Genetic and Physiological Characterization of Fructose-1,6-Bisphosphate Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase in the Crabtree-Negative Yeast Kluyveromyces lactis

Rosaura Rodicio,Jürgen J Heinisch,Hans-Peter Schmitz

doi:10.3390/ijms23020772

Abstract

The milk yeast Kluyveromyces lactis degrades glucose through glycolysis and the pentose phosphate pathway and follows a mainly respiratory metabolism. Here, we investigated the role of two reactions which are required for the final steps of glucose degradation from both pathways, as well as for gluconeogenesis, namely fructose-1,6-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In silico analyses identified one gene encoding the former (KlFBA1), and three genes encoding isoforms of the latter (KlTDH1, KlTDH2, KlGDP1). Phenotypic analyses were performed by deleting the genes from the haploid K. lactis genome. While Klfba1 deletions lacked detectable FBA activity, they still grew poorly on glucose. To investigate the in vivo importance of the GAPDH isoforms, different mutant combinations were analyzed for their growth behavior and enzymatic activity. KlTdh2 represented the major glycolytic GAPDH isoform, as its lack caused a slower growth on glucose. Cells lacking both KlTdh1 and KlTdh2 failed to grow on glucose but were still able to use ethanol as sole carbon sources, indicating that KlGdp1 is sufficient to promote gluconeogenesis. Life-cell fluorescence microscopy revealed that KlTdh2 accumulated in the nucleus upon exposure to oxidative stress, suggesting a moonlighting function of this isoform in the regulation of gene expression. Heterologous complementation of the Klfba1 deletion by the human ALDOA gene renders K. lactis a promising host for heterologous expression of human disease alleles and/or a screening system for specific drugs.

Highlights

Glycolysis has been studied for more than a century, with a crucial impact on biochemistry, ever since the observation of cell-free fermentation in extracts from the wine, beer, and baker’s yeast Saccharomyces cerevisiae [1]
A comparison in the yeast gene order browser revealed syntenic homologs of both FBA1 and TDH2 in the genomes of Saccharomyces cerevisiae and Kluyveromyces lactis
Contribute to glucose degradation in the milk yeast Kluyveromyces lactis [46], we decided to investigate the importance of the reactions of fructose-1,6bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydro-genase (GAPDH), which are situated at the interface of the two pathways (Figure 4)

Summary

Introduction

Glycolysis has been studied for more than a century, with a crucial impact on biochemistry, ever since the observation of cell-free fermentation in extracts from the wine, beer, and baker’s yeast Saccharomyces cerevisiae [1]. While S. cerevisiae may serve as a model organism to study the physiology of human cancer cells, it is less suited for comparison with central carbohydrate metabolism in normal mammalian cells The latter is more closely reflected by Crabtree-negative, Pasteur-positive yeasts, which favor respiration over fermentation, as a more effective way to generate energy in the presence of oxygen [12,13]. Despite these fundamental works on central carbohydrate metabolism in K. lactis, it remains less well studied by far than in S. cerevisiae In this context, the reactions of fructose-1,6-bisphosphate aldolase (FBA) and glyceraldehyde3-phosphate dehydrogenase (GAPDH) were only marginally addressed, they were of special interest for several reasons.

Deletions of KlFBA1 or KlTDH2 Affect Growth on Glucose

Heterologous Genes Complement the Growth Defects of a Klfba1 Deficiency

Discussion

Strains and Growth Conditions

Fluorescence Microscopy

Preparation of Crude Extracts and Enzymatic Determination