A bio-based economy has the potential to provide sustainable substitutes for petroleum-based products and new chemical building blocks for advanced materials. We previously engineered Saccharomyces cerevisiae for industrial production of the isoprenoid artemisinic acid for use in antimalarial treatments. Adapting these strains for biosynthesis of other isoprenoids such as β-farnesene (CH), a plant sesquiterpene with versatile industrial applications, is straightforward. However, S. cerevisiae uses a chemically inefficient pathway for isoprenoid biosynthesis, resulting in yield and productivity limitations incompatible with commodity-scale production. Here we use four non-native metabolic reactions to rewire central carbon metabolism in S. cerevisiae, enabling biosynthesis of cytosolic acetyl coenzyme A (acetyl-CoA, the two-carbon isoprenoid precursor) with a reduced ATP requirement, reduced loss of carbon to CO-emitting reactions, and improved pathway redox balance. We show that strains with rewired central metabolism can devote an identical quantity of sugar to farnesene production as control strains, yet produce 25% more farnesene with that sugar while requiring 75% less oxygen. These changes lower feedstock costs and dramatically increase productivity in industrial fermentations which are by necessity oxygen-constrained. Despite altering key regulatory nodes, engineered strains grow robustly under taxing industrial conditions, maintaining stable yield for two weeks in broth that reaches >15% farnesene by volume. This illustrates that rewiring yeast central metabolism is a viable strategy for cost-effective, large-scale production of acetyl-CoA-derived molecules.

The Arabidopsis thaliana resistance gene RPW8 triggers the hypersensitive response (HR) to restrict powdery mildew infection via the salicylic acid-dependent signaling pathway. To further understand how RPW8 signaling is regulated, we have conducted a genetic screen to identify mutations enhancing RPW8-mediated HR-like cell death (designated erh). Here, we report the isolation and characterization of the Arabidopsis erh1 mutant, in which the At2g37940 locus is knocked out by a T-DNA insertion. Loss of function of ERH1 results in salicylic acid accumulation, enhanced transcription of RPW8 and RPW8-dependent spontaneous HR-like cell death in leaf tissues, and reduction in plant stature. Sequence analysis suggests that ERH1 may encode the long-sought Arabidopsis functional homolog of yeast and protozoan inositolphosphorylceramide synthase (IPCS), which converts ceramide to inositolphosphorylceramide. Indeed, ERH1 is able to rescue the yeast aur1 mutant, which lacks the IPCS, and the erh1 mutant plants display reduced (;53% of wild type) levels of leaf IPCS activity, indicating that ERH1 encodes a plant IPCS. Consistent with its biochemical function, the erh1 mutation causes ceramide accumulation in plants expressing RPW8. These data reinforce the concept that sphingolipid metabolism (specifically, ceramide accumulation) plays an important role in modulating plant programmed cell death associated with defense.

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The sphingoid long chain bases (LCBs) and their phosphorylated derivatives (LCB-Ps) are important signaling molecules in eukaryotic organisms. The cellular levels of LCB-Ps are tightly controlled by the coordinated action of the LCB kinase activity responsible for their synthesis and the LCB-P phosphatase and lyase activities responsible for their catabolism. Although recent studies have implicated LCB-Ps as regulatory molecules in plants, in comparison with yeast and mammals, much less is known about their metabolism and function in plants. To investigate the functions of LCB-Ps in plants, we have undertaken the identification and characterization of Arabidopsis genes that encode the enzymes of LCB-P metabolism. In this study the Arabidopsis At1g27980 gene was shown to encode the only detectable LCB-P lyase activity in Arabidopsis. The LCB-P lyase activity was characterized, and mutant plant lines lacking the lyase were generated and analyzed. Whereas in other organisms loss of LCB-P lyase activity is associated with accumulation of high levels of LCB/LCB-Ps and developmental abnormalities, the sphingolipid profiles of the mutant plants were remarkably similar to those of wild-type plants, and no developmental abnormalities were observed. Thus, these studies indicate that the lyase plays a minor role in maintenance of sphingolipid metabolism during normal plant development and growth. However, a clear role for the lyase was revealed upon perturbation of sphingolipid synthesis by treatment with the inhibitor of ceramide synthase, fumonisin B 1 .Sphingolipids are ubiquitous membrane components that are critical for normal membrane function. Sphingolipid metabolites, including sphingoid long chain bases (LCBs), 3 phosphorylated LCBs (LCB-Ps), and ceramides, also function as signaling molecules in eukaryotic cells (1-4). For example, sphingosine 1-phosphate, a key sphingolipid second messenger, regulates proliferation, invasiveness, and programmed cell death. These effects of LCB-Ps have been observed in organisms as diverse as yeast and humans (5). Although far less is known about sphingolipid functions in plants (6, 7), recent studies indicate that sphingolipid-derived metabolites also act as signaling molecules in plants. For example, sphingosine 1-phosphate and more recently phytosphingosine 1-phosphate have been implicated in abscisic aciddependent guard cell closure through a G-protein-mediated pathway (8 -10). In addition, disruption of a ceramide kinase gene has been found to cause enhanced apoptosis in the Arabidopsis ACD5 mutant, suggesting that ceramides regulate programmed cell death in plants (11). Similarly, inhibition of the ceramide synthase step of sphingolipid biosynthesis by the fungal toxins fumonisin and Alternaria alternata f.sp. lycopersici toxin has also been shown to promote programmed cell death (12-15).Despite their possible importance as signaling molecules, the synthesis and metabolism of LCB-Ps in plants and the role of LCB-Ps in plant physiology remain largely unknown. It is clear tha...

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For commercial protein therapeutics, Chinese hamster ovary (CHO) cells have an established history of safety, proven capability to express a wide range of therapeutic proteins and high volumetric productivities. Expanding global markets for therapeutic proteins and increasing concerns for broadened access of these medicines has catalyzed consideration of alternative approaches to this platform. Reaching these objectives likely will require an order of magnitude increase in volumetric productivity and a corresponding reduction in the costs of manufacture. For CHO‐based manufacturing, achieving this combination of targeted improvements presents challenges. Based on a holistic analysis, the choice of host cells was identified as the single most influential factor for both increasing productivity and decreasing costs. Here we evaluated eight wild‐type eukaryotic micro‐organisms with prior histories of recombinant protein expression. The evaluation focused on assessing the potential of each host, and their corresponding phyla, with respect to key attributes relevant for manufacturing, namely (a) growth rates in industry‐relevant media, (b) adaptability to modern techniques for genome editing, and (c) initial characterization of product quality. These characterizations showed that multiple organisms may be suitable for production with appropriate engineering and development and highlighted that yeast in general present advantages for rapid genome engineering and development cycles.

Platelets were labelled separately with six different, radioactive unsaturated fatty acids. The cells were isolated from the radioactive precursors and treated with and without 2 U/ml of thrombin. The formation of radioactive free fatty acid+oxygenated fatty acids and of radioactive radioactive phosphatidic acid+diacylglycerol was taken as a measure of the PLA(2) and PLC reactions, respectively. We found that that in intact platelets PLA(2) prefers phospholipid molecular species containing unsaturated acyls, most likely in the sn-2 position, in the priority order: 20:4>20:5>18:2 = 18:3 = 22:6>>18:1, while PLC prefers its substrates in the priority order 20:5>20:4>18:2>18:3 = 22:6>18:1.