Lacto-N-tetraose (Gal(beta 1-3)GlcNAc(beta 1-3)Gal(beta 1-4)Glc) is one of the most abundant oligosaccharide structures in human milk. We recently described the synthesis of lacto-N-tetraose by a whole-cell biotransformation with recombinant Escherichia coli cells. However, only about 5% of the lactose was converted into lacto-N-tetraose by this approach. The major product obtained was the intermediate lacto-N-triose II (GlcNAc(beta 1-3)Gal(beta 1-4)Glc). In order to improve the bioconversion of lactose to lacto-N-tetraose, we have investigated the influence of the carbon source on the formation of lacto-N-tetraose and on the intracellular availability of the glycosyltransferase substrates, UDP-N-acetylglucosamine and UDP-galactose. By growth of the recombinant E. coli cells on D-galactose, the yield of lacto-N-tetraose (810.8 mg L-1 culture) was 3.6-times higher compared to cultivation on D-glucose. Using fed-batch cultivation with galactose as sole energy and carbon source, a large-scale synthesis of lacto-N-tetraose was demonstrated. During the 26 h feeding phase the growth rate (mu = 0.05) was maintained by an exponential galactose feed. In total, 16 g L-1 lactose were fed and resulted in final yields of 12.72 +/- 0.21 g L-1 lacto-N-tetraose and 13.70 +/- 0.10 g L-1 lacto-N-triose II. In total, 173 g of lacto-N-tetraose were produced with a space-time yield of 0.37 g L-1 h(-1). (C) 2015 Elsevier Inc. All rights reserved.