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Corn stover is one of the most promising lignocellulosic biomass that can be utilized for producing 1,3-propanediol and 2,3-butanediol. The pretreatment and enzymatic hydrolysis steps are essential for the bioconversion of lignocellulosic biomass to diols. For optimizing the pretreatment step, temperature, time, and NaOH concentration were evaluated based on total sugar recovery. Enzymatic hydrolysis for cellulose and hemicellulose were investigated at different solid-to-liquid ratios. The optimum conditions were found to be alkaline pretreatment with 0.25 mol dm−3 NaOH for 1 h at 60 °C followed by enzymatic hydrolysis at 50 °C for 48 h, with a solid slurry concentration of 100 g dm−3. Under these conditions, conversion rates of 92.55% and 78.82% were obtained from glucan and xylan, respectively. Diol production from fermentable sugars was 14.8 g dm−3, with a conversion yield and productivity of 0.46 g g−1, and 0.98 g dm−3 h−1, respectively. Our results are similar for diol production obtained using pure sugars under the same conditions. Therefore, mild alkaline pretreatment of corn stover facilitates delignification, significantly improving the rate of enzymatic saccharification and sugar recovery.

Hexanoic acid can be used for diverse industrial applications and is a precursor for fine chemistry. Although some natural microorganisms have been screened and evolved to produce hexanoic acid, the construction of an engineered biosynthetic pathway for producing hexanoic acid in yeast has not been reported. Here we constructed hexanoic acid pathways in Kluyveromyces marxianus by integrating 5 combinations of seven genes (AtoB, BktB, Crt, Hbd, MCT1, Ter, and TES1), by which random chromosomal sites of the strain are overwritten by the new genes from bacteria and yeast. One recombinant strain, H4A, which contained AtoB, BktB, Crt, Hbd, and Ter, produced 154 mg/L of hexanoic acid from galactose as the sole substrate. However, the hexanoic acid produced by the H4A strain was re-assimilated during the fermentation due to the reverse activity of AtoB, which condenses two acetyl-CoAs into a single acetoacetyl-CoA. This product instability could be overcome by the replacement of AtoB with a malonyl CoA-acyl carrier protein transacylase (MCT1) from Saccharomyces cerevisiae. Our results suggest that Mct1 provides a slow but stable acetyl-CoA chain elongation pathway, whereas the AtoB-mediated route is fast but unstable. In conclusion, hexanoic acid was produced for the first time in yeast by the construction of chain elongation pathways comprising 5–7 genes in K. marxianus.

Functional expression of a cellodextrin transporter and an intracellular β-glucosidase from Neurospora crassa in Saccharomyces cerevisiae enables simultaneous co-fermentation of cellobiose and non-glucose sugars such as xylose. Here we investigate the functional role of aldose 1-epimerase (AEP) in engineered cellobiose utilization. One AEP (Gal10) and two putative AEPs (Yhr210c and Ynr071c sharing 50.6% and 51.0% amino acid identity with Gal10, respectively) were selected. Deletion of GAL10 led to complete loss of both AEP activity and cell growth on cellobiose, while GAL10 complementation restored the AEP activity and cell growth. In addition, deletion of YHR210C or YNR071C resulted in improved cellobiose utilization. These results suggest that the intracellular mutarotation of β-glucose to α-glucose might be a rate controlling step and Gal10 play a crucial role in cellobiose fermentation by engineered S. cerevisiae.

Amylosucrase (AS), maltooligosyltrehalose synthase (MTS), and maltooligosyltrehalose trehalohydrolase (MTH) were used in combined cross-linked enzyme aggregates (combi-CLEAs) to achieve one-step bioconversion of sucrose to trehalose. Combi-CLEAs of three enzymes were successfully established with acetone and glutaraldehyde (GA) as a precipitant and a cross-linker, respectively. The optimum enzyme ratio was 8:0.5:0.5 (AS, 4 mg:MTS, 0.25 mg:MTH, 0.25 mg). To improve trehalose production, bovine serum albumin was co-aggregated with enzymes as a proteic feeder. The trehalose production yield of combi-CLEAs was about 8% in each cycle on the basis of substrate added up to 400 mM. Finally, the combi-CLEAs used in this experiment showed reusability of about five cycles without any activity loss.

As Saccharomyces cerevisiae cannot utilize xylose as a carbon source, expression of XYL1 coding for xylose reductase (XR) from Scheffersomyces (Pichia) stipitis enabled production of xylitol from xylose with a high yield. However, insufficient supply of NAD(P)H for XR and inhibition of xylose uptake by glucose are identified as major constraints for achieving high xylitol productivity. To overcome these problems, we engineered S. cerevisiae capable of converting xylose into xylitol through simultaneous utilization of xylose and cellobiose. An engineered S. cerevisiae (D-10-BT) expressing XR, cellodextrin transporter (cdt-1) and intracellular β-glucosidase (gh1-1) produced xylitol via simultaneous utilization of cellobiose and xylose. The D-10-BT strain exhibited 40% higher volumetric xylitol productivity with co-consumption of cellobiose and xylose compared to sequential utilization of glucose and xylose. Furthermore, the overexpression of S. cerevisiae ALD6, IDP2, or S. stipitis ZWF1 coding for cytosolic NADP(+)-dependent dehydrogenases increased the intracellular NADPH availability of the D-10-BT strain, which resulted in a 37-63% improvement in xylitol productivity when cellobiose and xylose were co-consumed. These results suggest that co-utilization of cellobiose and xylose can lead to improved xylitol production through enhanced xylose uptake and efficient cofactor regeneration.

