Optimization of scleroglucan production by Sclerotium rolfsii by lowering pH during fermentation via oxalate metabolic pathway manipulation using CRISPR/Cas9

摘要

Sclerotium rolfsii is a potent producer of many secondary metabolites, one of which like scleroglucan is an exopolysaccharide (EPS) appreciated as a multipurpose compound applicable in many industrial fields. Aspartate transaminase (AAT1) catalyzes the interconversion of aspartate and α-ketoglutarate to glutamate and oxaloacetate. We selected AAT1 in the oxalate metabolic pathway as a target of CRISPR/Cas9. Disruption of AAT1 leads to the accumulation of oxalate, rather than its conversion to α-ketoglutarate (AKG). Therefore, AAT1-mutant serves to lower the pH (pH 3–4) so as to increase the production of the pH-sensitive metabolite scleroglucan to 21.03 g L−1 with a productivity of up to 0.25 g L−1·h−1. We established a platform for gene editing that could rapidly generate and select mutants to provide a new beneficial strain of S. rolfsii as a scleroglucan hyper-producer, which is expected to reduce the cost of controlling the optimum pH condition in the fermentation industry.