No abstract

Economic bioconversion of plant cell wall hydrolysates into fuels and chemicals has been hampered mainly due to the inability of microorganisms to efficiently co-ferment pentose and hexose sugars, especially glucose and xylose, which are the most abundant sugars in cellulosic hydrolysates. Saccharomyces cerevisiae cannot metabolize xylose due to a lack of xylose-metabolizing enzymes. We developed a rapid and efficient xylose-fermenting S. cerevisiae through rational and inverse metabolic engineering strategies, comprising the optimization of a heterologous xylose-assimilating pathway and evolutionary engineering. Strong and balanced expression levels of the XYL1, XYL2, and XYL3 genes constituting the xylose-assimilating pathway increased ethanol yields and the xylose consumption rates from a mixture of glucose and xylose with little xylitol accumulation. The engineered strain, however, still exhibited a long lag time when metabolizing xylose above 10 g/l as a sole carbon source, defined here as xylose toxicity. Through serial-subcultures on xylose, we isolated evolved strains which exhibited a shorter lag time and improved xylose-fermenting capabilities than the parental strain. Genome sequencing of the evolved strains revealed that mutations in PHO13 causing loss of the Pho13p function are associated with the improved phenotypes of the evolved strains. Crude extracts of a PHO13-overexpressing strain showed a higher phosphatase activity on xylulose-5-phosphate (X-5-P), suggesting that the dephosphorylation of X-5-P by Pho13p might generate a futile cycle with xylulokinase overexpression. While xylose consumption rates by the evolved strains improved substantially as compared to the parental strain, xylose metabolism was interrupted by accumulated acetate. Deletion of ALD6 coding for acetaldehyde dehydrogenase not only prevented acetate accumulation, but also enabled complete and efficient fermentation of xylose as well as a mixture of glucose and xylose by the evolved strain. These findings provide direct guidance for developing industrial strains to produce cellulosic fuels and chemicals.

The dengue virus (DENV) NS3 protein is essential for viral polyprotein processing and RNA replication. It contains an N-terminal serine protease region (residues 1-168) joined to an RNA helicase (residues 180 -618) by an 11-amino acid linker (169 -179). The structure at 3.15 Å of the soluble NS3 protein from DENV4 covalently attached to 18 residues of the NS2B cofactor region (NS2B 18 NS3) revealed an elongated molecule with the protease domain abutting subdomains I and II of the helicase (Luo, D., Xu, T., Hunke, C., Grüber, G., Vasudevan, S. G., and Lescar, J. (2008) J. Virol. 82, 173-183). Unexpectedly, using similar crystal growth conditions, we observed an alternative conformation where the protease domain has rotated by ϳ161°with respect to the helicase domain. We report this new crystal structure bound to ADP-Mn 2؉ refined to a resolution of 2.2 Å . The biological significance for interdomain flexibility conferred by the linker region was probed by either inserting a Gly residue between Glu 173 and Pro 174 or replacing Pro 174 with a Gly residue. Both mutations resulted in significantly lower ATPase and helicase activities. We next increased flexibility in the linker by introducing a Pro 176 to Gly mutation in a DENV2 replicon system. A 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis were observed. Our results indicate that the linker region has evolved to an optimum length to confer flexibility to the NS3 protein that is required both for polyprotein processing and RNA replication. Dengue virus (DENV)4 is an important vector-borne virus that causes a spectrum of illnesses in humans ranging from asymptomatic to severe disease, including dengue hemorrhagic fever and dengue shock syndrome. More than half of the ϳ70 members of the flavivirus genus that includes the four serotypes of DENV (DENV1-4), yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, and West Nile virus, are important human pathogens (1). The positive-sense flavivirus RNA genome of ϳ11 kb contains a single open reading frame that is translated into a polyprotein precursor, consisting of the structural proteins C, prM, and E and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. During viral replication, the polyprotein is cleaved by host proteases in the endoplasmic reticulum lumen and viral proteases at the cytoplasmic face (1). The NS3 protein (618 amino acids in DENV4) contains a serine-protease domain at its N terminus (whose activity requires the formation of a noncovalent complex with the central 40-residue hydrophilic segment of the membrane-bound NS2B protein cofactor) and an ATP-driven helicase and RNA triphosphatase at its C-terminal end. Atomic structures for the active NS3 protease domain (NS2B 47 NS3pro) (2-4), the ATPase/helicase domain (NS3hel) (5-9), and the full-length NS3 molecule fused to 18 residues of the NS2B cofactor (NS2B 18 NS3) (10) have been reported and reviewed (11). Together, these structures provide an explana...

New mosquito control strategies are vitally needed to address established arthropod-borne infectious diseases such as dengue and yellow fever and emerging diseases such as Zika and chikungunya, all of which are transmitted by the disease vector mosquito Aedes aegypti. In this investigation, Saccharomyces cerevisiae (baker’s yeast) was engineered to produce short hairpin RNAs (shRNAs) corresponding to the Aedes aegypti orthologs of fasciculation and elongation protein zeta 2 (fez2) and leukocyte receptor cluster (lrc) member, two genes identified in a recent screen for A. aegypti larval lethal genes. Feeding A. aegypti with the engineered yeasts resulted in silenced target gene expression, disrupted neural development, and highly significant larval mortality. Larvicidal activities were retained following heat inactivation and drying of the yeast into tabular formulations that induced >95% mortality and were found to attract adult females to oviposit. These ready-to-use inactivated yeast interfering RNA tablets may one day facilitate the seamless integration of this new class of lure-and-kill species-specific biorational mosquito larvicides into integrated mosquito control programs.

The anticipation for substituting conventional fossil fuels with cellulosic biofuels is growing in the face of increasing demand for energy and rising concerns of greenhouse gas emissions. However, commercial production of cellulosic biofuel has been hampered by inefficient fermentation of xylose and the toxicity of acetic acid, which constitute substantial portions of cellulosic biomass. Here we use a redox balancing strategy to enable efficient xylose fermentation and simultaneous in situ detoxification of cellulosic feedstocks. By combining a nicotinamide adenine dinucleotide (NADH)-consuming acetate consumption pathway and an NADH-producing xylose utilization pathway, engineered yeast converts cellulosic sugars and toxic levels of acetate together into ethanol under anaerobic conditions. The results demonstrate a breakthrough in making efficient use of carbon compounds in cellulosic biomass and present an innovative strategy for metabolic engineering whereby an undesirable redox state can be exploited to drive desirable metabolic reactions, even improving productivity and yield.