Enabling glucose/xylose co-transport in yeast through the directed evolution of a sugar transporter

The capacity to co-transport glucose and xylose into yeast has remained a technical challenge in the field. While significant efforts have been made in transporter engineering to increase xylose transport rates, glucose-based inhibition still limit most of these transporters. To address this issue, we further engineer sugar transporter proteins to remove glucose inhibition and enable glucose/xylose co-transport. Specifically, we start with our previously derived CiGXS1 FIM mutant strain and subjugate it to several rounds of mutagenesis and selection in a hexose metabolism null strain. Through this effort, we identify several mutations including N326H, a truncation in the C-terminal tail, I171F, and M40V as additionally dominant for reducing glucose inhibition. The resulting transporter shows substantially improved xylose transport rates in the presence of high quantities of glucose including up to 70 g/L glucose. Moreover, the resulting transporter enables co-utilization of glucose and xylose with glucose rates on par with a wild-type transporter and xylose rates exceeding that of glucose. These results demonstrate that major facilitator superfamily hexose transporters can be rewired into glucose-xylose co-transporters without functional inhibition by either substrate. These results enhance the potential of using lignocellulosic biomass as a feedstock for yeast.     

Genetic transformation of the yeast Rhodotorula gracilis ATCC 26217 by electroporation

We examined the genetic transformation of the biotechnologically relevant yeast Rhodotorula gracilis ATCC 26217 by electroporation. To evaluate the yeast transformation, we created a genomic integration cassette that was targeted to the yeast orotidine-5′-phosphate decarboxylase gene (URA3 gene) locus and composed of the zeocin-resistant gene (Sh ble gene) with the URA3 promoter and terminator of the yeast. The yeast was unable to grow on medium containing 2.0 μg/mL zeocin, even with the inoculation of a large number of cells (approximately 1.0 × 10⁸ cells/plate). Using the integrative cassette and zeocin-containing medium, we successfully obtained yeast transformants by electroporation, and the highest transformation efficiency of approximately 40 colony-forming units/μg DNA was obtained with a 0.6-kV electrical pulse. No homologous integration of the cassette at the URA3 gene locus was detected by the analyses of uracil auxotrophy and genomic PCR of transformants, suggesting that this method is a useful tool for randomly mutating the yeast genome.     

Occurrence and taxonomic aspects of proton movements coupled to sugar transport in the yeast genusKluyveromyces

The occurrence of proton symport mechanisms for the transport of glucose, galactose, fructose, raffinose and sucrose in 21 yeast strains representing the species of the genusKluyveromyces was surveyed. Proton symport of one or more sugars occurred in 57% of the strains. Similarly, all the sugars investigated were transported by symports by several strains. Symport systems for non-utilisable sugars were rare. Starvation of cells frequently resulted in the appearance of a symport absent in non-starved glucose-grown cells, indicating that repression of proton symports by glucose and subsequent derepression by starvation is a general phenomenon in members ofKluyveromyces. The addition of a sugar to cell suspensions resulted in acidification in 80% of cases, indicating the activity of a membrane-bound ATPase. Acidification was also observed with a number of sugars that cannot be utilised by the particular species. Interesting correlations between the number of proton symports and the abundance of other phenotypic characteristics in members of the genus emerged. Most members of the infertile group of species showing an increase in the number of small chromosomes, inability to produce well-developed pseudomycelium, linoleic and linolenic acid, a decrease in the number of carbon compounds utilised and inability to utilise ethylamine also had no proton symports, whereas most members of the interfertile species produced one or more proton symports. It was concluded that the distribution of the number of proton symports amongstKluyveromyces species coincided with that of other positive characteristics and may therefore be of taxonomic value.     

Temperature dependence of the energy-linked monosaccharide transport across the cell membrane of Rhodotorula gracilis.

The temperature dependence of the active monosaccharide transport across the cell membrane of the yeast Rhodotorula gracilis has been studied between 0 and 55 degrees C with D-xylose as the transported substrate: (i) Between 0 and 10 degrees C there is virtually no transport. (ii) The initial velocity of transport increases exponentially from 15 to 30 degrees C (deltaE equal to 32 plus or minus 2 kcal/mol). (iii) At 30 degrees C a sharp "break" occurs in the Arrhenius plot and with increasing temperature the transport becomes inactivated, with a positive slope of the corresponding straight line ("deltaE equal to minus 15 kcal/mol"). (iv) In the temperature range of 50-55 degrees C, both the transport and the metabolic activity cease. In order to account for the abrupt changes of the membrane permeability, we attempted to ascribe them to phase transitions in the membrane structure: the first one, between 10 and 15 degrees C, to the crystalline: liquid-crystalline phase change; the second one, around 30 degrees C, to a change from highly ordered (low entropy) to less ordered (high entropy) membrane structure. Whereas the former phase transition is reversible, the latter appears to be irreversible. Arrhenius plots of the cell respiration exhibit a "break" at 30 degrees C, as well. However, at higher temperatures there is no thermal inactivation of the respiratory activity. The importance of a proper organization of the cell membrane constituents for the efficient transport function is discussed.     