The gene encoding sucrose isomerase from Enterobacter sp. FMB-1 species (ESI) was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using a glycosylphosphatidylinositol (GPI) anchor attachment signal sequence. Fluorescence activated cell sorting (FACS) analysis and immunofluorescence microscopy confirmed the localization of ESI on the yeast cell surface. The displayed ESI (dESI) was stable at a broad range of temperatures (35-55 °C) and pHs (pH 5-7) with optimal temperature and pH at 45 °C and pH 7.0, respectively. In addition, the thermostability of the dESI was significantly enhanced compared with the recombinant ESI expressed in Escherichia coli. Biotransformation of sucrose to isomaltulose was observed in various ranges of substrate concentrations (50-250 mM) with a 6.4-7.4% conversion yield. It suggested that the bioconversion of sucrose to isomaltulose can be successfully performed by the dESI on the surface of host S. cerevisiae.

Xylose-fermenting Saccharomyces strains are needed for commercialization of ethanol production from lignocellulosic biomass. Engineered Saccharomyces cerevisiae strains expressing XYL1, XYL2 and XYL3 from Pichia stipitis, however, utilize xylose in an oxidative manner, which results in significantly lower ethanol yields from xylose as compared to glucose. As such, we hypothesized that reconfiguration of xylose metabolism from oxidative into fermentative manner might lead to efficient ethanol production from xylose. To this end, we generated a respiration-deficient (RD) mutant in order to enforce engineered S. cerevisiae to utilize xylose only through fermentative metabolic routes. Three different repeated-batch fermentations were performed to characterize characteristics of the respiration-deficient mutant. When fermenting glucose as a sole carbon source, the RD mutant exhibited near theoretical ethanol yields (0.46 g g(-1)) during repeated-batch fermentations by recycling the cells. As the repeated-batch fermentation progressed, the volumetric ethanol productivity increased (from 7.5 to 8.3 g L(-1)h(-1)) because of the increased biomass from previous cultures. On the contrary, the mutant showed decreasing volumetric ethanol productivities during the repeated-batch fermentations using xylose as sole carbon source (from 0.4 to 0.3 g L(-1)h(-1)). The mutant did not grow on xylose and lost fermenting ability gradually, indicating that the RD mutant cannot maintain a good fermenting ability on xylose as a sole carbon source. However, the RD mutant was capable of fermenting a mixture of glucose and xylose with stable yields (0.35 g g(-1)) and productivities (0.52 g L(-1)h(-1)) during the repeated-batch fermentation. In addition, ethanol yields from xylose during the mixed sugar fermentation (0.30 g g(-1)) were higher than ethanol yields from xylose as a sole carbon source (0.21 g g(-1)). These results suggest that a strategy for increasing ethanol yield through respiration-deficiency can be applied for the fermentation of lignocellulosic hydrolyzates containing glucose and xylose.

Arbutin (Ab, 4-hydroxyphenyl β-glucopyranoside) is a glycosylated hydroquinone known to prevent the formation of melanin by inhibiting tyrosinase. An arbutin-α-glucoside was synthesized by the transglycosylation reaction of amylosucrase (AS) of Deinococcus geothermalis (DGAS) using arbutin and sucrose as an acceptor and a donor, respectively. The maximum yield of the arbutin transglycosylation product was determined to be over 98% with a 1:0.5 molar ratio of donor and acceptor molecules (sucrose and arbutin), in 50 mM sodium citrate buffer pH 7 at 35 °C. TLC and HPLC analyses revealed that only one transglycosylation product was observed, supporting the result that the transglycosylation reaction of DGAS was very specific. The arbutin transglycosylation product was isolated by preparative recycling HPLC. The structural analyses using 13 C and 1 H NMR proved that the transglycosylated product was 4-hydroxyphenyl β-maltoside (Ab-α-glucoside), in which a glucose molecule was linked to arbutin via an α-(1 → 4)-glycosidic linkage.

Amylosucrase (ASase, EC 2.4.1.4) is a member of family 13 of the glycoside hydrolases that catalyze the synthesis of an alpha-(1-->4)-linked glucan polymer from sucrose instead of an expensive activated sugar, such as ADP- or UDP-glucose. Transglycosylation reactions mediated by the ASases of Deinococcus geothermalis (DGAS) and Neisseria polysaccharea (NPAS) were applied to the synthesis of salicin glycosides with sucrose serving as the glucopyranosyl donor and salicin as the acceptor molecule. Two salicin glycoside transfer products were detected by TLC and HPLC analyses. The synthesis of salicin glycosides was very efficient with NPAS with a yield of over 90%. In contrast, DGAS specifically synthesized only one salicin transglycosylation product. The transglycosylation products were identified as alpha-d-glucopyranosyl-(1-->4)-salicin (glucosyl salicin) and alpha-D-glucopyranosyl-(1-->4)-alpha-D-glucopyranosyl-(1-->4)-salicin (maltosyl salicin) by NMR analysis. The ratio between donor and acceptor had a significant effect on the type of product that resulted from the transglycosylation reaction. With more acceptors present in the reaction, more glucosyl salicin and less maltosyl salicin were synthesized.