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Development of a flow cytometry-based plating-free system for strain engineering in industrial fungi.

Yu-Jing YangYin LiuDan-Dan LiuWen-Zhu GuoLi-Xian WangXing-Ji WangHe-Xin LvYang YangQian LiuChao-Guang Tian
Published in: Applied microbiology and biotechnology (2021)
Recent technical advances regarding filamentous fungi have accelerated the engineering of fungal-based production and benefited basic science. However, challenges still remain and limit the speed of fungal applications. For example, high-throughput technologies tailored to filamentous fungi are not yet commonly available for genetic modification. The currently used fungal genetic manipulations are time-consuming and laborious. Here, we developed a flow cytometry-based plating-free system to directly screen and isolate the transformed protoplasts in industrial fungi Myceliophthora thermophila and Aspergillus niger. This system combines genetic engineering via the 2A peptide and the CRISPR-Cas9 system, strain screening by flow cytometry, and direct sorting of colonies for deep-well-plate incubation and phenotypic analysis while avoiding culturing transformed protoplasts in plates, colony picking, conidiation, and cultivation. As a proof of concept, we successfully applied this system to generate the glucoamylase-hyperproducing strains MtYM6 and AnLM3 in M. thermophila and A. niger, respectively. Notably, the protein secretion level and enzyme activities in MtYM6 were 17.3- and 25.1-fold higher than in the host strain. Overall, these findings suggest that the flow cytometry-based plating-free system can be a convenient and efficient tool for strain engineering in fungal biotechnology. We expect this system to facilitate improvements of filamentous fungal strains for industrial applications. KEY POINTS: • Development of a flow cytometry-based plating-free (FCPF) system is presented. • Application of FCPF system in M. thermophila and A. niger for glucoamylase platform. • Hyper-produced strains MtYM6 and AnLM3 for glucoamylase production are generated.
Keyphrases
  • flow cytometry
  • high throughput
  • crispr cas
  • escherichia coli
  • cell wall
  • heavy metals
  • wastewater treatment
  • genome wide
  • copy number
  • genome editing
  • public health
  • risk assessment
  • single cell
  • binding protein