Repression vs. activation ofMOX,FMD,MPP1andMAL1promoters by sugars inHansenula polymorpha: the outcome depends on cell's ability to phosphorylate sugar

Abstract

A high-throughput approach was used to assess the effect of mono- and disaccharides on MOX, FMD, MPP1 and MAL1 promoters in Hansenula polymorpha. Site-specifically designed strains deficient for (1) hexokinase, (2) hexokinase and glucokinase, (3) maltose permease or (4) maltase were used as hosts for reporter plasmids in which β-glucuronidase (Gus) expression was controlled by these promoters. The reporter strains were grown on agar plates containing varied carbon sources and Gus activity was measured in permeabilized cells on microtitre plates. We report that monosaccharides (glucose, fructose) repress studied promoters only if phosphorylated in the cell. Glucose-6-phosphate was proposed as a sugar repression signalling metabolite for H. polymorpha. Intriguingly, glucose and fructose strongly activated expression from these promoters in strains lacking both hexokinase and glucokinase, indicating that unphosphorylated monosaccharides have promoter-derepressing effect. We also show that maltose and sucrose must be internalized and split into monosaccharides to exert repression on MOX promoter. We demonstrate that at yeast growth on glucose-containing agar medium, glucose-limitation is rapidly created that promotes derepression of methanol-specific promoters and that derepression is specifically enhanced in hexokinase-negative strain. We recommend double kinase-negative and hexokinase-negative mutants as hosts for heterologous protein production from MOX and FMD promoters.

Maltase protein of Ogataea (Hansenula) polymorpha is a counterpart to the resurrected ancestor protein ancMALS of yeast maltases and isomaltases

“…We cloned the maltase gene MAL1 of Op about 15 years ago (Liiv et al , ) and later studied its regulation (Alamäe et al , ; Suppi et al , ). A study of crude cell extracts of MAL1 ‐expressing E. coli (Liiv et al , ) showed that the Op maltase hydrolysed not only maltose and sucrose but also α ‐MG (for structure, see supporting information, Figure S1), which is a synthetic analogue of isomaltose and a substrate for isomaltases (Yamamoto et al ., ; Teste et al , ).…”

Carbon source dependent promoters in yeasts

“…This is consistent with the phenotype of a HXT deletion strain [14], and also with the observation that the AMP/ATP ratio reflects the glucose level inside the cell (a high AMP/ATP ratio leads to activation of Snf1 [9], a kinase directly involved in gene regulation by carbon sources). However, most likely the processed metabolite of monosaccharides in the cell–glucose-6-phosphate–is the main signal that activates glucose repression [15]. …”

Genome Mining of Non-Conventional Yeasts: Search and Analysis of MAL Clusters and Proteins

“…Our data on Op indicated that monosaccharides (glucose and fructose) produced at intracellular hydrolysis of disaccharides repress the MAL1 promoter and their phosphorylation is obligatory for the repression. At the same time, temporary accumulation of unphosphorylated hydrolysis products was shown to activate the promoter [ 11 ]. The growth of Op on disaccharides is assumingly complicated [ 11 ], because hydrolysis products of disaccharides that promote initial derepression of MAL genes will later cause repression when their phosphorylated species accumulate.…”

“…At the same time, temporary accumulation of unphosphorylated hydrolysis products was shown to activate the promoter [ 11 ]. The growth of Op on disaccharides is assumingly complicated [ 11 ], because hydrolysis products of disaccharides that promote initial derepression of MAL genes will later cause repression when their phosphorylated species accumulate. Therefore, expression of AGT and AG proteins has to be finely adjusted with further glycolytic flux to provide an appropriate expression level of MAL genes.…”

“…We showed that one of these species— Ogataea polymorpha ( Op )—can grow on maltose and sucrose using maltase (an α-glucosidase) for their hydrolysis [ 5 , 6 ]. In following studies, we focused on genetics, biochemistry, and regulation of utilization of α-glucosidic sugars in Op [ 7 , 8 , 9 , 10 , 11 , 12 ].…”