A genetic overhaul of Saccharomyces cerevisiae 424A(LNH-ST) to improve xylose fermentation.

Robust microorganisms are necessary for economical bioethanol production. However, such organisms must be able to effectively ferment both hexose and pentose sugars present in lignocellulosic hydrolysate to ethanol. Wild type Saccharomyces cerevisiae can rapidly ferment hexose, but cannot ferment pentose sugars. Considerable efforts were made to genetically engineer S. cerevisiae to ferment xylose. Our genetically engineered S cerevisiae yeast, 424A(LNH-ST), expresses NADPH/NADH xylose reductase (XR) that prefer NADPH and NAD(+)-dependent xylitol dehydrogenase (XD) from Pichia stipitis, and overexpresses endogenous xylulokinase (XK). This strain is able to ferment glucose and xylose, as well as other hexose sugars, to ethanol. However, the preference for different cofactors by XR and XD might lead to redox imbalance, xylitol excretion, and thus might reduce ethanol yield and productivity. In the present study, genes responsible for the conversion of xylose to xylulose with different cofactor specificity (1) XR from N. crassa (NADPH-dependent) and C. parapsilosis (NADH-dependent), and (2) mutant XD from P. stipitis (containing three mutations D207A/I208R/F209S) were overexpressed in wild type yeast. To increase the NADPH pool, the fungal GAPDH enzyme from Kluyveromyces lactis was overexpressed in the 424A(LNH-ST) strain. Four pentose phosphate pathway (PPP) genes, TKL1, TAL1, RKI1 and RPE1 from S. cerevisiae, were also overexpressed in 424A(LNH-ST). Overexpression of GAPDH lowered xylitol production by more than 40%. However, other strains carrying different combinations of XR and XD, as well as new strains containing the overexpressed PPP genes, did not yield any significant improvement in xylose fermentation.

Effect of acetic acid and pH on the cofermentation of glucose and xylose to ethanol by a genetically engineered strain of Saccharomyces cerevisiae.

A current challenge of the cellulosic ethanol industry is the effect of inhibitors present in biomass hydrolysates. Acetic acid is an example of one such inhibitor that is released during the pretreatment of hemicellulose. This study examined the effect of acetic acid on the cofermentation of glucose and xylose under controlled pH conditions by Saccharomyces cerevisiae 424A(LNH-ST), a genetically engineered industrial yeast strain. Acetic acid concentrations of 7.5 and 15 g L(-1), representing the range of concentrations expected in actual biomass hydrolysates, were tested under controlled pH conditions of 5, 5.5, and 6. The presence of acetic acid in the fermentation media led to a significant decrease in the observed maximum cell biomass concentration. Glucose- and xylose-specific consumption rates decreased as the acetic acid concentration increased, with the inhibitory effect being more severe for xylose consumption. The ethanol production rates also decreased when acetic acid was present, but ethanol metabolic yields increased under the same conditions. The results also revealed that the inhibitory effect of acetic acid could be reduced by increasing media pH, thus confirming that the undissociated form of acetic acid is the inhibitory form of the molecule.

Establishment of L-arabinose fermentation in glucose/xylose co-fermenting recombinant Saccharomyces cerevisiae 424A(LNH-ST) by genetic engineering.

Cost-effective and efficient ethanol production from lignocellulosic materials requires the fermentation of all sugars recovered from such materials including glucose, xylose, mannose, galactose, and L-arabinose. Wild-type strains of Saccharomyces cerevisiae used in industrial ethanol production cannot ferment D-xylose and L-arabinose. Our genetically engineered recombinant S. cerevisiae yeast 424A(LNH-ST) has been made able to efficiently ferment xylose to ethanol, which was achieved by integrating multiple copies of three xylose-metabolizing genes. This study reports the efficient anaerobic fermentation of L-arabinose by the derivative of 424A(LNH-ST). The new strain was constructed by over-expression of two additional genes from fungi L-arabinose utilization pathways. The resulting new 424A(LNH-ST) strain exhibited production of ethanol from L-arabinose, and the yield was more than 40%. An efficient ethanol production, about 72.5% yield from five-sugar mixtures containing glucose, galactose, mannose, xylose, and arabinose was also achieved. This co-fermentation of five-sugar mixture is important and crucial for application in industrial economical ethanol production using lignocellulosic biomass as the feedstock.

Biologic activity of spores and dried powder from Ganoderma lucidum for the inhibition of highly invasive human breast and prostate cancer cells.

OBJECTIVE: Ganoderma lucidum has been used in East Asia as a home remedy to prevent or cure cancer. Furthermore, Ganoderma lucidum is one of the herbs in the herbal mixture PC-SPES that has become an alternative herbal therapy for prostate cancer. Because the dried powder of ganoderma is commercially available as a dietary supplement itself, the purpose of this study was to evaluate the biologic activity of samples of Ganoderma lucidum from different sources. METHODS: Samples of Ganoderma lucidum were characterized morphologically and evaluated for their ability to inhibit cell migration of highly invasive breast cancer MDA-MB-231 cells and prostate cancer PC-3 cells. Because the inhibition of cell motility is directly linked to the inhibition of the signaling pathway for constitutively active NF-kappaB in breast and prostate cancer cells, we determined how different samples of Ganoderma lucidum inhibit constitutively active NF-kappaB in a reporter gene assay. RESULTS: Some of the samples of Ganoderma lucidum demonstrated strong inhibition of cancer cell migration comparable to the inhibition of constitutively active NF-kappaB, whereas other samples showed less or no activity in highly invasive estrogen receptor-negative breast cancer cells or androgen receptor-negative prostate cancer cells, respectively. Interestingly, we did not find any correlation between the purity and composition (spores versus powder) of Ganoderma lucidum and biologic activity. CONCLUSIONS: Ganoderma lucidum has demonstrated strong activity against breast and prostate cancer cells. Nevertheless, the composition of samples did not correlate with their ability to inhibit cell migration and activation of NF-kappaB in vitro.

