Energetic benefits and rapid cellobiose fermentation by Saccharomyces cerevisiae expressing cellobiose phosphorylase and mutant cellodextrin transporters

Anaerobic bacteria assimilate cellodextrins from plant biomass by using a phosphorolytic pathway to generate glucose intermediates for growth. The yeast Saccharomyces cerevisiae can also be engineered to ferment cellobiose to ethanol using a cellodextrin transporter and a phosphorolytic pathway. However, strains with an intracellular cellobiose phosphorylase initially fermented cellobiose slowly relative to a strain employing an intracellular β-glucosidase. Fermentations by the phosphorolytic strains were greatly improved by using cellodextrin transporters with elevated rates of cellobiose transport. Furthermore under stress conditions, these phosphorolytic strains had higher biomass and ethanol yields compared to hydrolytic strains. These observations suggest that, although cellobiose phosphorolysis has energetic advantages, phosphorolytic strains are limited by the thermodynamics of cellobiose phosphorolysis (ΔG°=+3.6 kJ mol⁻¹). A thermodynamic “push” from the reaction immediately upstream (transport) is therefore likely to be necessary to achieve high fermentation rates and energetic benefits of phosphorolysis pathways in engineered S. cerevisiae.     

Co-fermentation of xylose and cellobiose by an engineered Saccharomyces cerevisiae

We have integrated and coordinately expressed in Saccharomyces cerevisiae a xylose isomerase and cellobiose phosphorylase from Ruminococcus flavefaciens that enables fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. The native xylose isomerase was active in cell-free extracts from yeast transformants containing a single integrated copy of the gene. We improved the activity of the enzyme and its affinity for xylose by modifications to the 5′-end of the gene, site-directed mutagenesis, and codon optimization. The improved enzyme, designated RfCO*, demonstrated a 4.8-fold increase in activity compared to the native xylose isomerase, with a K_m for xylose of 66.7?mM and a specific activity of 1.41?μmol/min/mg. In comparison, the native xylose isomerase was found to have a K_m for xylose of 117.1?mM and a specific activity of 0.29?μmol/min/mg. The coordinate over-expression of RfCO* along with cellobiose phosphorylase, cellobiose transporters, the endogenous genes GAL2 and XKS1, and disruption of the native PHO13 and GRE3 genes allowed the fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. Interestingly, this strain was unable to utilize xylose or cellobiose as a sole carbon source for growth under anaerobic conditions, thus minimizing yield loss to biomass formation and maximizing ethanol yield during their fermentation.     

Construction of a flocculent Saccharomyces cerevisiae fermenting lactose

A flocculent Saccharomyces cerevisiae strain with the ability to express both the LAC4 (coding for β-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus was constructed. This recombinant strain is not only able to grow on lactose, but it can also ferment this substrate. To our knowledge this is the first time that a recombinant S. cervisiae has been found to ferment lactose in a way comparable to that of the existing lactose-fermenting yeast strains. Moreover, the flocculating capacity of the strain used in this work gives the process several advantages. On the one hand, it allows for operation in a continuous mode at high cell concentration, thus increasing the system's overall productivity; on the other hand, the biomass concentration in the effluent is reduced, thus decreasing product separation/purification costs. Received: 2 October 1998 / Received revision: 15 January 1999 / Accepted: 17 January 1999     

Cofermentation of cellobiose and galactose by an engineered Saccharomyces cerevisiae strain

We demonstrate improved ethanol yield and productivity through cofermentation of cellobiose and galactose by an engineered Saccharomyces cerevisiae strain expressing genes coding for cellodextrin transporter (cdt-1) and intracellular β-glucosidase (gh1-1) from Neurospora crassa. Simultaneous fermentation of cellobiose and galactose can be applied to producing biofuels from hydrolysates of marine plant biomass.     

Co-expression of a mammalian accessory trafficking protein enables functional expression of the rat MCT1 monocarboxylate transporter in Saccharomyces cerevisiae

We have developed a new heterologous expression system for monocarboxylate transporters. The system is based on a Saccharomyces cerevisiae pyk1 mae1 jen1 triple-deletion strain that is auxotrophic for pyruvate and deficient in monocarboxylate uptake. Growth of the yeast cells on ethanol medium supplemented with pyruvate or lactate was dependent on the expression of a suitable monocarboxylate transporter. We have used the system to characterize the functional significance of interactions between the rat MCT1 transporter and its ancillary protein CD147. CD147 was shown to improve trafficking of MCT1 to the plasma membrane and its uptake activity. Our results demonstrate a new strategy for the production of properly folded and correctly targeted membrane proteins in a microbial expression system by co-expression of appropriate accessory proteins.     

