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Living organisms carry out a wide range of remarkable functions, including the synthesis of thousands of simple and complex chemical structures for cellular growth and maintenance. The manipulation of this reaction network has allowed for the genetic engineering of cells for targeted chemical synthesis, but it remains challenging to alter the program underlying their fundamental chemical behavior. By taking advantage of the unique ability of living systems to use evolution to find solutions to complex problems, we have achieved ~95% theoretical yield of three C4commodity chemicals,n-butanol, 1,3-butanediol, and 4-hydroxy-2-butanone. Genomic sequencing of the evolved strains identifiedpcnBandrpoBCas two gene loci that are able to alter carbon flow by remodeling the transcriptional landscape of the cell, highlighting the potential of synthetic pathways as a tool to identify metabolic control points.

Targeting of most integral membrane proteins to the endoplasmic reticulum is controlled by the signal recognition particle, which recognizes a hydrophobic signal sequence near the protein N terminus. Proper folding of these proteins is monitored by the unfolded protein response and involves protein degradation pathways to ensure quality control. Here, we identify a new pathway for quality control of major facilitator superfamily transporters that occurs before the first transmembrane helix, the signal sequence recognized by the signal recognition particle, is made by the ribosome. Increased rates of translation elongation of the N-terminal sequence of these integral membrane proteins can divert the nascent protein chains to the ribosome-associated complex and stress-seventy subfamily B chaperones. We also show that quality control of integral membrane proteins by ribosome-associated complex-stress-seventy subfamily B couples translation rate to the unfolded protein response, which has implications for understanding mechanisms underlying human disease and protein production in biotechnology.

The economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.

Atovaquone is a front-line antimalarial drug that functions by competitively inhibiting binding of coenzyme Q10 to the cytochrome bc1 complex. Atovaquone is administered orally, but has low solubility and is poorly absorbed with high variability in bioavailability. In vivo binding of human serum albumin has been cited as the major transporter of atovaquone in plasma. The research presented herein demonstrates that saposin B, a known binder/transporter of coenzyme Q10, also binds to atovaquone in a 1 : 1 ratio and with comparably high affinity at pH 5.5.

The use of coenzyme Q10 (CoQ10) has been increasing rapidly during recent years due to its postulated beneficial properties in human health, providing energy and antioxidant protection. There are no known negative side effects of CoQ10 even at very high levels. Recently, native saposin B (sapB) has been shown to bind CoQ10 and subsequently be excreted. It is thought that this interaction between sapB and CoQ10 could be a mechanism to avoid any possible CoQ10 toxicity. The interaction between sapB and CoQ10 is poorly understood. Here we present an increased fermentative yield of recombinant sapB and demonstrate that recombinant sapB will bind CoQ10 in a pH-dependent manner similar to sapB binding with other lipids. SapB was coated onto an IMAC (immobilized metal affinity chromatography) resin and successfully bound CoQ10 at pH 5.0 with release of the CoQ10 at pH 9.0.

Background Economical production of fuels and chemicals from plant biomass requires the efficient use of sugars derived from the plant cell wall. Neurospora crassa, a model lignocellulosic degrading fungus, is capable of breaking down the complex structure of the plant cell wall. In addition to cellulases and hemicellulases, N. crassa secretes lytic polysaccharide monooxygenases (LPMOs), which cleave cellulose by generating oxidized sugars—particularly aldonic acids. However, the strategies N. crassa employs to utilize these sugars are unknown. Results We identified an aldonic acid utilization pathway in N. crassa, comprised of an extracellular hydrolase (NCU08755), cellobionic acid transporter (CBT-1, NCU05853) and cellobionic acid phosphorylase (CAP, NCU09425). Extracellular cellobionic acid could be imported directly by CBT-1 or cleaved to gluconic acid and glucose by a β-glucosidase (NCU08755) outside the cells. Intracellular cellobionic acid was further cleaved to glucose 1-phosphate and gluconic acid by CAP. However, it remains unclear how N. crassa utilizes extracellular gluconic acid. The aldonic acid pathway was successfully implemented in Saccharomyces cerevisiae when N. crassa gluconokinase was co-expressed, resulting in cellobionic acid consumption in both aerobic and anaerobic conditions. Conclusions We successfully identified a branched aldonic acid utilization pathway in N. crassa and transferred its essential components into S. cerevisiae, a robust industrial microorganism. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0303-2) contains supplementary material, which is available to authorized users.

