Won Heong Lee

Enhanced xylitol production through simultaneous co-utilization of cellobiose and xylose by engineered Saccharomyces cerevisiae

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.

Engineering of NADPH regenerators in Escherichia coli for enhanced biotransformation

Efficient regeneration of NADPH is one of the limiting factors that constrain the productivity of biotransformation processes. In order to increase the availability of NADPH for enhanced biotransformation by engineered Escherichia coli, modulation of the pentose phosphate pathway and amplification of the transhydrogenases system have been conventionally attempted as primary solutions. Recently, other approaches for stimulating NADPH regeneration during glycolysis, such as replacement of native glyceradehdye-3-phosphate dehydrogenase (GAPDH) with NADP-dependent GAPDH from Clostridium acetobutylicum and introduction of NADH kinase catalyzing direct phosphorylation of NADH to NADPH from Saccharomyces cerevisiae, were attempted and resulted in remarkable impacts on NADPH-dependent bioprocesses. This review summarizes several metabolic engineering approaches used for improving the NADPH regenerating capacity in engineered E. coli for whole-cell-based bioprocesses and discusses the key features and progress of those attempts.

Simultaneous saccharification and fermentation by engineered Saccharomyces cerevisiae without supplementing extracellular β-glucosidase.

Simultaneous saccharification and fermentation (SSF) has been considered a promising and economical process for cellulosic ethanol production. Further cost savings could be gained by reducing enzyme loading and engineering host strain for ethanol production. In this study, we demonstrate efficient ethanol production by SSF without supplementation of β-glucosidase using an engineered Saccharomyces cerevisiae strain expressing a cellodextrin transporter and an intracellular β-glucosidase from Neurospora crassa. Ethanol production profiles by the engineered yeast without supplementation of β-glucosidase and by a parental strain with supplementation of β-glucosidase were examined under various fermentation conditions. When initial cell mass concentrations were low, the traditional SSF with supplementation of β-glucosidase showed better ethanol production than SSF with the engineered strain without supplementing β-glucosidase. However, the engineered strain without supplementation of β-glucosidase showed almost the same or even better ethanol productivity than the parental strain with supplementation of β-glucosidase when initial cell mass concentrations were elevated. Our results suggest that efficient ethanol production by SSF could be achieved by engineered yeast capable of fermenting cellobiose without addition of extracellular β-glucosidase, leading to economic production of cellulosic ethanol.

Analysis of cellodextrin transporters from Neurospora crassa in Saccharomyces cerevisiae for cellobiose fermentation

Saccharomyces cerevisiae can be engineered to ferment cellodextrins produced by cellulases as a product of cellulose hydrolysis. Direct fermentation of cellodextrins instead of glucose is advantageous because glucose inhibits cellulase activity and represses the fermentation of non-glucose sugars present in cellulosic hydrolyzates. To facilitate cellodextrin utilization by S. cerevisiae, a fungal cellodextrin-utilizing pathway from Neurospora crassa consisting of a cellodextrin transporter and a cellodextrin hydrolase has been introduced into S. cerevisiae. Two cellodextrin transporters (CDT-1 and CDT-2) were previously identified in N. crassa, but their kinetic properties and efficiency for cellobiose fermentation have not been studied in detail. In this study, CDT-1 and CDT-2, which are hypothesized to transport cellodextrin with distinct mechanisms, were introduced into S. cerevisiae along with an intracellular β-glucosidase (GH1-1). Cellobiose transport assays with the resulting strains indicated that CDT-1 is a proton symporter while CDT-2 is a simple facilitator. A strain expressing CDT-1 and GH1-1 (DCDT-1G) showed faster cellobiose fermentation than the strain expressing CDT-2 and GH1-1 (DCDT-2G) under various culture conditions with different medium compositions and aeration levels. While CDT-2 is expected to have energetic benefits, the expression levels and kinetic properties of CDT-1 in S. cerevisiae appears to be optimum for cellobiose fermentation. These results suggest CDT-1 is a more effective cellobiose transporter than CDT-2 for engineering S. cerevisiae to ferment cellobiose.

Construction of an efficient xylose-fermenting diploid Saccharomyces cerevisiae strain through mating of two engineered haploid strains capable of xylose assimilation.

Saccharomyces cerevisiae can be engineered for xylose fermentation through introduction of wild type or mutant genes (XYL1/XYL1 (R276H), XYL2, and XYL3) coding for xylose metabolic enzymes from Scheffersomyces stipitis. The resulting engineered strains, however, often yielded undesirable phenotypes such as slow xylose assimilation and xylitol accumulation. In this study, we performed the mating of two engineered strains that exhibit suboptimal xylose-fermenting phenotypes in order to develop an improved xylose-fermenting diploid strain. Specifically, we obtained two engineered haploid strains (YSX3 and SX3). The YSX3 strain consumed xylose rapidly and produced a lot of xylitol. On the contrary, the SX3 strain consumed xylose slowly with little xylitol production. After converting the mating type of SX3 from alpha to a, the resulting strain (SX3-2) was mated with YSX3 to construct a heterozygous diploid strain (KSM). The KSM strain assimilated xylose (0.25gxyloseh(-1)gcells(-1)) as fast as YSX3 and accumulated a small amount of xylitol (0.03ggxylose(-1)) as low as SX3, resulting in an improved ethanol yield (0.27ggxylose(-1)). We found that the improvement in xylose fermentation by the KSM strain was not because of heterozygosity or genome duplication but because of the complementation of the two xylose-metabolic pathways. This result suggested that mating of suboptimal haploid strains is a promising strategy to develop engineered yeast strains with improved xylose fermenting capability.

