Abstract
BackgroundThe filamentous fungus Trichoderma reesei, the most widely used cellulase producer, also has promising applications in lignocellulose-based biorefinery: consolidated bioprocessing for the production of high value-added products. However, such applications are thwarted by the time-consuming metabolic engineering processes (design–build–test–learn cycle) for T. reesei, resulted from (i) the spore separation-mediated purification as the multinucleate hyphae, (ii) transformant screening for high expression levels since unavailable of episomal expression system, and (iii) cases of inexpressible heterologous proteins.ResultsIn this study, a GFP-fusion coupled fluorescence-activated cell sorting (FACS) platform was established to speed up the build and test process of the DBTL cycle, by enabling rapid selection for expressible heterologous genes and bypassing both laborious spore separation and transformant screening. Here, the feasibility of flow cytometry in analyzing and sorting T. reesei cells harboring GFP-fused expressible protein was proven, as well as the application of the platform for constitutive promoter strength evaluation. As a proof-of-concept, the platform was employed to construct the first T. reesei strain producing fatty alcohol, resulting in up to 2 mg hexadecanol being produced per gram biomass. Pathway construction was enabled through rapid selection of functional fatty acyl-CoA reductase encoding gene Tafar1 from three candidate genes and strains with high expression level from spore pools. As a result of using this method, the total costed time for the build and test cycle using T. reesei, subsequently, reduced by approx. 75% from 2 months to 2 weeks.ConclusionThis study established the GFP-fusion coupling FACS platform and the first filamentous fungal fatty alcohol-producing cell factory, and demonstrated versatile applications of the platform in the metabolic engineering of filamentous fungi, which can be harnessed to potentially advance the application of filamentous fungi in lignocellulose-based biorefinery.
Highlights
The filamentous fungus Trichoderma reesei, the most widely used cellulase producer, has promising applications in lignocellulose-based biorefinery: consolidated bioprocessing for the production of high valueadded products
Flow cytometry as a robust tool for analyzing filamentous fungi in high throughput To test the feasibility of flow cytometry in analyzing filamentous fungi, we tested T. reesei based on both its cell shape and fluorescence, as well as testing the gene expressibility, we applied flow cytometry to analyze spores and protoplasts of pyr4-TU-6 strain and Trpdi2gfp-TU-6 strain expressing GFP-fused homologous TrPDI2
In contrast to the control strain pyr4TU-6, wherein no GFP was present, the cell population of Trpdi2-gfp-TU-6 showed much higher fluorescence levels (Fig. 2). This suggested that flow cytometry was able to identify T. reesei cells and discern according to their difference in fluorescence intensity, consistent with the previous studies proving the application feasibility of flow cytometry on T. reesei germinating spores [26, 27]
Summary
The filamentous fungus Trichoderma reesei, the most widely used cellulase producer, has promising applications in lignocellulose-based biorefinery: consolidated bioprocessing for the production of high valueadded products. Due to the advantage of a well-understood cellular metabolism and a comprehensive genetic manipulation platform, both Escherichia coli [8–10] and Saccharomyces cerevisiae [11–13] predominate as hosts for rapid cell factory construction Their application in industry may be limited by expensive feedstock consumption (glucose, glycerol, etc.), especially when economic pressures on TRY (titer, rate, and yield) are high, such as in the case of low-priced bulk chemical and fuel production. The Sordariomycete fungus Trichoderma reesei is the most widely used cellulase producer in both academic investigations and industrial applications [22] As it demonstrates a great capability for degrading cellulose, T. reesei, subsequently, has huge potential in consolidated bioprocessing, to convert recalcitrant and abundant cellulose into value-added products via single microorganism-based fermentation [23]
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