Abstract
In this study, a thermophilic cellulolytic filamentous fungus Thermothelomyces heterothallica was engineered for direct production of cellobionic acid (CBA) from alkali-pretreated wheat straw without the addition of external cellulases. The CBA bioproduction pathway was established through sequential disruption of genes involved in cellobiose and CBA catabolism, including eight β-glucosidases, one cellobiose/cellobionate phosphorylase, and two putative cellobionate transporters using a ribonucleoprotein (RNP)-based CRISPR-Cas9 system. The resulting T. heterothallica strain, TH11, was able to produce cellobionate from cellobiose and pretreated wheat straw at a high yield. Further optimization of fermentation pH using 50 mM citrate buffer (pH 6.0) increased the final cellobionate concentration. Fermentation temperature optimization showed that 43 °C accelerated substrate conversion without significantly affecting final titers. The engineered strain, TH11 produces 64 mM of cellobionate in 5 days. The cellulose conversion was about 88%, and the yield of the cellobionate from the consumed cellulose was about 96%. The new strain provides a promising platform for direct bioconversion of lignocellulosic biomass into value-added aldonic acids without any enzyme addition.