A GFP-fusion coupling FACS platform for advancing the metabolic engineering of filamentous fungi

Biotechnology for Biofuels
Guokun WangDongyuan Zhang

Abstract

The 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. In 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 ...Continue Reading

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Citations

Jan 8, 2020·Critical Reviews in Biotechnology·Huaxiang DengYujie Cai
Jun 23, 2019·Applied Microbiology and Biotechnology·Yan-Zhen MeiChuan-Chao Dai
Dec 19, 2021·Applied Microbiology and Biotechnology·Yu-Jing YangChao-Guang Tian

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Datasets Mentioned

BETA
APQ46081
EU280149
JN638549

Methods Mentioned

BETA
fluorescence-activated cell sorting
FACS
flow cytometry
flow
spore collection
PCR

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