Metabolic engineering for direct lactose utilization by Saccharomyces cerevisiae

A recombinant strain of Saccharomyces cerevisiae, secreting -galactosidase from Kluyveromyces lactis, grew efficiently with more than 60 g lactose l^–1. The growth rate (0.23 h^–1) in a cheese-whey medium was close to the highest reported hitherto for other recombinant S. cerevisiae strains that express intracellular -galactosidase and lactose-permease genes. The conditions for growth and -galactosidase secretion in this medium were optimized in a series of factorial experiments. Best results were obtained at 23 °C for 72 h. Since the recombinant strain produced less than 3% ethanol from the lactose, it was also assayed for the production of fructose 1,6-bisphosphate from cheese whey, and 0.06 g l^–1 h^–1 were obtained.     

Characterization of Schizosaccharomyces pombe malate permease by expression in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, L-malic acid transport is not carrier mediated and is limited to slow, simple diffusion of the undissociated acid. Expression in S. cerevisiae of the MAE1 gene, encoding Schizosaccharomyces pombe malate permease, markedly increased L-malic acid uptake in this yeast. In this strain, at pH 3.5 (encountered in industrial processes), L-malic acid uptake involves Mae1p-mediated transport of the monoanionic form of the acid (apparent kinetic parameters: Vmax = 8.7 nmol/mg/min; Km = 1.6 mM) and some simple diffusion of the undissociated L-malic acid (Kd = 0.057 min(-1)). As total L-malic acid transport involved only low levels of diffusion, the Mae1p permease was further characterized in the recombinant strain. L-Malic acid transport was reversible and accumulative and depended on both the transmembrane gradient of the monoanionic acid form and the DeltapH component of the proton motive force. Dicarboxylic acids with stearic occupation closely related to L-malic acid, such as maleic, oxaloacetic, malonic, succinic and fumaric acids, inhibited L-malic acid uptake, suggesting that these compounds use the same carrier. We found that increasing external pH directly inhibited malate uptake, resulting in a lower initial rate of uptake and a lower level of substrate accumulation. In S. pombe, proton movements, as shown by internal acidification, accompanied malate uptake, consistent with the proton/dicarboxylate mechanism previously proposed. Surprisingly, no proton fluxes were observed during Mae1p-mediated L-malic acid import in S. cerevisiae, and intracellular pH remained constant. This suggests that, in S. cerevisiae, either there is a proton counterflow or the Mae1p permease functions differently from a proton/dicarboxylate symport.     

Photoaffinity labelling of the purine-cytosine permease of Saccharomyces cerevisiae

8-Azidoadenine was used as a photoaffinity reagent to characterize the purine-cytosine permease of Saccharomyces cerevisiae. It is a potent competitive inhibitor of cytosine uptake and irradiation of the cells incubated with the label induced the irreversible inactivation of cytosine uptake. Addition of excess cytosine prevented this labelling which was restricted to the outer face of the plasma membrane since it was not accumulated by the cells. In the strain with the amplified purine-cytosine permease gene the maximum cytosine uptake rate was increased 4-5-fold relative to wild type without a modification of the Michaelis constant of uptake (Kt); no uptake could be measured in the deleted strain. The relative amounts of specific labelling determined for the cells and for membrane preparations were 0, 1 and 4 for the null, the wild-type and the amplified strains, respectively. One major band specifically labelled by [3H]azidoadenine, corresponding to a polypeptide with an apparent molecular mass of 45 kDa, was observed in the wild type, amplified in the strain carrying the multicopy plasmid and not detected in the deleted strain. Therefore this polypeptide corresponds to the purine-cytosine permease.     

Kinetics of lactose fermentation using a recombinant Saccharomyces cerevisiae strain

This work presents a multi-route, non-structural kinetic model for interpretation of ethanol fermentation of lactose using a recombinant flocculent Saccharomyces cerevisiae strain expressing both the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces lactis. In this model, the values of different metabolic pathways are calculated applying a modified Monod equation rate in which the growth rate is proportional to the concentration of a key enzyme controlling the single metabolic pathway. In this study, three main metabolic routes for S. cerevisiae are considered: oxidation of lactose, reduction of lactose (producing ethanol), and oxidation of ethanol. The main bioprocess variables determined experimentally were lactose, ethanol, biomass, and dissolved oxygen concentrations. Parameters of the proposed kinetic model were established by fitting the experimental data obtained in a small lab-scale fermentor with the initial lactose concentrations ranging from 5 g/dm3 to 50 g/dm3. A very good agreement between experimental data and simulated profiles of the main variables (lactose, ethanol, biomass, and dissolved oxygen concentrations) was achieved.     

Inactivation of maltose permease and maltase in sporulating Saccharomyces cerevisiae

Maltose transport and maltase activities were inactivated during sporulation of a MAL constitutive yeast strain harboring different MAL loci. Both activities were reduced to almost zero after 5 h of incubation in sporulation medium. The inactivation of maltase and maltose permease seems to be related to optimal sporulation conditions such as a suitable supply of oxygen and cell concentration in the sporulating cultures, and occurs in the fully derepressed conditions of incubation in the sporulation acetate medium. The inactivation of maltase and maltose permease under sporulation conditions in MAL constitutive strains suggests an alternative mechanism for the regulation of the MAL gene expression during the sporulation process.     

Continuous ethanol fermentation of lactose by a recombinant flocculating Saccharomyces cerevisiae strain.

