Journal
CARBOHYDRATE POLYMERS
Volume 315, Issue -, Pages -Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.carbpol.2023.121028
Keywords
Human milk oligosaccharides; Lacto-N-triose II; Lacto-N-tetraose; Lacto-N-fucopentaose I; alpha 1,2-Fucosyltransferase
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An efficient LNFP I-producing strain without by-product 2'-FL was developed by stepwise de novo pathway construction. LNTri II-producing strains were created by integrating beta 1,3-N-acetylglucosaminyltransferase, which can be further converted to LNT by LNT-producing beta 1,3-galactosyltransferase. The de novo and salvage pathways of GDP-fucose were introduced into highly efficient LNT-producing chassis. Specific alpha 1,2-fucosyltransferase was verified to eliminate by-product 2'-FL.
Lacto-N-fucopentaose I (LNFP I) is an abundant and important fucosylated human milk oligosaccharide (HMO). Here, an efficient LNFP I-producing strain without by-product 2'-fucosyllactose (2'-FL) was developed by advisable stepwise de novo pathway construction in Escherichia coli. Specifically, the genetically stable lacto-N-triose II (LNTri II)-producing strains were constructed by the multicopy integration of beta 1,3-N-acetylglucosaminyltransferase. LNTri II can be further converted to lacto-N-tetraose (LNT) by LNT-producing beta 1,3-galactosyltransferase. The de novo and salvage pathways of GDP-fucose were introduced into highly efficient LNT-producing chassis. Specific alpha 1,2-fucosyltransferase was verified to eliminate by-product 2'-FL, and binding free energy of the complex was analyzed to explain the product distribution. Subsequently, further attempts aiming to improve alpha 1,2-fucosyltransferase activity and the supply of GDP-fucose were carried out. Our engineering strategies enabled the stepwise de novo construction of strains that produced up to 30.47 g/L of extracellular LNFP I, without accumulation of 2'-FL, and with only minor intermediates residue.
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