Inhibition of microbial biofuel production in drought-stressed switchgrass hydrolysate

Additional file 2. Maps of significant gene ontology terms for chemical genomics data. Untreated biomass composition. Detailed hydrolysate composition

Engineering and Two-Stage Evolution of a Lignocellulosic Hydrolysate-Tolerant <i>Saccharomyces cerevisiae</i> Strain for Anaerobic Fermentation of Xylose from AFEX Pretreated Corn Stover

<div><p>The inability of the yeast <i>Saccharomyces cerevisiae</i> to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of <i>S. cerevisiae</i> 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. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting <i>S. cerevisiae</i> strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent <i>S. cerevisiae</i> strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in <i>GRE3</i>, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust <i>S. cerevisiae</i> strain with the ability to ferment xylose anaerobically from ACSH.</p></div

Second stage anaerobic adaptation on xylose enabled rapid xylose fermentation by evolved GLBRCY128 isolate.

<p>Average fermentation kinetic profiles of the GLBRCY127 strain cultured in bioreactors containing YPDX media and sparged with nitrogen from biological duplicates are shown (<b>A</b>). Average concentrations with standard deviations of indicated compounds were quantified from media samples at times from initial inoculation. In (<b>B</b>), the percentage of xylose consumed and change in cell density per day is plotted for each transfer during the anaerobic adaptation of Y127 in YP media containing 0.1% glucose and 2% xylose. In the first two transfers (hatched bars), Tween-80 and ergosterol were added to the media. In (<b>C</b>), evolved isolate Y128 was cultured in biological duplicate under the same conditions as in (<b>A</b>), and samples measurements taken in an identical manner.</p

Phenotypic screening of wild and domesticated <i>S. cerevisiae</i> strains identifies NRRL YB-210 with tolerance to hydrolysates made from a variety of pretreated lignocellulose.

<p>In (<b>A</b>), 117 <i>S. cerevisiae</i> strains (including some in duplicate) were cultured in 96-well plates and monitored for changes cell density and growth rates calculated as described in Materials and Methods. All strains in each condition were then ranked from 1 (highest growth rate in yellow) to 117 (lowest growth rate, or no growth, in blue) and hierarchically clustered. Arrows indicate clustered rows for BY4741 (green), CEN.PK2 (black) in duplicate microtiter wells, and NRRL YB-210/GLBRCY0 (red). Representative growth data for the YB-210/GLBRCY0 strain in the indicated media from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107499#pone-0107499-g002" target="_blank">Fig. 2A</a> are plotted (<b>B–C</b>). CS, corn stover; SG, switchgrass; YP; Yeast Extract and Peptone supplementation, 6%; 6% glucan loading ACSH, 9%; 9% glucan loading ACSH, Dtx.; Detoxified.</p

The xylose consumption phenotypes of the evolved Y127 and Y128 strains are dependent upon <i>CpxylA</i> and <i>ScTAL1.</i>

<p>Extracellular xylose concentrations (solid lines) and cell density (dashed lines) were measured by YSI instrument and OD₆₀₀ readings, respectively, from cultures containing KanMX marker rescued versions of (<b>A</b>) GLBRCY127 (Y132) and GLBRCY132 <i>xylAΔ</i> or (<b>B</b>) Y132 and Y132 <i>tal1Δ</i> strains inoculated in aerobic YPX media. In (<b>C</b>), extracellular xylose concentrations (solid lines) and cell density (dashed lines) were measured as in (<b>A</b>, <b>B</b>) for KanMX marker rescued GLBRCY128 (Y133) and two independent GLBRCY133 <i>xylAΔ</i> strains inoculated in anaerobic YPX media. These selection marker-rescued Y128 strains were cultured in YPD media and total RNA isolated from a single time point. Expression of <i>CpxylA</i> was then quantified and normalized to <i>ScERV25</i> RNA levels by qPCR. The bar graph in (<b>D</b>) displays the average values and standard deviations for <i>CpxylA</i> RNA from three independent biological replicates.</p