Efficient and Cost-Effective Synthesis of N-Acetyllactosamine by Sequential Modular Enzymatic Cascade Reactions Involving NTP Regeneration

Runmiao Jiao; Liming Zhang; Ran You; Xinlv Peng; Caixia Pei; Bowen Jiang; Meirong Hu; Jianjun Li; Yuguang Du; Eika W Qian

doi:10.1021/acs.jafc.4c08638

Efficient and Cost-Effective Synthesis of N-Acetyllactosamine by Sequential Modular Enzymatic Cascade Reactions Involving NTP Regeneration

J Agric Food Chem. 2024 Dec 3. doi: 10.1021/acs.jafc.4c08638. Online ahead of print.

Authors

Runmiao Jiao^{1

2

3}, Liming Zhang⁴, Ran You⁵, Xinlv Peng^{2

3

6}, Caixia Pei^{1

2

3}, Bowen Jiang^{1

2

3}, Meirong Hu⁵, Jianjun Li^{2

3

7}, Yuguang Du^{2

3

7}, Eika W Qian¹

Affiliations

¹ Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Nakacho 2-24-16, Koganei, Tokyo 1848588, Japan.
² State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
³ Key Laboratory of Biopharmaceutical Preparation and Delivery, Chinese Academy of Sciences, Beijing 100190, China.
⁴ College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang 330045, China.
⁵ Institute of Microbiology Chinese Academy of Sciences, Beijing 100101, China.
⁶ School of Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, China.
⁷ Innovation Academy for Green Manufacture Institute, Chinese Academy of Sciences, Beijing 100190, China.

PMID: 39625714
DOI: 10.1021/acs.jafc.4c08638

Abstract

Human milk oligosaccharides (HMOs) play important roles in the development of infants, which are the third most abundant component in human milk. N-Acetyllactosamine (LacNAc) is an important intermediate for the biosynthesis of other HMOs and antigens. Since currently appropriate synthetic methods for large-scale production of LacNAc are not available, it is urgently needed to develop an efficient and cost-effective synthetic pathway for LacNAc preparation. In this study, a cost-effective pathway of LacNAc synthesis involving regeneration of adenosine triphosphate (ATP) and uridine 5'-triphosphate (UTP) was established. After optimizing the reaction conditions, LacNAc was synthesized at a yield of >90% via a sequential one-pot multienzyme (OPME) method with crude enzymes at 100 mM substrates. Finally, LacNAc was produced efficiently and cost-effectively at a 5 L scale. This strategy would possibly meet the requirements of potential industrial production of LacNAc and would provide guidance for the production of other structurally complex HMOs or functional oligosaccharides in the future.

Keywords: N-acetyllactosamine; NTP regeneration; human milk oligosaccharides; multienzymatic synthesis; scale-up.