Transport of gluconate in Rhodotorula glutinis. Inactivation by glucose of the uptake system.

Transport of gluconate has been studied in a wild-type strain of Rhodotorula glutinis and in a mutant derived from it which has acquired the ability to grow on gluconate as the only carbon and energy source. The transport is energy dependent. It shows the same K_m for gluconate (0.1 mm) between pH 4.7 and 7, which suggests that the negatively charged gluconate is the true substrate for the transport system. The rate of gluconate uptake is much lower in the wild type than in the mutant. The mutant grown on gluconate transports gluconate much faster than if grown on other carbon sources. Glucose rapidly and irreversibly inactivates the transport system. This inactivation can also be effected by δ-gluconolactone and to a lesser extent by acetate; it is not prevented by gluconate and occurs also in the presence of cycloheximide.     

Different proton-sugar stoichiometries for the uptake of glucose analogues by Chlorella vulgaris. Evidence for sugar-dependent proton uptake without concomitant sugar uptake by the proton-sugar symport system.

The uptake of hexoses by Chlorella vulgaris is accompanied by the uptake of protons. For 6-deoxyglucose a stoichiometry of one proton taken up per sugar molecule has been measured, whereas for 1-deoxyglucose approximately two protons are taken up per sugar molecule. It was found that in the presence of 1-deoxyglucose a considerable proportion of "carrier" catalyzes the transport of protons without the concomitant transport of sugar. Presumably, the binding of sugar initiates the translocation of the carrier-proton-sugar complex, but whereas 1-deoxyglucose can still dissociate from the complex at the external side of the cytoplasmic membrane, the translocation of the carrier-proton complex continues. This conclusion was reached since (a) the composition of the translocated carrier-proton-sugar complex is the same for both sugar. Its formation is a first order reaction with respect to protons. (b) When 6-deoxyglucose, present inside cells, is exchanged for external sugar, the exchange ratio is two to one when the external sugar is 1-deoxyglucose, two molecules of 6-deoxyglucose are lost for each molecule of 1-deoxyglucose entering. This result indicates that during uptake of 1-deoxyglucose statistically only each second carrier molecule appearing at the internal side of the cytoplasmic membrane is carrying sugar.     

Evidence for rapid uptake of D-galacturonic acid in the yeast Saccharomyces cerevisiae by a channel-type transport system

D-Galacturonic acid is a major component of pectins but cannot be metabolized by Saccharomyces cerevisiae. It is assumed not to be taken up. We show that yeast displays surprisingly rapid low-affinity uptake of D-galacturonic acid, strongly increasing with decreasing extracellular pH and without saturation up to 1.5 M. There was no intracellular concentration above the extracellular level and transport was reversible. Among more than 160 single and multiple deletion mutants in channels and transporters, no strain was affected in D-galacturonic acid uptake. The uptake was not inhibited by any compound tested as candidate competitive inhibitor, including D-glucuronic acid, which was also transported. The characteristics of D-galacturonic acid uptake are consistent with involvement of a channel-type system, probably encoded by multiple genes.     

Restricted sugar uptake by sugar-induced internalization of the yeast lactose/galactose permease Lac12

Kluyveromyces lactis Lac12 permease mediates lactose and low-affinity galactose transports. In this study we investigated the effects of carbon sources on internalization of Lac12 using a LAC12-GFP fusion construct. When galactose- or lactose-grown cells are shifted to a fresh sugar medium, Lac12-GFP is removed from the plasma membrane and is localized intracellularly. Surprisingly, either galactose or lactose in the new media caused the internalization, and cells responded differently to these two sugars. Our results reveal that this process is dependent on sugar species and also sugar concentration. Lac12-GFP internalization causes reduction of [C(14) ]lactose uptake rates and also occurs in a Klsnf1 mutant strain; it is therefore independent of KlSnf1 activity. We suggest that glucose-6-phosphate is the intracellular signal, as internalization was induced by 2-deoxyglucose, and inhibition of phosphoglucomutase by lithium prevented galactose- but not lactose- or glucose-induced internalization. Lac12-GFP internalization was not triggered by 6-deoxyglucose, and was irreversible in the absence of protein synthesis.     

