New Papiliotrema laurentii UFV-1 strains with improved acetic acid tolerance selected by adaptive laboratory evolution

The production of yeast oil from lignocellulosic biomasses is impaired by inhibitors formed during the pre-treatment step, mainly acetic acid. Herein, we applied Adaptive Laboratory Evolution (ALE) to select three Acetic acid Tolerant Strains (ATS) of P. laurentii UFV-1. Different phenotypes emerged alongside evolution. The ATS II presented trade-offs in the absence of acetic acid, suggesting that it displays a specialized phenotype of tolerance to growth on organic acids. On the other hand, ATS I and ATS III presented phenotypes associated with the behavior of generalists. ATS I was considered the most promising evolved strain as it displayed the oleaginous phenotype in all conditions tested. Thus, we applied whole-genome sequencing to detect the mutations that emerged in this strain during the ALE. We found alterations in genes encoding proteins involved in different cellular functions, including multidrug resistance (MDR) transporters, energy metabolism, detoxification, co-enzyme recycling, and cell envelope remodeling. To evaluate acetic acid stress responses, both parental and ATS I strains were cultivated in chemostat mode in the absence and presence of acetic acid. In contrast to ATS I, the parental strain presented alterations in the cell envelope and cell size under acetic acid stress conditions. Furthermore, the parental strain and the ATS I presented differences regarding acetic acid assimilation. Contrary to the parental strain, the ATS I displayed an increase in unsaturated fatty acid content irrespective of acetic acid stress, which might be related to improved tolerance to acetic acid. Altogether, these results provided insights into the mechanisms involved with the acetic acid tolerance displayed by ATS I and the responses of P. laurentii to this stressful condition.

Automated summary

In this study, three acetic acid tolerant strains (ATS) of P. laurentii UFV-1 were selected using Adaptive Laboratory Evolution (ALE). Phenotypic analysis and whole-genome sequencing revealed that ATS I strain showed the most promising evolved characteristics, displaying oleaginous phenotype in all conditions. The study also discovered changes in cell envelope and cell size of ATS I strain under acetic acid stress conditions. These findings provided insights into the acetic acid tolerance mechanism and stress response of P. laurentii.