Identification of a thermotolerant strain of Aureobasidium melanogenum DA22 and functional analysis of Spt23 as a key transcriptional regulator in high-temperature polymalic acid fermentation

Abstract

Industrial application of Aureobasidium pullulans in organic acid production is limited by mesophilic characteristics under thermal stress. Physiological screening of 120 wild-type isolates identified a naturally robust strain, Aureobasidium melanogenum DA22, which maintained near-optimal growth and preserved 93.8% of its maximum polymalic acid titer (24.55 g/L, calculated as l-malic acid) at 35°C. To confer thermotolerance to a high-yield chassis, candidate thermoprotective genes-including heat shock proteins, antioxidant enzymes, and transcription factor-were identified from strain DA22. Overexpression of the membrane-associated transcription factor gene SPT23 increased cell growth by 20.0% and enhanced malic acid production by 37.7% (reaching 39.86 g/L) at 35°C in shake flasks. Scale-up batch fermentations in 5-L bioreactors achieved 56.08 g/L of l-malic acid at 35°C. In contrast, SPT23 deletion caused morphological defects and metabolic repression. RT-qPCR analysis and physiological indicators also indicated that SPT23 overexpression coordinately upregulated genes involved in lipid and ergosterol biosynthesis to maintain membrane integrity under thermal stress and elevated key enzymes in the reductive tricarboxylic cycle to redirect carbon flux toward polymalic acid synthesis. Engineering with Spt23 presents an effective synthetic biology strategy to construct a thermotolerant microbial cell factory suitable for organic acid production under industrial thermal-stress conditions.

Keywords: Aureobasidium; Spt23; l-malic acid; membrane-associated transcription factor; polymalic acid; thermotolerance.