Biosynthesis of Lacto-N-biose I from starch and N-acetylglucosamine via an in vitro synthetic enzymatic biosystem

Lijie Chen; Yanmei Qin; Long Ma; Dongdong Meng; Chun You

doi:10.1016/j.synbio.2023.08.004

Biosynthesis of Lacto-N-biose I from starch and N-acetylglucosamine via an in vitro synthetic enzymatic biosystem

Synth Syst Biotechnol. 2023 Aug 18;8(3):555-562. doi: 10.1016/j.synbio.2023.08.004. eCollection 2023 Sep.

Authors

Lijie Chen^{1

2}, Yanmei Qin^{2

3}, Long Ma¹, Dongdong Meng², Chun You^{2

3

4}

Affiliations

¹ College of Bioengineering, Tianjin University of Science and Technology, Tianjin, 300457, China.
² Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
³ University of Chinese Academy of Sciences, Beijing, 100049, China.
⁴ National Center of Technology Innovation for Synthetic Biology, Tianjin, 300308, China.

Abstract

Human milk oligosaccharides (HMOs) are very distinctive components in human milk and are beneficial for infant health. Lacto-N-biose I (LNB) is the core structural unit of HMOs, which could be used for the synthesis of other HMOs. In this study, an ATP-free in vitro synthetic enzymatic biosystem contained four thermostable enzymes (alpha-glucan phosphorylase from Thermococcus kodakarensis, UDP-glucose-hexose-1-phosphate uridylyltransferase from Thermotoga maritima, UDP-glucose 4-epimerase from T. maritima, lacto-N-biose phosphorylase from Clostridium thermobutyricum) were constructed for the biosynthesis of LNB from starch and N-acetylglucosamine (GlcNAc). Under the optimal conditions, 0.75 g/L and 2.23 g/L LNB was produced from 1.1 g/L and 4.4 g/L GlcNAc and excess starch, with the molar yield of 39% and 29% based on the GlcNAc concentration, respectively, confirming the feasibility of this in vitro synthetic enzymatic biosystem for LNB synthesis and shedding light on the biosynthesis of other HMOs using LNB as the core structural unit from low cost polysaccharides.

Keywords: Human milk oligosaccharide; In vitro synthetic enzymatic biosystem; Lacto-N-biose I; Thermostable enzyme.