Reprogramming the Metabolic Network in Kluyveromyces lactis with a Transcriptional Switch for De Novo Lacto-N-biose Synthesis

Abstract

Lacto-N-biose (LNB) is a member of the human milk oligosaccharide (HMO) family and is synthesized via an enzymatic reaction in vitro with N-acetylglucosamine (GlcNAc) and cofactors. In this study, LNB was synthesized using a cell factory for the first time. First, three modules were constructed in Kluyveromyces lactis for producing LNB from lactose and GlcNAc without the addition of cofactors. Second, a de novo pathway was constructed in K. lactis for producing LNB from lactose without adding GlcNAc. Finally, a transcriptional switch was introduced into K. lactis to reprogram its metabolic network for improving the flux from GlcNAc-6-P to GlcNAc in the de novo pathway. Subsequently, a final LNB yield of 10.41 g/L, similar to the salvage pathway yield, was achieved through the de novo pathway. The engineered K. lactis provides a promising technology platform for the industrial scale production of LNB.

Efficient Biosynthesis of Lacto-N-Biose I, a Building Block of Type I Human Milk Oligosaccharides, by a Metabolically Engineered Escherichia coli

“…By comparison, the fed-batch cultivation of the engineered K. lactis via de novo GlcNAc pathway produced 10.41 g/L LNB, which was 3.34 times that of shake-flask cultivation …”

“…The dual sugar phosphorylase system has not been used for in vivo biosynthesis of LNB yet. The GalK-LnbP route has been developed for in vivo biosynthesis of LNB by engineered Kluyveromyces Lactis; however, the salvage pathway of GlcNAc should be introduced for enhancing the productivity …”

“…By comparison, the initial LNB titer was only 0.60 g/L via de novo GlcNAc pathway. The Agm1-On/Off switch was developed to enhance the LNB titer to up to 3.12 g/L . In this study, we developed a new and simpler pathway for in vivo LNB biosynthesis.…”

“…In addition, Li et al recently constructed a metabolically engineered Kluyveromyces lactis for in vivo LNB biosynthesis via LnbP as the last pathway enzyme. Three modules were designed for LNB biosynthesis, including Gal-1-P synthesis, GlcNAc supply strengthening, and LNB production via LnbP; however, efficient biosynthesis of LNB was significantly dependent on the salvage pathway of GlcNAc …”

Recent Advances in the Biosynthesis of Natural Sugar Substitutes in Yeast

“…2′-Fucosyllactose (2′-FL) is one of the oligosaccharides found in human milk (human milk oligosaccharide, HMO), which plays a crucial role in protecting infants against immunological diseases [ 20 , 21 ]. Hence, 2′-FL is not only a sugar substitute but also a milk analog [ 22 ].…”