Evolved alkaline fungal laccase secreted by Saccharomyces cerevisiae as useful tool for the synthesis of C–N heteropolymeric dye

Enzymatic production of C–N heteropolymeric dyes at alkaline pHs is an attractive process for the textile industry. In this work, we have designed a fungal laccase by directed evolution so that it may be used at alkaline pHs for the synthesis of C–N heteropolymeric dyes (C–N polydye) from catechol and 2,5-diaminobenzenesulfonic acid (2,5-DABSA). Firstly, several medium- and high-redox potential fungal laccases from previous laboratory evolution campaigns were benchmarked for the synthesis of the C–N polydye at pH 8.0, choosing an alkaline laccase mutant from Myceliophthora thermophila as the departure point for further engineering. Mutant libraries were then constructed, expressed in Saccharomyces cerevisiae and screened using a high-throughput colorimetric assay for the detection of the C–N polydye. By combining directed and focused molecular evolution, a novel, strongly expressed alkaline laccase variant was identified. This laccase was secreted at 37 mg/L and its catalytic efficiency for the oxidation of catechol and 2,5-DABSA at pH 8.0 was enhanced 3.5-fold relative to that of the wild-type, promoting the synthesis of the C–N polydye at basic pHs. While the improved expression was mostly the result of accumulating mutations that favor the yeast’s codon usage together with the recovery of a secretion mutation, the enhanced C–N polydye synthetic activity of the mutant laccase was dependent on the alkaline mutations it inherited. Readily secreted, this laccase mutant would appear to be a valuable platform for organic synthesis at basic pHs.