Production of ethanol from cellulosic biomass hydrolysates using genetically engineered Saccharomyces yeast capable of cofermenting glucose and xylose.

Recent studies have proven ethanol to be the ideal liquid fuel for transportation, and renewable lignocellulosic materials to be the attractive feedstocks for ethanol fuel production by fermentation. The major fermentable sugars from hydrolysis of most cellulosic biomass are D-glucose and D-xylose. The naturally occurring Saccharomyces yeasts that are used by industry to produce ethanol from starches and cane sugar cannot metabolize xylose. Our group at Purdue University succeeded in developing genetically engineered Saccharomyces yeasts capable of effectively cofermenting glucose and xylose to ethanol, which was accomplished by cloning three xylose-metabolizing genes into the yeast. In this study, we demonstrated that our stable recombinant Saccharomyces yeast, 424A(LNH-ST), which contains the cloned xylose-metabolizing genes stably integrated into the yeast chromosome in high copy numbers, can efficiently ferment glucose and xylose present in hydrolysates from different cellulosic biomass to ethanol.

Bioethanol from lignocellulosic biomass.

China is suffering from a sustained shortage of crude oil supply, making fuel ethanol and other biofuels alternative solutions for this issue. However, taking into account the country's large population and dwindling arable land due to rapid urbanization, it is apparent that current fuel ethanol production from grain-based feedstocks is not sustainable, and lignocellulosic biomass, particularly agricultural residues that are abundantly available in China, is the only choice for China to further expand its fuel ethanol production, provided economically viable processes can be developed. In this chapter, cutting edge progress in bioethanol is reviewed, with a focus on the understanding of the molecular structure of the feedstock, leading pretreatment technologies, enzymatic hydrolysis of the cellulose component and strategies for the co-fermentation of the C5 and C6 sugars with engineered microorganisms. Finally, process integration and optimization is addressed with a case study on the COFCO Corporation's pilot plant, and challenges and perspectives for commercial production of bioethanol are highlighted.

Characterization of the effectiveness of hexose transporters for transporting xylose during glucose and xylose co-fermentation by a recombinant Saccharomyces yeast.

We have developed recombinant Saccharomyces yeasts that can effectively co-ferment glucose and xylose to ethanol. However, these yeasts still ferment glucose more efficiently than xylose. The transport of xylose could be one of the steps limiting the fermentation of xylose. In this study, we characterized the changes in the expression pattern of the hexose transporter and related genes during co-fermentation of glucose and xylose using one of our recombinant yeasts, Saccharomyces cerevisiae 424A(LNH-ST). The transcription of the hexose transporter and related genes was strongly influenced by the presence of glucose; HXT1, HXT2 and HXT3 were greatly activated by glucose and HXT5, HXT7 and AGT1 were significantly repressed by glucose. We also examined the effectiveness of individual transporters encoded by HXT1, HXT2, HXT4, HXT5, HXT7 and GAL2 genes for transporting xylose during co-fermentation of glucose and xylose in a Saccharomyces hxt degrees mutant (RE700A). We compared these hxt degrees derivatives to RE700A wild-type strain (S. cerevisiae MC996A) where all of them contained the same xylose metabolizing genes present in our xylose-fermenting yeasts such as 424A(LNH-ST). Our results showed that recombinant RE700A containing the cloned HXT7 or HXT5 were substantially more effective for fermenting xylose to ethanol. In addition, we found that the efficiency of transporters for intracellular accumulation of xylose was as follows: HXT7 > HXT5 > GAL2 > WT > HXT1 > HXT4 > > > RE700A. Furthermore, we provided evidence that the Saccharomyces galactose transporter system could be a highly effective xylose transporter. The information reported here should be of great importance for improving the Saccharomyces yeast transport of xylose.

Ganoderma lucidum suppresses motility of highly invasive breast and prostate cancer cells.

A dried powder from basidiomycetous fungi, Ganoderma lucidum, has been used in East Asia in therapies for several different diseases, including cancer. However, the molecular mechanisms involved in the biological actions of Ganoderma are not well understood. We have recently demonstrated that phosphatidylinositol 3-kinase (PI 3-kinase) and nuclear factor-kappaB (NF-kappaB) regulate motility of highly invasive human breast cancer cells by the secretion of urokinase-type plasminogen activator (uPA). In this study, we investigated the effect of G. lucidum on highly invasive breast and prostate cancer cells. Here we show that spores or dried fruiting body of G. lucidum inhibit constitutively active transcription factors AP-1 and NF-kappaB in breast MDA-MB-231 and prostate PC-3 cancer cells. Furthermore, Ganoderma inhibition of expression of uPA and uPA receptor (uPAR), as well secretion of uPA, resulted in the suppression of the migration of MDA-MB-231 and PC-3 cells. Our data suggest that spores and unpurified fruiting body of G. lucidum inhibit invasion of breast and prostate cancer cells by a common mechanism and could have potential therapeutic use for cancer treatment.