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

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.

Mutability‐Landscape‐Guided Engineering of l‐Threonine Aldolase Revealing the Prelog Rule in Mediating Diastereoselectivity of C−C Bond Formation

“…Although a flip of the aldehyde was observed to enable the C anion to attack its si ‐ or re ‐face to yield racemic products in a pyruvate‐dependent aldolase, [29] it might not happen in Cp LTA as the bulky substrate MTB causes steric hindrance, which hinders its rotation, as evidenced by the high diastereoselectivity of Cp LTA variants and the conformations with a single orientation of the substrate in MD simulations. The path hypothesis and Prelog rule are actually common in the enzymes promoting the formation C−C bonds, such as coenzyme‐independent aldolase, [2a] transaldolases, [2b] transketolases [22] and so forth. For example, two substrate access tunnels ( S ‐pathway and R ‐pathway) of thiamine diphosphate‐dependent pyruvate decarboxylase were identified, and the hot regions in the tunnels were engineered for asymmetric synthesis of ( S )‐benzoin [30] .…”

Mutability‐Landscape‐Guided Engineering of l‐Threonine Aldolase Revealing the Prelog Rule in Mediating Diastereoselectivity of C−C Bond Formation

“…Although a flip of the aldehyde was observed to enable the C anion to attack its si ‐ or re ‐face to yield racemic products in a pyruvate‐dependent aldolase, [29] it might not happen in Cp LTA as the bulky substrate MTB causes steric hindrance, which hinders its rotation, as evidenced by the high diastereoselectivity of Cp LTA variants and the conformations with a single orientation of the substrate in MD simulations. The path hypothesis and Prelog rule are actually common in the enzymes promoting the formation C−C bonds, such as coenzyme‐independent aldolase, [2a] transaldolases, [2b] transketolases [22] and so forth. For example, two substrate access tunnels ( S ‐pathway and R ‐pathway) of thiamine diphosphate‐dependent pyruvate decarboxylase were identified, and the hot regions in the tunnels were engineered for asymmetric synthesis of ( S )‐benzoin [30] .…”

Morphological, Biochemical, and Proteomic Analyses to Understand the Promotive Effects of Plant-Derived Smoke Solution on Wheat Growth under Flooding Stress

“…In this study, FBPA and GAPDH decreased in wheat leaves by the application of plant-derived smoke under flooding ( Figure 7 ). FBPA and GAPDH are located at the key intersections between glycolysis and the pentose-phosphate pathway, which are required for both pathways and are essential for the synthesis of glucose [ 27 ]. Additionally, both FBPA and GAPDH exert so-called “moonlighting” functions in yeast and other organisms, which are biological activities in addition to their catalytic role in glycolysis and gluconeogenesis [ 28 ].…”