achieved by introduction of a second copy of the native HAA1 gene. Dissection of the contribution of the two point mutations to the phenotype showed that the major improvement was caused by an amino acid exchange at position 135 (serine to phenylalanine). In order to further study the mechanisms underlying the tolerance phenotype, Haa1 translocation and transcriptional activation of Haa1 target genes was compared between Haa1 mutant, overproduction and wild-type strains. While the rapid Haa1 translocation from the cytosol to the nucleus in response to acetic acid was not affected in the Haa1S135F mutant strain, the levels of transcriptional activation of four selected Haa1-target genes by acetic acid were significantly higher in cells of the mutant strain as compared to cells of the wild-type strain. Interestingly, the time-course of transcriptional activation in response to acetic acid was comparable for the mutant and wild-type strain whereas the maximum mRNA levels obtained correlate with each strain’s tolerance level.ConclusionOur data confirms that engineering of the regulon-specific transcription factor Haa1 allows the improvement of acetic acid tolerance in S. cerevisiae. It was also shown that the beneficial S135F mutation identified in the current work did not lead to an increase of HAA1 transcript level, suggesting that an altered protein structure of the Haa1S135F mutant protein led to an increased recruitment of the transcription machinery to Haa1 target genes.
Highlights
Besides being a major regulator of the response to acetic acid in Saccharomyces cerevisiae, the tran‐ scription factor Haa1 is an important determinant of the tolerance to this acid
Mutant alleles of the HAA1 coding sequence were created by error-prone Polymerase chain reaction (PCR), after which the PCR products were placed between the native HAA1 promoter and terminator in the backbone of the low-copy (CEN/ARS) pRS416 plasmid by recombination-based cloning in the strain CEN.PK113-13D haa1Δ
After four rounds of enrichment, all cultures showed a clear improvement in acetic acid tolerance, the improvement was more pronounced for the library cultures than for the control culture (Fig. 2a)
Summary
Besides being a major regulator of the response to acetic acid in Saccharomyces cerevisiae, the tran‐ scription factor Haa is an important determinant of the tolerance to this acid. The yeast Saccharomyces cerevisiae is a favorite microorganism in industrial biotechnology, mainly due to its robustness under real process conditions, its tolerance to low pH, and its impressive accessibility and versatility for metabolic engineering. Bioethanol is still the largest-scale product in industrial biotechnology, research and development programs on engineering S. cerevisiae for the production of other valuable compounds are underway, and production processes for a few biofuels, bulk and fine chemicals have already been commercialized. Examples of the latter are isobutanol, farnesene, succinic acid and resveratrol [1]. Most previous studies (including those mentioned in the paragraphs) have been conducted in comparably low acetic acid concentrations that allowed the majority of cells to resume proliferation after acetic acid exposure but still significantly influenced the duration of the latency phase
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
