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

J Agric Food Chem. 2023 Jun 14;71(23):9031-9039. doi: 10.1021/acs.jafc.3c01779. Epub 2023 Jun 1.

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.

Keywords: Kluyveromyces lactis; de novo pathway; lacto-N-biose; microbial cell factory; transcriptional switch.

MeSH terms

  • Humans
  • Kluyveromyces* / genetics
  • Kluyveromyces* / metabolism
  • Lactose*
  • Metabolic Networks and Pathways
  • Oligosaccharides / metabolism

Substances

  • Lactose
  • Oligosaccharides

Supplementary concepts

  • Kluyveromyces lactis