Glucose repression can be alleviated by reducing glucose phosphorylation rate in Saccharomyces cerevisiae

Abstract

Microorganisms commonly exhibit preferential glucose consumption and diauxic growth when cultured in mixtures of glucose and other sugars. Although various genetic perturbations have alleviated the effects of glucose repression on consumption of specific sugars, a broadly applicable mechanism remains unknown. Here, we report that a reduction in the rate of glucose phosphorylation alleviates the effects of glucose repression in Saccharomyces cerevisiae. Through adaptive evolution under a mixture of xylose and the glucose analog 2-deoxyglucose, we isolated a mutant strain capable of simultaneously consuming glucose and xylose. Genome sequencing of the evolved mutant followed by CRISPR/Cas9-based reverse engineering revealed that mutations in the glucose phosphorylating enzymes (Hxk1, Hxk2, Glk1) were sufficient to confer simultaneous glucose and xylose utilization. We then found that varying hexokinase expression with an inducible promoter led to the simultaneous utilization of glucose and xylose. Interestingly, no mutations in sugar transporters occurred during the evolution, and no specific transporter played an indispensable role in simultaneous sugar utilization. Additionally, we demonstrated that slowing glucose consumption also enabled simultaneous utilization of glucose and galactose. These results suggest that the rate of intracellular glucose phosphorylation is a decisive factor for metabolic regulations of mixed sugars.The baker's yeast Saccharomyces cerevisiae has long served as a model for studying glucose repression, the multi-layer process by which glucose is consumed before all other carbon sources 1,2 . A wide variety of interconnected mechanisms contribute to yeast's ability to sense, respond, and optimize internal metabolism to preferentially consume glucose 3,4 . Transcriptional repressors such as Mig1, Cat8, and the Ssn6/Tup1 complex prevent transcription of glucose-repressed genes, such as those involved in gluconeogenesis and metabolism of alternative carbon sources [5][6][7][8] . The activities of these repressors are then mediated by kinases and phosphatases such as Snf1 and Glc7/Reg1, respectively 6,9,10 . Beyond these intracellular sensing mechanisms, membrane sensors such as Snf3 and Rgt2 allow yeast to sense extracellular sugar concentrations and internalize signals 11 . In sum, the S. cerevisiae glucose repression pathway is a complex network of signals and regulations comprising significant amounts of research and a continuously growing base of knowledge.Recently, a new layer of glucose repression of galactose consumption has been reported to be linked to the kinetic properties of sugar transporters 12 . Because sugars compete for cellular uptake, relative transport efficiency between two sugars will depend on extracellular sugar concentrations as well as transporter affinities (K m values) for each sugar. Consequently, it was reported that the extracellular sugar concentrations coupled with transporter substrate affinity determine the intracellular sugar concentrations 12 . As GAL ge...

Glycolytic flux inSaccharomyces cerevisiaeis dependent on RNA polymerase III and its negative regulator Maf1

“…When assessed in cells grown on glycerol, expression of HXT1–lacZ reporter was decreased in all strains (Fig 5 B) as expected [40]. Consistent with the reports on mutants in genes of the glycolytic pathway, which are blocked in glycolysis [59], the HXT1 expression was reduced in RNAP III compromised yeast suggesting that in this mutant, supply of glucose for a functional glycolytic pathway cannot be maintained.…”

“…However, HXT2 transcription is not dependent on Gcn4 transcriptional activity [84] in the rpc160-122 mutant. HXT2 overexpression in rpc128-1007 is most likely related to lower glycolytic efficiency in the mutant strain [59], but the direct regulator of the phenomenon still remains to be discovered.…”

Combined Cell Surface Display of β‐d‐Glucosidase (BGL), Maltose Transporter (MAL11), and Overexpression of Cytosolic Xylose Reductase (XR) in Saccharomyces cerevisiae Enhance Cellobiose/Xylose Coutilization for Xylitol Bioproduction from Lignocellulosic Biomass

“…Although higher Yxylitol/xylose (nearly 1 g g −1 xylose) after simultaneous cofermentation of cellobiose/xylose has already been reported in the literature, it has to be noticed that the present study significantly improved our system as the overexpression of Sc MAL11 yielded an increase of +30% of xylitol production (0.54–0.84 g g −1 xylose) in the engineered YPH499‐ Ss XR‐ Sc MAL11‐ Aa BGL recombinant strain compared to the YPH499‐ Ss XR‐ Aa BGL strain only. The engineered strain YPH499‐ Ss XR‐ Sc MAL11‐ Aa BGL was able to produce 1.1 g L −1 (Yxylitol/xylose = 0.13 g g −1 xylose) and 2.1 g L −1 (Yxylitol/xylose = 0.27 g g −1 xylose) of xylitol from pretreated KP and rice straw hydrolysate, respectively, which represents a very promising result for xylitol industrial bioproduction by this combined approach (Figure ; Table S5, Supporting Information).…”

“…Candida guilliermondii FTI‐20037 and Candida parapsilosis produced xylitol with yields ≈0.74 g g −1 xylose . While glucose is the most abundant sugar along with xylose in lignocellulosic biomass, it is not a suitable cosubstrate for xylitol bioproduction due to the glucose repression, which inhibits the transcriptional level of native XR . Overexpression of heterologous XR with a constitutive promoter circumvented the repression effect of glucose on XR expression.…”

“…As the lignocellulosic biomass is primarily composed of cellulose, hemicellulose, and lignin, depolymerization and hydrolysis processes yield hexoses and pentoses. Due to glucose repression of alternative carbon sources, engineered yeasts generally consume glucose before any other available carbon source . Baker's yeast ( Saccharomyces cerevisiae [ S. cerevisiae ]) is a valuable host for bioengineering as this microbe is generally recognized as safe (GRAS) and its genome is fully known.…”