Affordable Access

deepdyve-link
Publisher Website

Biosynthesis of the human milk oligosaccharide 3-fucosyllactose in metabolically engineered Escherichia coli via the salvage pathway through increasing GTP synthesis and β-galactosidase modification.

Authors
  • Choi, Yun Hee1, 2
  • Park, Bum Seok2, 3
  • Seo, Joo-Hyun4
  • Kim, Byung-Gee1, 2, 3
  • 1 Interdisciplinary Program in Bioengineering, Seoul National University, Seoul, Republic of Korea. , (North Korea)
  • 2 Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Republic of Korea. , (North Korea)
  • 3 School of Chemical and Biological Engineering, Institute of Engineering Research, Seoul National University, Seoul, Republic of Korea. , (North Korea)
  • 4 Department of Bio and Fermentation Convergence Technology, Kookmin University, Seoul, Republic of Korea. , (North Korea)
Type
Published Article
Journal
Biotechnology and Bioengineering
Publisher
Wiley (John Wiley & Sons)
Publication Date
Dec 01, 2019
Volume
116
Issue
12
Pages
3324–3332
Identifiers
DOI: 10.1002/bit.27160
PMID: 31478191
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

3-Fucosyllactose (3-FL) is one of the major fucosylated oligosaccharides in human milk. Along with 2'-fucosyllactose (2'-FL), it is known for its prebiotic, immunomodulator, neonatal brain development, and antimicrobial function. Whereas the biological production of 2'-FL has been widely studied and made significant progress over the years, the biological production of 3-FL has been hampered by the low activity and insoluble expression of α-1,3-fucosyltransferase (FutA), relatively low abundance in human milk oligosaccharides compared with 2'-FL, and lower digestibility of 3-FL than 2'-FL by bifidobacteria. In this study, we report the gram-scale production of 3-FL using E. coli BL21(DE3). We previously generated the FutA quadruple mutant (mFutA) with four site mutations at S46F, A128N, H129E, Y132I, and its specific activity was increased by nearly 15 times compared with that of wild-type FutA owing to the increase in kcat and the decrease in Km . We overexpressed mFutA in its maximum expression level, which was achieved by the optimization of yeast extract concentration in culture media. We also overexpressed L-fucokinase/GDP- L-fucose pyrophosphorylase to increase the supply of GDP-fucose in the cytoplasm. To increase the mass of recombinant whole-cell catalysts, the host E. coli BW25113 was switched to E. coli BL21(DE3) because of the lower acetate accumulation of E. coli BL21(DE3) than that of E. coli BW25113. Finally, the lactose operon was modified by partially deleting the sequence of LacZ (lacZΔm15) for better utilization of D-lactose. Production using the lacZΔm15 mutant yielded 3-FL concentration of 4.6 g/L with the productivity of 0.076 g·L-1 ·hr-1 and the specific 3-FL yield of 0.5 g/g dry cell weight. © 2019 Wiley Periodicals, Inc.

Report this publication

Statistics

Seen <100 times