A 37-amino acid loop in the Yarrowia lipolytica hexokinase impacts its activity and affinity and modulates gene expression

Piotr Hapeta,Zbigniew Lazar,Cécile Neuvéglise,Patrycja Szczepańska

doi:10.1038/s41598-021-85837-8

Abstract

The oleaginous yeast Yarrowia lipolytica is a potent cell factory as it is able to use a wide variety of carbon sources to convert waste materials into value-added products. Nonetheless, there are still gaps in our understanding of its central carbon metabolism. Here we present an in-depth study of Y. lipolytica hexokinase (YlHxk1), a structurally unique protein. The greatest peculiarity of YlHxk1 is a 37-amino acid loop region, a structure not found in any other known hexokinases. By combining bioinformatic and experimental methods we showed that the loop in YlHxk1 is essential for activity of this protein and through that on growth of Y. lipolytica on glucose and fructose. We further proved that the loop in YlHxk1 hinders binding with trehalose 6-phosphate (T6P), a glycolysis inhibitor, as hexokinase with partial deletion of this region is 4.7-fold less sensitive to this molecule. We also found that YlHxk1 devoid of the loop causes strong repressive effect on lipase-encoding genes LIP2 and LIP8 and that the hexokinase overexpression in Y. lipolytica changes glycerol over glucose preference when cultivated in media containing both substrates.

Highlights

The oleaginous yeast Yarrowia lipolytica is a potent cell factory as it is able to use a wide variety of carbon sources to convert waste materials into value-added products
YlHxk[1] successfully substitutes hexokinase II from S. cerevisiae (ScHxk2) in glucose catabolite repression of invertase, what indicates the bifunctionality of this protein[13]
A substantial progress was achieved by showing that the 37-amino acid extra-loop in the central region of YlHxk[1] is essential for activity of this protein and through that on growth on glucose and fructose

Summary

Introduction

The oleaginous yeast Yarrowia lipolytica is a potent cell factory as it is able to use a wide variety of carbon sources to convert waste materials into value-added products. Wild strains of Y. lipolytica utilize only a handful of sugar substrates, namely glucose, fructose and mannose These hexoses are transported inside the cell via hexose transporters and incorporated to the central carbon metabolism after their phosphorylation by hexose kinases. D343 (D386 in YlHxk1) is a residue in the heart of the large lobe, away from the sugar binding cleft while E457 (E500 in YlHxk1) is a part of the highly conserved motif in hexokinase family proteins 457EDGSGAGAAV466 (500EDGSGVGAAL509 in YlHxk1) at the C-terminal end[24] Mutations of these residues severely affect catalytic activity and substrate recognition and were reported to change the derepression of SUC2 by glucose as in S. cerevisiae hxk[2] null mutant[24]. These residues are conserved in YlHxk[1], no reports on their functions were provided so far

Methods

Results

Discussion

Conclusion