A novel rhodamine-based fluorescent pH probe for high-throughput screening of high-yield polymalic acid strains from random mutant libraries

Abstract

Polymalic acid (PMA) is produced from the yeast-like fungus Auerobasidium pullulans, and is a water-soluble biopolymer with many useful properties for pharmaceutical applications. Its monomer, L-malic acid, is a potential C4 platform chemical and organic acid for food industry. Owing to the A. pullulans complex genetic background, random mutagenesis is still an effective tool for enhancing the production of PMA; however, ineffective screening methods for random mutant libraries still hamper this process. In this study, a high-throughput screening procedure was developed to solve this problem. A novel rhodamine-based fluorescent pH probe R was synthesized and characterized, and it showed a good quantitative relationship between fluorescent intensity and the acidic pH value (ranging from ∼2 to ∼5) of malic acid, the sole monomer of PMA, applied to evaluate the titer of PMA in fermentation broth. Based on this screening strategy, the mutant AH-21 was successfully selected from the atmospheric and room temperature plasma (ARTP) mutant libraries containing ∼1000 mutants. The titer of PMA was enhanced by 13.8% compared with that of wild type in a 3 L fermentor. Fed-batch fermentation in a 30 L fermentor achieved a high final PMA titer of 128.2 g L⁻¹ (145.4 g L⁻¹ of malic acid after hydrolysis) with the highest yield of 0.51 g g⁻¹ from glucose. The high-throughput screening process established with this study represents a rapid and accurate approach to enhance the production of other similar organic acids from random mutant libraries.