Importance of the pentose phosphate pathway for d-glucose catabolism in the obligatory aerobic yeast Rhodotorula gracilis

d-Glucose catabolism of a phosphofructokinase-deficient yeast Rhodotorula gracilis has been studied. By using d-glucose specifically ¹⁴C-labelled at different positions and measuring the distribution of the label in various fractions of cell metabolism, the following results were found. 1. The pentose phosphate pathway, being the main pathway of d-glucose catabolism, simultaneously converts glucose molecules into pentose phosphates oxidatively by using two NADP-linked dehydrogenases and via the non-oxidative transketolase–transaldolase pathway. 2. From the correlation of the ¹⁴CO₂ liberation and the d-glucose consumption and from the fact that the pentose phosphate moiety in nucleic acids is almost equally labelled from d-[1-¹⁴C]- and d-[6-¹⁴C]-glucose, it is concluded that of the glucose utilized about 80% undergoes transformation via the non-oxidative pentose phosphate pathway. Only about 20% of glucose is directly decarboxylated to pentose phosphate. 3. For further degradation it is postulated that the pentose phosphates are split into C₂ fragments and glyceraldehyde 3-phosphates. 4. All three loci of oxidative decarboxylation appear to be effective in Rh. gracilis, the oxidative part of the pentose phosphate pathway, the decarboxylation of pyruvate in the later part of the glycolytic pathway as well as the oxidation in the tricarboxylic acid cycle. 5. d-Glucose molecules taken up are only partially oxidized to CO₂: about four-fifths of each glucose molecule metabolized is incorporated into cell constituents. 6. The quantitative interrelations of the fluxes of d-glucose subunits along the catabolic pathways have been estimated and are discussed.     

A model for proton and potassium co-transport during the uptake of glutamine and sucrose by tomato internode disks

Internode disks of tomato (Lycopersicon esculentum cv. Moneymaker) were shaken in glutamine and sucrose solutions. At low external pH (<±5.5), the uptake of these substances was accompanied with K⁺ efflux, at high pH (>±5.5) with K⁺ influx. Low concentrations of external K⁺ (2 mmol l^-1) stimulated the uptake of glutamine, which was strongly inhibited by the supply of high K⁺ concentrations (20 mmol l^-1). The effect of K⁺ was particularly pronounced at high pH-values. Addition of CCCP in light reduced the uptake of glutamine to the same level as in the dark, and stopped the K⁺ fluxes which coincided with the uptake. A model is presented wherein the movements of K⁺ across the membrane are related to co-transport, depending on the membrane potential and the Nernst potential of K⁺.Abbreviation CCCP carbonylcyanide-m-chlorophenylhydrazone     

Kinetics of ion translocation across charged membranes mediated by a two-site transport mechanism. Effects of polyvalent cations upon rubidium uptake into yeast cells.

(1) The effect of surface charge upon the kinetics of monovalent cation translocation via a two-site mechanism is investigated theroretically. (2) According to the model dealt with, typical relations are expected for the dependence of the kinetic parameters of the translocation process upon the concentration of a polyvalent cation, differing essentially from those derived for the case in which the membrane carries no excess charge. (3) Even when a polyvalent cation does not compete with the substrate cation for binding to the translocation sites, apparently competitive inhibition may occur when the membrane is negatively charged. (4) The model is tested experimentally by studying the effects of the polyvalent cations Mg2+, Sr2+, Ca2+, Ba2+ and Al3+ upon Rb+ uptake into yeast cells at pH 4.5 A good applicability is found. (5) Equimolar concentrations of polyvalent cations reduce the rate of the Rb+ uptake into yeast cells in the order Mg2+ less than Sr2+ less than Ca2+ less than Ba2+ less than Al3+. (6) The conclusion is reached that the reduction in the rate of Rb+ uptake caused by the polyvalent cations applied results mainly from screening of the negative fixed charges on the membrane surface and binding to these negative sites rather than competition with Rb+ for the transport sites. (7) The results of our investigation indicate the affinity of the alkaline-earth cations for the negative fixed charges on the surface to the yeast cell membrane increases in the orther Mg2+ less than Sr2 less than Ca2+ less than Ba2+. (8) Probably mainly phosphoryl groups determine the net charge on the membrane of the yeast cell at a medium pH of 4.5.     

Different proton-sugar stoichiometries for the uptake of glucose analogues by Chlorella vulgaris: Evidence for sugar-dependent proton uptake without concomitant sugar uptake by the proton-sugar symport system

The uptake of hexoses by Chlorella vulgaris is accompanied by the uptake of protons. For 6-deoxyglucose a stoichiometry of one proton taken up per sugar molecule has been measured, whereas for 1-deoxyglucose approximately two protons are taken up per sugar molecule.It was found that in the presence of 1-deoxyglucose a considerable proportion of “carrier” catalyzes the transport of protons without the concomitant transport of sugar. Presumably the binding of sugar initiates the translocation of the carrier-proton-sugar complex, but whereas 1-deoxyglucose can still dissociate from the complex at the external side of the cytoplasmic membrane, the translocation of the carrier-proton complex continues.This conclusion was reached since (a) the composition of the translocated carrier-proton-sugar complex is the same for both sugar. Its formation is a first order reaction with respect to protons.(b) When 6-deoxyglucose, present inside cells, is exchanged for external sugar, the exchange ratio is two to one when the external sugar is 1-deoxyglucose, two molecules of 6-deoxyglucose are lost for each molecule of 1-deoxyglucose entering. This result indicates that during uptake of 1-deoxyglucose statistically only each second carrier molecule appearing at the internal side of the cytoplasmic membrane is carrying sugar.