Alcohol fermentation of lactose was investigated using a recombinant flocculating Saccharomyces cerevisiae, expressing the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus. Data on yeast fermentation and growth on a medium containing lactose as the sole carbon source are presented. In the range of studied lactose concentrations, total lactose consumption was observed with a conversion yield of ethanol close to the expected theoretical value. For the continuously operating bioreactor, an ethanol productivity of 11 g L(-1) h(-1) (corresponding to a feed lactose concentration of 50 g L(-1) and a dilution rate of 0.55 h(-1)) was obtained, which is 7 times larger than the continuous conventional systems. The system stability was confirmed by keeping it in operation for 6 months.     

纤维二糖磷酸化酶和纤维寡糖磷酸化酶的研究与应用

自然界中一些厌氧的纤维素降解菌能够产生纤维二糖磷酸化酶(Cellobiose Phosphorylase,CBP)和纤维寡糖磷酸化酶(Cellodextrin Phosphorylase,CDP)磷酸化裂解纤维二糖和纤维寡糖.CBP和CDP属于糖苷水解酶94家族(Glycoside Hydrolase Family 9...     

Engineering heterologous molybdenum-cofactor-biosynthesis and nitrate-assimilation pathways enables nitrate utilization by Saccharomyces cerevisiae

Metabolic capabilities of cells are not only defined by their repertoire of enzymes and metabolites, but also by availability of enzyme cofactors. The molybdenum cofactor (Moco) is widespread among eukaryotes but absent from the industrial yeast Saccharomyces cerevisiae. No less than 50 Moco-dependent enzymes covering over 30 catalytic activities have been described to date, introduction of a functional Moco synthesis pathway offers interesting options to further broaden the biocatalytic repertoire of S. cerevisiae. In this study, we identified seven Moco biosynthesis genes in the non-conventional yeast Ogataea parapolymorpha by SpyCas9-mediated mutational analysis and expressed them in S. cerevisiae. Functionality of the heterologously expressed Moco biosynthesis pathway in S. cerevisiae was assessed by co-expressing O. parapolymorpha nitrate-assimilation enzymes, including the Moco-dependent nitrate reductase. Following two-weeks of incubation, growth of the engineered S. cerevisiae strain was observed on nitrate as sole nitrogen source. Relative to the rationally engineered strain, the evolved derivatives showed increased copy numbers of the heterologous genes, increased levels of the encoded proteins and a 5-fold higher nitrate-reductase activity in cell extracts. Growth at nM molybdate concentrations was enabled by co-expression of a Chlamydomonas reinhardtii high-affinity molybdate transporter. In serial batch cultures on nitrate-containing medium, a non-engineered S. cerevisiae strain was rapidly outcompeted by the spoilage yeast Brettanomyces bruxellensis. In contrast, an engineered and evolved nitrate-assimilating S. cerevisiae strain persisted during 35 generations of co-cultivation. This result indicates that the ability of engineered strains to use nitrate may be applicable to improve competitiveness of baker's yeast in industrial processes upon contamination with spoilage yeasts.     

Isolation and characterization of lactose permease mutants with an enhanced recognition of maltose and diminished recognition of cellobiose

In the present study, lactose permease mutants were isolated which have an enhanced recognition toward maltose (an alpha-glucoside) and diminished recognition for cellobiose (a beta-glucoside). Nine mutants were isolated from a strain encoding a wild-type permease (pTE18) and nine from a strain encoding a mutant permease which recognizes maltose (pB15). All 18 mutants were subjected to DNA sequencing, and it was found that all mutations are single base substitutions within the lac Y gene effecting single amino acid substitutions within the protein. From the pTE18 parent, substitutions involved Tyr-236 to Phe or His; Ser-306 to Thr; and six independent mutants in which Ala-389 was changed to Pro. From pB15, Tyr-236 was changed to Phe or Asn, Ser-306 to Thr or Leu, Lys-319 to Asn, and His-322 to Tyr, Asn, or Gln. All 18 mutants exhibited enhanced recognition for maltose (compared with the pTE18 strain) and a diminished recognition for cellobiose. In addition, all mutants showed a diminished recognition toward beta-galactosides as well. The Phe-236, His-236, Leu-306, Asn-319, Tyr-322, Asn-322, and Gln-322 mutants were completely defective in the uphill accumulation of methyl-beta-D-thiogalactopyranoside whereas the Asn-236, Thr-306, and Pro-389 mutants could effectively accumulate methyl-beta-D-thiogalactopyranoside against a concentration gradient. The mutants obtained in this study, together with previous lactose permease mutants, tend to be found on transmembrane segments, and those which are on the same transmembrane segment are often found three or four amino acids away from each other. This pattern is consistent with a protein structure in which important amino acid side chains project from several transmembrane segments in such a way as to form a hydrophilic channel for the recognition and transport of H+ and galactosides. It is proposed that the mechanism for H+/lactose cotransport is consistent with a "flanking gate" model in which the protein contains a single recognition site for galactosides within the channel which is flanked on either side by gates.