Adaptive mutation of Acetobacter pasteurianus SKU1108 enhances acetic acid fermentation ability at high temperature

• We performed in vitro adaptation of A. pasteurianus at higher growth temperature.
• Thermally adapted strains were obtained with high acetic acid fermentation ability.
• Two strains adapted separately had common mutations in three specific genes.
• Two of the three genes were disrupted and confirmed to be involved in thermotolerance.
• The adapted strains may be acquired by assembling several single gene mutations.

In vitro adaptation is one of the most challenging subjects in biology to understand adaptive evolution. Microbial adaptation to temperature is not only interesting in terms of understanding the adaptation mechanism, but also useful for industrial applications. In this study, we attempted the in vitro adaptation of Acetobacter pasteurianus SKU1108 by repeating its cultivation under high-temperature acetic acid fermentation conditions. As a result, thermo-adapted strains having the higher fermentation ability than the wild-type strain were obtained. Mutations and/or disruptions in several proteins of the adapted strains were detected with NGS sequencing technology. In particular, two different adapted strains had mutations or disruptions in three specific genes in common, suggesting that these genes are essential for thermotolerance or fermentation at higher temperature. In order to clarify their involvement in thermotolerance, two of the three genes were disrupted and their phenotype was examined. The results showed that mutations of the two proteins, MarR and an amino acid transporter, are partly responsible for higher fermentation ability and/or thermotolerance. Thus, it was suggested that these elevated abilities of the adapted strains are acquired by assembling several single gene mutations including the above two mutations.