Biosynthesis of a rosavin natural product in Escherichia coli by glycosyltransferase rational design and artificial pathway construction

• Published in: Metabolic engineering Vol. 69; pp. 15 - 25
• Main Authors: Bi, Huiping, Qu, Ge, Wang, Shuai, Zhuang, Yibin, Sun, Zhoutong, Liu, Tao, Ma, Yanhe
• Format: Journal Article
• Language: English
• Published: Belgium Elsevier Inc 01.01.2022
• Subjects: Biological Products - metabolism; Disaccharides - chemistry; Disaccharides - metabolism; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Glycosyltransferase; Glycosyltransferases - genetics; Glycosyltransferases - metabolism; Rational design; Rosavin natural product; UDP-xylose; Rational design; Rosavin natural product; Glycosyltransferase; UDP-xylose; Escherichia coli
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2021.10.010

Phytochemicals are rich resources for pharmaceutical and nutraceutical agents. A key challenge of accessing these precious compounds can present significant bottlenecks for development. The cinnamyl alcohol disaccharides also known as rosavins are the major bioactive ingredients of the notable medicinal plant Rhodiola rosea L. Cinnamyl-(6′-O-β-xylopyranosyl)-O-β-glucopyranoside (rosavin E) is a natural rosavin analogue with the arabinopyranose unit being replaced by its diastereomer xylose, which was only isolated in minute quantity from R. rosea. Herein, we described the de novo production of rosavin E in Escherichia coli. The 1,6-glucosyltransferase CaUGT3 was engineered into a xylosyltransferase converting cinnamyl alcohol monoglucoside (rosin) into rosavin E by replacing the residue T145 with valine. The enzyme activity was further elevated 2.9 times by adding the mutation N375Q. The synthesis of rosavin E from glucose was achieved with a titer of 92.9 mg/L by combining the variant CaUGT3T145V/N375Q, the UDP-xylose synthase from Sinorhizobium meliloti 1021 (SmUXS) and enzymes for rosin biosynthesis into a phenylalanine overproducing E. coli strain. The production of rosavin E was further elevated by co-overexpressing UDP-xylose synthase from Arabidopsis thaliana (AtUXS3) and SmUXS, and the titer in a 5 L bioreactor with fed-batch fermentation reached 782.0 mg/L. This work represents an excellent example of producing a natural product with a disaccharide chain by glycosyltransferase engineering and artificial pathway construction. •The 1,6-glucosyltransferase CaUGT3 was altered into a xylosyltransferase by switching T145 into valine.•The activity of CaUGT3T145V was further elevated by engineering N375 into glutamine.•Escherichia coli was engineered to produce a rosavin natural product by recruiting CaUGT3T145V/N375Q.•782.0 mg/L of the rosavin natural product was produced in a bioreactor.