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

Lacto-N-biose (LNB)is a member of the human milkoligosaccharide (HMO) family and is synthesized via an enzymatic reactionin vitro with N-acetylglucosamine (GlcNAc) and cofactors.In this study, LNB was synthesized using a cell factory for the firsttime. First, three modules were constructed in Kluyveromyceslactis for producing LNB from lactose and GlcNAc withoutthe addition of cofactors. Second, a de novo pathway was constructedin K. lactis for producing LNB fromlactose without adding GlcNAc. Finally, a transcriptional switch wasintroduced into K. lactis to reprogramits metabolic network for improving the flux from GlcNAc-6-P to GlcNAcin the de novo pathway. Subsequently, a final LNB yield of 10.41 g/L,similar to the salvage pathway yield, was achieved through the denovo pathway. The engineered K. lactis provides a promising technology platform for the industrial scaleproduction of LNB.

Automated summary

In this study, LNB was synthesized using a cell factory for the first time. Through the construction of three modules in Kluyveromyces lactis, LNB was successfully produced from lactose and GlcNAc without the addition of cofactors. The engineered K. lactis provides a promising technology platform for the industrial scale production of LNB.