Fuente: Microorganisms - Revista científica (MDPI) Microorganisms, Vol. 14, Pages 1029: CRISPR-Mediated Metabolic Engineering of Escherichia coli W for Selective Biopurification of Stachyose from Soybean Molasses
Microorganisms doi: 10.3390/microorganisms14051029
Authors:
Haotian Wang
Guoyu Liu
Jia Liu
Yifei Zhu
Jingmei Huang
Shiwei Liu
Huaping Pan
Yafang Li
Yan Zou
Xueying Zeng
Guankai Hao
Haizhi Li
Shufan Yang
Shenglin Duan
Juxiu Li
Peng Yuan

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.