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.     

Influence of the presence of Zymomonas anaerobia on the conversion of cellobiose, glucose, and xylose to ethanol by Clostridium saccharolyticum

To convert sugar mixtures containing cellobiose, glucose, and xylose to ethanol in a single step, the possibility of using a coculture consisting of Clostridium saccharolyticum and Zymomonas anaerobia was studied. In monoculture, C. saccharolyticum utilized all three sugars; however, it preferentially utilized glucose and produced acetic acid in addition to ethanol. The formation of acetic acid from the metabolism of glucose inhibited the growth of C. saccharolyticum and, consequently, the utilization of cellobiose and xylose. In monoculture, Z. anaerobia utilized glucose at a rate of 50 g/L day, but it did not ferment cellobiose or xylose. In coculture, Z. anaerobia converted most of the glucose to ethanol during the lag phase of growth of C. saccharolyticum, which then converted cellobiose and xylose to ethanol. The use of this coculture increased both the rate and the efficiency of the conversion of these three sugars to ethanol, and produced relatively small amounts of acetic acid.     

Co-fermentation of cellobiose and xylose using beta-glucosidase displaying diploid industrial yeast strain OC-2

The co-utilization of sugars, particularly xylose and glucose, during industrial fermentation is essential for economically feasible processes with high ethanol productivity. However, the major problem encountered during xylose/glucose co-fermentation is the lower consumption rate of xylose compared with that of glucose fermentation. Here, we therefore attempted to construct high xylose assimilation yeast by using industrial yeast strain with high β-glucosidase activity on the cell surface. We first constructed the triple auxotrophic industrial strain OC2-HUT and introduced four copies of the cell-surface-displaying β-glucosidase (BGL) gene and two copies of a xylose-assimilating gene into its genome to generate strain OC2-ABGL4Xyl2. It was confirmed that the introduction of multiple copies of the BGL gene increased the cell-surface BGL activity, which was also correlated to the observed increase in xylose-assimilating ability. The strain OC2-ABGL4Xyl2 was able to consume xylose during cellobiose/xylose co-fermentation (0.38 g/h/g-DW) more rapidly than during glucose/xylose co-fermentation (0.18 g/h/g-DW). After 48 h, 5.77% of the xylose was consumed despite the co-fermentation conditions, and the observed ethanol yield was 0.39 g-ethanol/g-total sugar. Our results demonstrate that a BGL-displaying and xylose-assimilating industrial yeast strain is capable of efficient xylose consumption during the co-fermentation with cellobiose. Due to its high performance for fermentation of mixtures of cellobiose and xylose, OC2-ABGL4Xyl2 does not require the addition of β-glucosidase and is therefore a promising yeast strain for cost-effective ethanol production from lignocellulosic biomass.     

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.     

Transport of xylose and glucose in the xylose-fermenting yeast Pichia stipitis

Summary A low-affinity and a high-affinity sylose proton symport operated simultaneously in both starved and non-starved cells of Pichia stipitis. Glucose competed with xylose for transport by the low-affinity system and inhibited xylose transport by the high-affinity system non-competitively. The low affinity system was subject to substrate inhibition when glucose but not when xylose was the substrate. The differences between the characteristics of monosaccharide transport by Pichia stipitis and its imperfect state, Candida shehatae, are discussed.     

Improved fermentation of cellobiose,glucose, and xylose to ethanol by Zymomonas anaerobia and a high ethanol tolerant strain of Clostridium saccharolyticum

Summary To improve single step conversion of sugar mixtures containing cellobiose, glucose, and xylose to ethanol by a coculture of Zymomonas anaerobia and Clostridium saccharolyticum, an ethanol tolerant mutant of C. saccharolyticum was obtained. The mutant obtained by the enrichment procedure was able to grow in the presence of 75 g·l^-1 ethanol, with improved ability to utilize cellobiose, and little or no change in its ability to convert xylose to ethanol. This mutant in coculture with Zymomonas anaerobia produced over 50 g·l^-1 ethanol in media containing 130 g·l^-1 total sugars comprising of 60% glucose, 20% cellobiose, and 20% xylose.Issued as NRCC No. 23936     

Engineered Escherichia coli capable of co-utilization of cellobiose and xylose

Natural ability to ferment the major sugars (glucose and xylose) of plant biomass is an advantageous feature of Escherichia coli in biofuel production. However, excess glucose completely inhibits xylose utilization in E. coli and decreases yield and productivity of fermentation due to sequential utilization of xylose after glucose. As an approach to overcome this drawback, E. coli MG1655 was engineered for simultaneous glucose (in the form of cellobiose) and xylose utilization by a combination of genetic and evolutionary engineering strategies. The recombinant E. coli was capable of utilizing approximately 6 g/L of cellobiose and 2 g/L of xylose in approximately 36 h, whereas wild-type E. coli was unable to utilize xylose completely in the presence of 6 g/L of glucose even after 75 hours. The engineered strain also co-utilized cellobiose with mannose or galactose; however, it was unable to metabolize cellobiose in the presence of arabinose and glucose. Successful cellobiose and xylose co-fermentation is a vital step for simultaneous saccharification and co-fermentation process and a promising step towards consolidated bioprocessing.     

