Abstract
BackgroundIn the biofuel industry, cellulase plays an indispensable role in hydrolyzing cellulose into fermentable glucose. Trichoderma reesei is a popular filamentous fungus with prominent ability to produce cellulase. While classical mutagenesis and modern multiplex genome engineering are both effective ways to improve cellulase production, successful obtaining of strains with improved cellulase-producing ability requires screening a large number of strains, which is time-consuming and labor intensive.ResultsHerein, we developed a versatile method coupling expression of the red fluorescence protein (DsRed) in T. reesei and fluorescence-assisted cell sorting (FACS) of germinated spores. This method was first established by expressing DsRed intracellularly under the control of the major cellulase cbh1 promoter in T. reesei, which allowed us to rapidly isolate cellulase hyperproducers from T. reesei progenies transformed with a dedicated transcriptional activator ace3 and from an atmospheric and room temperature plasma-created mutant T. reesei library. Since intracellularly expressed DsRed was expected to isolate mutations mainly affecting cellulase transcription, this method was further improved by displaying DsRed on the T. reesei cell surface, enabling isolation of strains with beneficial genetic alterations (overexpressing hac1 and bip1) affecting regulatory stages beyond transcription. Using this method, T. reesei cellulase hyperproducers were also successfully isolated from an Agrobacterium-mediated random insertional mutant library.ConclusionsThe coupled DsRed-FACS high-throughput screening method proved to be an effective strategy for fast isolation of T. reesei cellulase hyperproducers and could also be applied in other industrially important filamentous fungi.
Highlights
In the biofuel industry, cellulase plays an indispensable role in hydrolyzing cellulose into fermentable glucose
To correlate the expression of DsRed to that of the T. reesei cellulase, the DsRed gene should be placed under the control of a major cellulase promoter
The chimeric gene was placed between the cbh1 promoter and terminator (Fig. 1b)
Summary
Cellulase plays an indispensable role in hydrolyzing cellulose into fermentable glucose. Trichoderma reesei is a popular filamentous fungus with prominent ability to produce cellulase. The maximal extracellular protein concentration of T. reesei cellulase has been reported to reach 100 g/L [1]. On the route towards lignocellulosic biofuels, cellulase plays an indispensable role by hydrolyzing cellulose into fermentable glucose. To improve production of cellulase widely useful in the biofuel industries, chemical and physical mutagenesis were initially carried out, which successfully generated hyperproducer mutants such as RUT-C30 [13] and CL-847 [14]. It is noticed that multiplex genome engineering, i.e., manipulating more than one gene at a time, creates rich biological diversity from which a cellulase hyperproducer can be isolated [16]. The concurring higher complexity demands a larger amount of transformants to be analyzed
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