Ancestral bacterial laccase enables efficient biodetoxification of inhibitory wheat straw hydrolysate for sustainable microbial protein production

Abstract

The valorization of lignocellulosic hydrolysates is frequently impeded by the presence of fermentation inhibitors, particularly phenolic compounds. In this study, we report the discovery of an efficient ancestral bacterial laccase for detoxifying a high-phenol dilute-acid hydrolysate (DAH7) derived from wheat straw. DAH7 contains 49 g L⁻¹ of sugars, predominantly xylose, as well as 1.302 g L⁻¹ of total phenolics, which severely inhibit the cell growth of xylose-utilizing yeasts Candida utilis ATCC 22023 and Cutaneotrichosporon dermatis ZZ-46 at 30% (v/v). Through the screening of nine ancestral bacterial laccases, Anc44 was identified as the most effective biocatalyst. Utilizing a whole-cell catalysis system, Anc44 degraded over 50% of four representative phenolic inhibitors (syringaldehyde, ferulic acid, p-coumaric acid, caffeic acid) under initial screening conditions. When applied to the DAH7, Anc44 completely removed ferulic acid and reduced the total phenolic content by 41%, thereby eliminating the need for enzyme purification and immobilization. Consequently, the detoxified DAH7 served as the sole carbon source for C. utilis to convert these sugars into single-cell protein with a cellular protein content of 40.28%, comparable to synthetic medium results. Experiments utilizing purified Anc44 further demonstrated its broad substrate specificity. Mechanistic investigation coupled with quantitative mass balance revealed a bifurcated radical pathway: phenoxy radicals derived from syringyl and guaiacyl units underwent oxidative ring cleavage (e.g., 3-methyl-2,5-furandione), whereas p-hydroxyphenyl units formed dimers via radical coupling. This work not only provides an efficient strategy for detoxifying lignocellulosic hydrolysates but also holds great potential for enhancing the economic viability of lignocellulosic bioconversion processes.