Improved ethanol production by engineered Saccharomyces cerevisiae expressing a mutated cellobiose transporter during simultaneous saccharification and fermentation

Although simultaneous saccharification and fermentation (SSF) of cellulosic biomass can offer efficient hydrolysis of cellulose through alleviating feed-back inhibition of cellulases by glucose, supplementation of β-glucosidase is necessary because most fermenting microorganisms cannot utilize cellobiose. Previously, we observed that SSF of cellulose by an engineered Saccharomyces cerevisiae expressing a cellobiose transporter (CDT-1) and an intracellular β-glucosidase (GH1-1) without β-glucosidase could not be performed as efficiently as the traditional SSF with extracellular β-glucosidase. However, we improved the ethanol production from SSF of cellulose by employing a further engineered S. cerevisiae expressing a mutant cellobiose transporter [CDT-1 (F213L) exhibiting higher VMAX than CDT-1] and GH1-1 in this study. Furthermore, limitation of cellobiose formation by reducing the amounts of cellulases mixture in SSF could lead the further engineered strain to produce ethanol considerably better than the parental strain with β-glucosidase. Probably, better production of ethanol by the further engineered strain seemed to be due to a higher affinity to cellobiose, which might be attributed to not only 2-times lower Monod constant (KS) for cellobiose than KS of the parental strain for glucose but also 5-times lower KS than Michaelis-Menten constant (KM) of the extracellular β-glucosidase for glucose. Our results suggest that modification of the cellobiose transporter in the engineered yeast to transport lower level of cellobiose enables a more efficient SSF for producing ethanol from cellulose.

Evaluation of ethanol production activity by engineered saccharomyces cerevisiae fermenting cellobiose through the phosphorolytic pathway in simultaneous saccharification and fermentation of cellulose

In simultaneous saccharification and fermentation (SSF) for production of cellulosic biofuels, engineered Saccharomyces cerevisiae capable of fermenting cellobiose has provided several benefits, such as lower enzyme costs and faster fermentation rate compared with wild-type S. cerevisiae fermenting glucose. In this study, the effects of an alternative intracellular cellobiose utilization pathway-a phosphorolytic pathway based on a mutant cellodextrin transporter (CDT-1 (F213L)) and cellobiose phosphorylase (SdCBP)-was investigated by comparing with a hydrolytic pathway based on the same transporter and an intracellular β-glucosidase (GH1-1) for their SSF performances under various conditions. Whereas the phosphorolytic and hydrolytic cellobiose-fermenting S. cerevisiae strains performed similarly under the anoxic SSF conditions, the hydrolytic S. cerevisiae performed slightly better than the phosphorolytic S. cerevisiae under the microaerobic SSF conditions. Nonetheless, the phosphorolytic S. cerevisiae expressing the mutant CDT-1 showed better ethanol production than the glucose-fermenting S. cerevisiae with an extracellular β-glucosidase, regardless of SSF conditions. These results clearly prove that introduction of the intracellular cellobiose metabolic pathway into yeast can be effective on cellulosic ethanol production in SSF. They also demonstrate that enhancement of cellobiose transport activity in engineered yeast is the most important factor affecting the efficiency of SSF of cellulose.

Enhanced cellobiose fermentation by engineered Saccharomyces cerevisiae expressing a mutant cellodextrin facilitator and cellobiose phosphorylase

To efficiently ferment intermediate cellodextrins released during cellulose hydrolysis, Saccharomyces cerevisiae has been engineered by introduction of a heterologous cellodextrin utilizing pathway consisting of a cellodextrin transporter and either an intracellular β-glucosidase or a cellobiose phosphorylase. Among two types of cellodextrin transporters, the passive facilitator CDT-2 has not enabled better cellobiose fermentation than the active transporter CDT-1, which suggests that the CDT-2 might be engineered to provide energetic benefits over the active transporter in cellobiose fermentation. We attempted to improve cellobiose transporting activity of CDT-2 through laboratory evolution. Nine rounds of a serial subculture of S. cerevisiae expressing CDT-2 and cellobiose phosphorylase on cellobiose led to the isolation of an evolved strain capable of fermenting cellobiose to ethanol 10-fold faster than the original strain. After sequence analysis of the isolated CDT-2, a single point mutation on CDT-2 (N306I) was revealed to be responsible for enhanced cellobiose fermentation. Also, the engineered strain expressing the mutant CDT-2 with cellobiose phosphorylase showed a higher ethanol yield than the engineered strain expressing CDT-1 and intracellular β-glucosidase under anaerobic conditions, suggesting that CDT-2 coupled with cellobiose phosphorylase may be better choices for efficient production of cellulosic ethanol with the engineered yeast.