Development of an Haa1-based biosensor for acetic acid sensing in Saccharomyces cerevisiae

Abstract

Acetic acid is one of the main inhibitors of lignocellulosic hydrolysates and acetic acid tolerance is crucial for the development of robust cell factories for conversion of biomass. As a precursor of acetyl-CoA, it also plays an important role in central carbon metabolism. Thus, monitoring acetic acid levels is a crucial aspect when cultivating yeast. Transcription factor-based biosensors represent useful tools to follow metabolite concentrations. Here, we present the development of an acetic acid biosensor based on the Saccharomyces cerevisiae transcription factor Haa1 that upon binding to acetic acid relocates to the nucleus. In the biosensor, a synthetic transcription factor consisting of Haa1 and BM3R1 from Bacillus megaterium was used to control expression of a reporter gene under a promoter containing BM3R1 binding sites. The biosensor did not drive expression under a promoter containing Haa1 binding sites and responded to acetic acid over a linear range spanning from 10 to 60 mM. To validate its applicability, the biosensor was integrated into acetic acid producing strains. A direct correlation between biosensor output and acetic acid production was detected. The developed biosensor enables high-throughput screening of strains producing acetic acid and could also be used to investigate acetic acid tolerant strain libraries.

Real-Time Monitoring of the Yeast Intracellular State During Bioprocesses With a Toolbox of Biosensors

“…The design of the YAP1-based biosensor for oxidative stress has been improved and showed potential applications also in the probiotic yeast S. boulardii (Dacquay and McMillen, 2021). Acid stress resulting from acids in the substrates and/or products used in bioindustries can be sensed with HAA1-based or acid-responsive-promoterbased biosensors and used for screening new acetic-acidproducing strains (Hahne et al, 2021;Mormino et al, 2021). New condition-specific biosensors can be developed thanks to the use of yeast native promoters (Xiong et al, 2018).…”

Identification of acetic acid sensitive strains through biosensor-based screening of a Saccharomyces cerevisiae CRISPRi library

“…The biosensor readout is expression of the red fluorescent protein mCherry, under the control of a synthetic promoter that includes binding sites for the BM3R1 DNA-binding protein enclosed in the biosensor. In Mormino et al [ 22 ] we demonstrated that this biosensor was able to report both acetic acid added to the medium and acetic acid produced by the cells themselves.…”

“…The expression of the biosensor reporter can be translated to describe the concentration of the target molecule. Commonly, yeast biosensors exploit heterologous prokaryotic TFs [ 19 ], but endogenous eukaryotic TFs have also been successfully used for monitoring the NADPH/NADP + ratios [ 33 ] or sensing acetic acid [ 22 ]. Several parameters may describe the performance of a biosensor, including specificity, sensitivity, dynamic and operational range.…”

“…We recently described the design, characterization and application of a TF-based biosensor reporting acetic acid production in S. cerevisiae [ 22 ]. This biosensor is based on the endogenous zinc-finger TF Haa1 which has been reported to relocate from the cytoplasm to the nucleus upon direct binding of acetate [ 36 , 37 ].…”

Assessment of fluorescent protein candidates for multi-color flow cytometry analysis of Saccharomyces cerevisiae

“…For example, Skjoedt et al., showed the possibility of real-time monitoring of cis,cis -muconic acid (CCM) production in yeast by introducing a transcription factor-based biosensor in which the transcriptional activator BenM controlled GFP expression [2] . Similarly, the production of acetic acid could be monitored by the development of a biosensor based on the transcription factor Haa1 [3] . Transcription factor-based biosensors have also been implemented in S. cerevisiae for measuring different cellular properties.…”