Enhancing photosynthetic production of glycogen-rich biomass for use as a fermentation feedstock.

Current sources of fermentation feedstocks, i.e. corn, sugar cane, or plant biomass, fall short of demand for liquid transportation fuels and commodity chemicals in the United States. Aquatic phototrophs including cyanobacteria have the potential to supplement the supply of current fermentable feedstocks. In this strategy, cells are engineered to accumulate storage molecules including glycogen, cellulose, and/or lipid oils that can be extracted from harvested biomass and fed to heterotrophic organisms engineered to produce desired chemical products. In this manuscript, we examine the production of glycogen in the model cyanobacteria, Synechococcus sp. strain PCC 7002, and subsequent conversion of cyanobacterial biomass by an engineered Escherichia coli to octanoic acid as a model product. In effort to maximize glycogen production, we explored the deletion of catabolic enzymes and overexpression of GlgC, an enzyme that catalyzes the first committed step towards glycogen synthesis. We found that deletion of glgP increased final glycogen titers when cells were grown in diurnal light. Overexpression of GlgC led to a temporal increase in glycogen content but not in an overall increase in final titer or content. The best strains were grown, harvested, and used to formulate media for growth of E. coli. The cyanobacterial media was able to support the growth of an engineered E. coli and produce octanoic acid at the same titer as common laboratory media.

Flux Balance Analysis Indicates that Methane Is the Lowest Cost Feedstock for Microbial Cell Factories.

The low cost of natural gas has driven significant interest in using C1 carbon sources (e.g. methane, methanol, CO, syngas) as feedstocks for producing liquid transportation fuels and commodity chemicals. Given the large contribution of sugar and lignocellulosic feedstocks to biorefinery operating costs, natural gas and other C1 sources may provide an economic advantage. To assess the relative costs of these feedstocks, we performed flux balance analysis on genome-scale metabolic models to calculate the maximum theoretical yields of chemical products from methane, methanol, acetate, and glucose. Yield calculations were performed for every metabolite (as a proxy for desired products) in the genome-scale metabolic models of three organisms: Escherichia coli (bacterium), Saccharomyces cerevisiae (yeast), and Synechococcus sp. PCC 7002 (cyanobacterium). The calculated theoretical yields and current feedstock prices provided inputs to create comparative feedstock cost surfaces. Our analysis shows that, at current market prices, methane feedstock costs are consistently lower than glucose when used as a carbon and energy source for microbial chemical production. Conversely, methanol is costlier than glucose under almost all price scenarios. Acetate feedstock costs could be less than glucose given efficient acetate production from low-cost syngas using nascent biological gas to liquids (BIO-GTL) technologies. Our analysis suggests that research should focus on overcoming the technical challenges of methane assimilation and/or yield of acetate via BIO-GTL to take advantage of low-cost natural gas rather than using methanol as a feedstock.

A molecular transporter engineering approach to improving xylose catabolism in Saccharomyces cerevisiae.

Traditional metabolic pathway engineering rarely considers the influence of molecular transport. Here, we describe the directed evolution of two heterologous transporters, Candida intermedia GXS1 and Scheffersomyces stipitis XUT3. Growth rate on xylose was improved up to 70% by mutant transporter expression. Most mutants were found to exhibit vastly improved V(max) values and display an increase in high cell density sugar consumption rates. Mixed glucose and xylose fermentations reveal that mutant transporters can alter the diauxic shift dynamics and the simultaneous sugar utilization capacity of the host strain. Analysis of mutations highlights several important residues influencing transporter function including point mutations at F40 of C. intermedia GXS1 and at E538 of S. stipitis XUT3. This work is the first to demonstrate that molecular transporter proteins can be improved for biotechnological applications through directed evolution in yeast.