Construction of a recombinant yeast strain converting xylose and glucose to ethanol

Candida shehatae gene xyl1 and Pichia stipitis gene xyl2, encoding xylose reductase (XR) and xylitol dehydrogenase (XD) respectively, were amplified by PCR. The genes xyl1 and xyl2 were placed under the control of promoter GAL in vector pYES2 to construct the recombinant expression vector pYES2-P12. Subsequently the vector pYES2-P12 was transformed into S. cerevisiae YS58 by LiAc to produce the recombinant yeast YS58-12. The alcoholic ferment indicated that the recombinant yeast YS58-12 could convert xylose to ethanol with the xylose consumption rate of 81.3%. __________ Translated from Microbiology, 2006, 33(3): 104–108 [译自:微生物学通报]     

Construction of a recombinant yeast strain converting xylose and glucose to ethanol

Candida shehatae gene xyll and Pichia stipitis gene xyl2,encoding xylose reductase (XR) and xylitol dehydrogenase (XD) respectively,were amplified by PCR.The genes xyl1 and xyl2 were placed under the control of promoter GAL in vector pYES2 to construct the recombinant expression vector pYES2-PI2.Subsequently the vector pYES2-P12 was transformed into S.cerevisiae YS58 by LiAc to produce the recombinant yeast YS58-12.The alcoholic ferment indicated that the recombinant yeast YS58-12 could convert xylose to ethanol with the xylose consumption rate of 81.3%.     

Three new beetle-associated yeast species in the Pichia guilliermondii clade

New yeasts in the Pichia guilliermondii clade were isolated from the digestive tract of basidiocarp-feeding members of seven families of Coleoptera. A molecular phylogeny and unique traits placed eight isolates in Candida fermentati and three undescribed taxa in the genus Candida. The new species and type strains are C. smithsonii (type strain NRRL Y-27642^T), C. athensensis (type strain NRRL Y-27644^T), and C. elateridarum (type strain NRRL Y-27647^T). Based on comparison of small-and large-subunit rDNA sequences, C. smithsonii and C. athensensis form a statistically well-supported subclade with P. guilliermondii, C. xestobii, and C. fermentati; C. elateridarum is basal to this subclade.     

Antifungal cellobiose lipid secreted by the epiphytic yeast Pseudozyma graminicola

The yeast Pseudozyma graminicola isolated from plants inhibited growth of almost all ascomycetes and basidiomycetes tested (over 270 species of ca. 100 genera) including pathogenic species. This yeast secreted a fungicidal agent, which was identified as a glycolipid composed of cellobiose residue with two O-substituents (acetyl and 3-hydroxycaproic acid) and 2,15,16-trihydroxypalmitic acid. The release of ATP from the glycolipid-treated cells indicated that this glycolipid impaired the permeability of the cytoplasmic membrane. Basidiomycetes were more sensitive to the cellobiose lipid than ascomycetes.     

Spathaspora brasiliensis sp. nov., Spathaspora suhii sp. nov., Spathaspora roraimanensis sp. nov. and Spathaspora xylofermentans sp. nov., four novel d-xylose-fermenting yeast species from Brazilian Amazonian forest

Four new d-xylose fermenting yeast species of the clade Spathaspora were recovered from rotting-wood samples in a region of Amazonian forest, Northern Brazil. Three species produced unconjugated asci with a single elongated ascospore with curved ends. These species are described as Spathaspora brasiliensis, Spathaspora suhii and Spathaspora roraimanensis. Two isolates of an asexually reproducing species belonging to the Spathaspora clade were also obtained and they are described as Spathaspora xylofermentans. All these species are able to ferment d-xylose during aerobic batch growth in rich YP (1 % yeast extract, 2 % peptone and 2 % D-xylose) medium, albeit with differing efficiencies. The type strains are Spathaspora brasiliensis sp. nov UFMG-HMD19.3 (=CBMAI 1425=CBS 12679), Spathaspora suhii sp. nov. UFMG-XMD16.2 (=CBMAI 1426=CBS 12680), Spathaspora roraimanensis sp. nov. UFMG-XMD23.2 (CBMAI 1427=CBS 12681) and Spathaspora xylofermentans sp. nov. UFMG-HMD23.3 (=CBMAI 1428=CBS 12682).     

Fermentation of glucose, lactose, galactose, mannitol, and xylose by bifidobacteria

For six strains of Bifidobacterium bifidum (Lactobacillus bifidus), fermentation balances of glucose, lactose, galactose, mannitol, and xylose were determined. Products formed were acetate, l(+)-lactate, ethyl alcohol, and formate. l(+)-Lactate dehydrogenase of all strains studied was found to have an absolute requirement for fructose-1,6-diphosphate. The phosphoroclastic enzyme could not be demonstrated in cell-free extracts. Cell suspensions fermented pyruvate to equimolar amounts of acetate and formate. Alcohol dehydrogenase was shown in cell-free extracts. Possible explanations have been suggested for the differences in fermentation balances found for different strains and carbon sources. By enzyme determinations, it was shown that bifidobacteria convert mannitol to fructose-6-phosphate by an inducible polyol dehydrogenase and fructokinase. For one strain of B. bifidum, molar growth yields of glucose, lactose, galactose, and mannitol were determined. The mean value of Y (ATP), calculated from molar growth yields and fermentation balances, was 11.3.