Metabolic Engineering of Escherichia coli for Efficient Biosynthesis of Lacto-N-tetraose Using a Novel β-1,3-Galactosyltransferase from Pseudogulbenkiania ferrooxidans

Abstract

Human milk oligosaccharides (HMOs) attract considerable interest in recent years because of their particular role in infant health. Lacto-N-tetraose (LNT), one of the most abundant HMOs, has been commercially added in the infant formula as a functional fortifier. In this study, a novel LNT-producing β-1,3-galactosyltransferase (β-1,3-GalT) from Pseudogulbenkiania ferrooxidans was screened from 14 putative candidates, and a highly LNT-producing metabolically engineered Escherichia coli strain was constructed based on a previously constructed lacto-N-triose II (LNT II)-producing strain, by strengthening UDP-galactose synthesis and introduction of P. ferrooxidans β-1,3-GalT. The engineered strain produced 3.11 and 25.49 g/L LNT in shake-flask and fed-batch cultivation, with the molar conversion ratio of LNT II to LNT of 88.15 and 85.09%, respectively. The productivity and specific yield of LNT in fed-batch cultivation were measured to be 0.61 g/L·h and 0.76 g/g dry cell weight, respectively. To the best of our knowledge, it is the highest LNT yield ever reported.

Pathway Optimization and Uridine 5′-Triphosphate Regeneration for Enhancing Lacto-N-Tetraose Biosynthesis in Engineered Escherichia coli

“…The media and culture conditions were almost the same as those previously reported. 21 Luria−Bertani (LB) and glycerol-defined medium were utilized for seed culture preparation and LNT production, respectively. LB medium was composed of 0.5% (w/v) yeast extract, 1% (w/v) tryptone, and 1% (w/v) NaCl by adjusting the pH to 7.0.…”

“…The detailed HPAEC conditions were as the same as the previously reported. 21 ■ RESULTS AND DISCUSSION Strengthening the Biosynthetic Pathway for UDP-Galactose Synthesis by Integration of Critical Gene galE in Genome. UDP-galactose and LNTri II are two direct precursors for LNT biosynthesis via metabolic engineering approach (Figure 1).…”

“…23 Previously, we constructed a highly efficient LNTri II-producing engineered E. coli strain E42-ΔWNL 20 and used it to further construct an LNT-producing engineered strain EL03 by introducing a new β-1,3-GalT from P. ferrooxidans and overexpressing GalE from E. coli MG1655, which produced 25.49 g/L LNT in fed-batch fermentation. 21 In this study, to stabilize the critical gene galE expression, the galE expression cassette was integrated into the chromosome first. The CRISPR-Cas9 system was used to insert galE into the recA locus of E. coli E42-ΔWNL strain guided by specific sgRNA, generating an engineered strain EN0.…”

“…When introducing the P. ferrooxidans β-1,3-GalT for LNT synthesis and overexpressing UDP glucose 4-epimerase (GalE, EC 5.1.3.2) from E. coli MG1655 for strengthening UDP-galactose synthesis in the previously constructed LNTri II-generating E. coli strain, the LNT yield reached 25.49 g/L by fed-batch fermentation. 21 In this study, to further improve LNT biosynthesis, galE strengthening was performed by both genomic integration and plasmid expression, various genes related to uridine 5′-triphosphate (UTP) regeneration were regulated for improving UDP-galactose supply, and fed-batch fermentation was performed for large-scale production.…”

Recent Advances on Lacto-N-neotetraose, a Commercially Added Human Milk Oligosaccharide in Infant Formula

“…The highest LNnT yield reported is 5.41 g/L by fed-batch fermentation; however, by comparison, the highest LNT yield reaches 25.49 g/L. 120 Thus, there is much room for the improvement of LNT biosynthesis by metabolic engineering approach.…”