A5IL97 production in <i>A</i>. <i>niger</i>.

<p>A5IL97 production in <i>A</i>. <i>niger</i>.</p

Expression of naturally ionic liquid-tolerant thermophilic cellulases in <i>Aspergillus niger</i>

<div><p>Efficient deconstruction of plant biomass is a major barrier to the development of viable lignocellulosic biofuels. Pretreatment with ionic liquids reduces lignocellulose recalcitrance to enzymatic hydrolysis, increasing yields of sugars for conversion into biofuels. However, commercial cellulases are not compatible with many ionic liquids, necessitating extensive water washing of pretreated biomass prior to hydrolysis. To circumvent this issue, previous research has demonstrated that several thermophilic bacterial cellulases can efficiently deconstruct lignocellulose in the presence of the ionic liquid, 1-ethyl-3-methylimadizolium acetate. As promising as these enzymes are, they would need to be produced at high titer in an industrial enzyme production host before they could be considered a viable alternative to current commercial cellulases. <i>Aspergillus niger</i> has been used to produce high titers of secreted enzymes in industry and therefore, we assessed the potential of this organism to be used as an expression host for these ionic liquid-tolerant cellulases. We demonstrated that 29 of these cellulases were expressed at detectable levels in a wild-type strain of <i>A</i>. <i>niger</i>, indicating a basic level of compatibility and potential to be produced at high levels in a host engineered to produce high titers of enzymes. We then profiled one of these enzymes in detail, the β-glucosidase A5IL97, and compared versions expressed in both <i>A</i>. <i>niger</i> and <i>Escherichia coli</i>. This comparison revealed the enzymatic activity of A5IL97 purified from <i>E</i>. <i>coli</i> and <i>A</i>. <i>niger</i> is equivalent, suggesting that <i>A</i>. <i>niger</i> could be an excellent enzyme production host for enzymes originally characterized in <i>E</i>. <i>coli</i>, facilitating the transition from the laboratory to industry.</p></div

Zymography of A5IL97 produced in <i>E</i>. <i>coli</i> and <i>A</i>. <i>niger</i>.

<p>Zymography was performed with unpurified, non-denatured extracts of A5IL97, expressing strains of <i>E</i>. <i>coli</i> (Ec-A5IL97) and <i>A</i>. <i>niger</i> (An-A5IL97). Both β-glucosidase and cellobiohydrolase activities were analyzed on native gels, containing A) 4-methylumbelliferyl β-D-glucopyranoside (MUG) or B) 4-methylumbelliferyl β-D-cellobioside (MUC), respectively (top). The same gel was stained with Coomassie blue G250 after the zymography (bottom). Black arrows indicate the position of A5IL97.</p

A5IL97 production in <i>A</i>. <i>niger</i>.

<p>A5IL97 production in <i>A</i>. <i>niger</i>.</p

Thermostability of A5IL97 in <i>E</i>. <i>coli</i> and <i>A</i>. <i>niger</i>.

<p>Enzyme stability of A5IL97 using equal amounts of purified A5IL97 (200 μg/mL) from <i>E</i>. <i>coli</i> (Ec-A5IL97) and <i>A</i>. <i>niger</i> (An-A5IL97) at 70°C, 85°C, and 95°C. Error bars indicate the standard deviations from three replicates.</p

Bacterial and fungal cellulases expressed in <i>A</i>. <i>niger</i>.

<p>Bacterial and fungal cellulases expressed in <i>A</i>. <i>niger</i>.</p

Enzymatic activity profile of A5IL97 from <i>E</i>. <i>coli</i> and <i>A</i>. <i>niger</i>.

<p>Enzyme activity profiles were generated using purified Ec-A5IL97 and An-A5IL97 at temperatures between 70 and 95°C, pH between 4 and 8, and [C₂C₁lm][OAc] IL concentrations between 5 and 30% (v/v). The colorimetric substrate, <i>p</i>NPG, was used to measure the enzyme activity, represented in the color bars in the legend.</p

Saccharification of biomass using cellulase mixture containing A5IL97 from either <i>E</i>. <i>coli</i> or <i>A</i>. <i>niger</i>.

<p>Enzyme hydrolysis of IL-pretreated switchgrass using the JTherm cellulase mixture supplemented with either purified Ec-A5IL97 or An-A5IL97. The IL-pretreated switchgrass was washed free of IL prior to saccharification. Equivalent amounts of glucose and xylose were liberated over 72 hours. Error bars indicate the standard deviations from three replicates.</p