CRISPR-Mediated Metabolic Engineering of Escherichia coli W for Selective Biopurification of Stachyose from Soybean Molasses

Soybean molasses, a by-product of alcohol-based soy protein concentrate production, is rich in stachyose and other functional oligosaccharides, but its high sucrose content and other fermentable non-target sugars hinder the efficient purification of stachyose. In this study, the sugar-utilization patterns of four commonly used microbial chassis or production strains, Escherichia coli W, E. coli BL21, Saccharomyces pastorianus Weihenstephan 34/70, and Komagataella phaffii (formerly Pichia pastoris) GS115, were systematically compared to identify a suitable host for selective stachyose enrichment. Among them, E. coli W showed the best performance in rapidly consuming non-target sugars while retaining stachyose. Based on this strain, a CRISPR–Cas9 engineering strategy was applied by deleting the endogenous α-galactosidase gene melA and overexpressing the sucrose permease gene cscB. The resulting strain selectively and nearly completely removed sucrose and other non-target sugars from soybean molasses, increasing the proportion of stachyose from <30% to >90% of total soluble solids. Further optimization of nitrogen source level, inoculum size, and initial °Brix improved fermentation performance. These results demonstrate an effective biological pre-purification strategy for selective stachyose enrichment from soybean molasses.