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. To address this challenge, we developed a genetic approach for studying and engineering protein-secretion pathways in E. coli<i>.</i> Using the YebF pathway as a model, we demonstrate that direct fluorescent labeling of tetracysteine-motif-tagged secretory proteins with the biarsenical compound FlAsH is possible <i>in situ</i> without the need to recover the cell-free supernatant. High-throughput screening of a bacterial strain library yielded superior YebF expression hosts capable of secreting higher titers of YebF and YebF-fusion proteins into the culture medium. We also show that the method can be easily extended to other secretory pathways, including type II and type III secretion, directly in E. coli. Thus, our FlAsH-tetracysteine-based genetic assay provides a convenient, high-throughput tool that can be applied generally to diverse secretory pathways. This platform should help to shed light on poorly understood aspects of these processes as well as to further assist in the construction of engineered E. coli strains for efficient secretory-protein production

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

The GLBRCY127 strain developed by directed engineering with xylose isomerase coupled with batch evolution can rapidly consume xylose aerobically.

<p>Average sugar consumption and cell growth of unevolved GLBRCY22-3 strain engineered with <i>ScTAL1</i>, <i>CpxylA</i> and <i>SsXYL3</i> cultured in bioreactors containing YPDX media and sparged with air from biological duplicates is shown (<b>A</b>). Indicated components were quantified from media samples at times from initial inoculation. In (<b>B</b>), the average percentage of xylose consumed and change in cell density per day are plotted for each transfer during the adaption of the Y22-3 strain in YP media containing 0.1% glucose and 2% xylose. The pattern of lower % of xylose consumed and change in cell density per day during every third transfer is due to reaching saturated growth prior to transfer. Average extracellular xylose concentrations and cell density measurements from parental Y22-3 and evolved Y127 strains grown aerobically in culture tubes with YPX media from three independent biological replicates are plotted in (<b>C</b>). In (<b>D</b>), evolved isolate Y127 was cultured in the same conditions as in (<b>A</b>), and samples measurements taken in an identical manner.</p

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

Fermentation kinetic profiles for engineered and evolved <i>S. cerevisiae</i> strains.

<p>ND, Not Determined for aerobic conditions; ND*, Not Determined – no ethanol produced.</p>1<p>In g xylose consumed/L/h.</p>2<p>In g xylose consumed/g DCW/h.</p>3<p>Calculated from the maximum ethanol concentration produced divided by the consumed xylose concentration at that time.</p>4<p>Calculated from the ethanol concentration produced between two time points after glucose depletion.</p>5<p>Yield of g DCW/g glucose consumed calculated at or near the time of glucose depletion and prior to xylose consumption. No cell growth was observed during xylose consumption.</p>6<p>Yield of g glycerol/g glucose consumed calculated at or near the time of glucose depletion and prior to xylose consumption.</p><p>Fermentation kinetic profiles for engineered and evolved <i>S. cerevisiae</i> strains.</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

GLBRCY128 can anaerobically ferment xylose from ACSH.

<p>A diagram summarizing the engineering and evolution of the YB-210 strain into the evolved Y128 strain is provided in (<b>A</b>). Fermentation kinetic profiles of the Y127 (<b>B</b>) and Y128 (<b>C</b>) strains cultured in bioreactors containing ACSH and sparged with nitrogen from biological duplicates are shown. Average concentrations and standard deviations of indicated components were quantified from media samples at times from initial inoculation. Vertical colored bars indicate time points at which samples were taken for metabolomic analysis described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107499#pone-0107499-g007" target="_blank">Fig. 7A–D</a>.</p

GLBRCY128 can anaerobically ferment xylose from YPX medium into ethanol faster than its parental strains.

<p>Fermentation kinetic profiles comparing Y22-3, Y127 and Y128 strains cultured anaerobically in bioreactors containing YPX medium and sparged with nitrogen are shown. Average concentrations with standard deviations of xylose, ethanol and DCW concentrations in the media were calculated from independent biological triplicates.</p

Comparison of anaerobic fermentation kinetics for Y22-3, Y127 and Y128 in YPX medium.

<p>ND, Not Determined – low xylose consumption; ND*, Not Determined – no ethanol produced and low xylose consumption.</p>1<p>In g xylose consumed/L/h.</p>2<p>In g xylose consumed/g DCW/h.</p>3<p>Calculated from the maximum ethanol concentration produced divided by the consumed xylose concentration at that time.</p>4<p>Calculated from the maximum ethanol concentration produced divided by the starting xylose concentration.</p>5<p>In g ethanol/L.</p><p>Comparison of anaerobic fermentation kinetics for Y22-3, Y127 and Y128 in YPX medium.</p