Engineering and two-stage evolution of a lignocellulosic hydrolysate-tolerant Saccharomyces cerevisiae strain for anaerobic fermentation of xylose from AFEX pretreated corn stover.

The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen.

Universal genetic assay for engineering extracellular protein expression.

A variety of strategies now exist for the extracellular expression of recombinant proteins using laboratory strains of Escherichia coli . However, secreted proteins often accumulate in the culture medium at levels that are too low to be practically useful for most synthetic biology and metabolic engineering applications. The situation is compounded by the lack of generalized screening tools for optimizing the secretion process.

Isolation of improved free fatty acid overproducing strains of via Nile red based high-throughput screening.

Biological production of hydrocarbons is an attractive strategy to produce drop-in replacement transportation fuels. Several methods for converting microbially-produced fatty acids into reduced compounds compatible with petrodiesel have been reported. For these processes to become economically viable, microorganisms must be engineered to approach the theoretical yield of fatty acid products from renewable feedstocks such as glucose.

Development of an automated platform for high-throughput P1-phage transduction of Escherichia coli.

Synthetic biology depends on the ability to rapidly produce strains with improved phenotypes but is limited by the ability to rapidly produce strain collections with directed mutations. Here, we present a system capable of overcoming this limitation through automated P1-phage transductions of Escherichia coli. By combining the Keio collection of single-gene deletion E.

Chemical screening method for the rapid identification of microbial sources of marine invertebrate-associated metabolites.

Marine invertebrates have proven to be a rich source of secondary metabolites. The growing recognition that marine microorganisms associated with invertebrate hosts are involved in the biosynthesis of secondary metabolites offers new alternatives for the discovery and development of marine natural products. However, the discovery of microorganisms producing secondary metabolites previously attributed to an invertebrate host poses a significant challenge.

Cloning of casbene and neocembrene synthases from Euphorbiaceae plants and expression in Saccharomyces cerevisiae.

A large number of diterpenes have been isolated from Euphorbiaceae plants, many of which are of interest due to toxicity or potential therapeutic activity. Specific Euphorbiaceae diterpenes of medical interest include the latent HIV-1 activator prostratin (and related 12-deoxyphorbol esters), the analgesic resiniferatoxin, and the anticancer drug candidate ingenol 3-angelate. In spite of the large number of diterpenes isolated from these plants and the similarity of their core structures, there is little known about their biosynthetic pathways.

Identification of isopentenol biosynthetic genes from Bacillus subtilis by a screening method based on isoprenoid precursor toxicity.

We have developed a novel method to clone terpene synthase genes. This method relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype. When these precursors were consumed by a terpene synthase, normal growth was restored.

Biosynthesis and engineering of isoprenoid small molecules.

Isoprenoid secondary metabolites are a rich source of commercial products that have not been fully explored. At present, there are isoprenoid products used in cancer therapy, the treatment of infectious diseases, and crop protection. All isoprenoids share universal prenyl diphosphate precursors synthesized via two distinct pathways.

Production of the antimalarial drug precursor artemisinic acid in engineered yeast.

Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing.

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids.

Isoprenoids are the most numerous and structurally diverse family of natural products. Terpenoids, a class of isoprenoids often isolated from plants, are used as commercial flavor and fragrance compounds and antimalarial or anticancer drugs. Because plant tissue extractions typically yield low terpenoid concentrations, we sought an alternative method to produce high-value terpenoid compounds, such as the antimalarial drug artemisinin, in a microbial host.