Details

Autor(en) / Beteiligte

• Zhu, Yingying
• Chen, Roulin
• Wang, Hao
• Chen, Yihan
• Huang, Zhaolin
• Du, Zhihui
• Meng, Jiawei
• Zhou, Jingwen
• Mu, Wanmeng

Titel

De Novo Biosynthesis of Difucosyllactose by Artificial Pathway Construction and α1,3/4-Fucosyltransferase Rational Design in Escherichia coli

Ist Teil von

• Journal of agricultural and food chemistry, 2024-04, Vol.72 (16), p.9247-9258

Ort / Verlag

United States: American Chemical Society

Erscheinungsjahr

2024

Link zum Volltext

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• Difucosyllactose (DFL) is a significant and plentiful oligosaccharide found in human breast milk. In this study, an artificial metabolic pathway of DFL was designed, focusing on the de novo biosynthesis of GDP-fucose from only glycerol. This was achieved by engineering Escherichia coli to endogenously overexpress genes manB, manC, gmd, and wcaG and heterologously overexpress a pair of fucosyltransferases to produce DFL from lactose. The introduction of α-1,2-fucosyltransferase from Helicobacter pylori (FucT2) along with α-1,3/4-fucosyltransferase (HP3/4FT) addressed rate-limiting challenges in enzymatic catalysis and allowed for highly efficient conversion of lactose into DFL. Based on these results, molecular modification of HP3/4FT was performed based on computer-assisted screening and structure-based rational design. The best-performing mutant, MH5, containing a combination of five mutated sites (F49K/Y131D/Y197N/E338D/R369A) of HP3/4FT was obtained. The best strain BLC09-58 harboring MH5 yielded 45.81 g/L of extracellular DFL in 5-L fed-batch cultures, which was the highest titer reported to date.