Sustainable biofuel production from renewable biomass will require the efficient and complete use of all abundant sugars in the plant cell wall. Using the cellulolytic fungus Neurospora crassa as a model, we identified a xylodextrin transport and consumption pathway required for its growth on hemicellulose. Reconstitution of this xylodextrin utilization pathway in Saccharomyces cerevisiae revealed that fungal xylose reductases act as xylodextrin reductases, producing xylosyl-xylitol oligomers as metabolic intermediates. These xylosyl-xylitol intermediates are generated by diverse fungi and bacteria, indicating that xylodextrin reduction is widespread in nature. Xylodextrins and xylosyl-xylitol oligomers are then hydrolyzed by two hydrolases to generate intracellular xylose and xylitol. Xylodextrin consumption using a xylodextrin transporter, xylodextrin reductases and tandem intracellular hydrolases in cofermentations with sucrose and glucose greatly expands the capacity of yeast to use plant cell wall-derived sugars and has the potential to increase the efficiency of both first-generation and next-generation biofuel production. DOI: http://dx.doi.org/10.7554/eLife.05896.001, eLife digest Plants can be used to make ‘biofuels’, which are more sustainable alternatives to traditional fuels made from petroleum. Unfortunately, most biofuels are currently made from simple sugars or starch extracted from parts of plants that we also use for food, such as the grains of cereal crops. Making biofuels from the parts of the plant that are not used for food—for example, the stems or leaves—would enable us to avoid a trade-off between food and fuel production. However, most of the sugars in these parts of the plant are locked away in the form of large, complex carbohydrates called cellulose and hemicellulose, which form the rigid cell wall surrounding each plant cell. Currently, the industrial processes that can be used to make biofuels from plant cell walls are expensive and use a lot of energy. They involve heating or chemically treating the plant material to release the cellulose and hemicellulose. Then, large quantities of enzymes are added to break these carbohydrates down into simple sugars that can then be converted into alcohol (a biofuel) by yeast. Fungi may be able to provide us with a better solution. Many species are able to grow on plants because they can break down cellulose and hemicellulose into simple sugars they can use for energy. If the genes involved in this process could be identified and inserted into yeast it may provide a new, cheaper method to make biofuels from plant cell walls. To address this challenge, Li et al. studied how the fungus Neurospora crassa breaks down hemicellulose. This study identified a protein that can transport molecules of xylodextrin—which is found in hemicellulose—into the cells of the fungus, and two enzymes that break down the xylodextrin to make simple sugars, using a previously unknown chemical intermediate. When Li et al. inserted the genes that make the transport protein and the enzymes into yeast, the yeast were able to use plant cell wall material to make simple sugars and convert these to alcohol. The yeast used more of the xylodextrin when they were grown with an additional source of energy, such as the sugars glucose or sucrose. Li et al.'s findings suggest that giving yeast the ability to break down hemicellulose has the potential to improve the efficiency of biofuel production. The next challenge will be to improve the process so that the yeast can convert the xylodextrin and simple sugars more rapidly. DOI: http://dx.doi.org/10.7554/eLife.05896.002

The economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.

Sustainable biofuel production from renewable biomass will require the efficient and complete use of all abundant sugars in the plant cell wall. Using the cellulolytic fungusNeurospora crassaas a model, we identified a xylodextrin transport and consumption pathway required for its growth on hemicellulose. Successful reconstitution of this xylodextrin utilization pathway inSaccharomyces cerevisiaerevealed that fungal xylose reductases act as xylodextrin reductases, and together with two hydrolases, generate intracellular xylose and xylitol. Xylodextrin consumption using xylodextrin reductases and tandem intracellular hydrolases greatly expands the capacity of yeasts to use plant cell wall-derived sugars, and should be adaptable to increase the efficiency of both first-generation and next-generation biofuel production.

Altering the hardwired stoichiometry of central metabolism in yeast enables efficient synthesis of the isoprenoid β-farnesene.