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The F-box protein gene exo-1 is a target for reverse engineering enzyme hypersecretion in filamentous fungi

Journal Article · · Proceedings of the National Academy of Sciences of the United States of America
 [1];  [2];  [3];  [4];  [5];  [6];  [7];  [8];  [9];  [1];  [10];  [11];  [3];  [10];  [12];  [13];  [2]
  1. Lawrence Berkeley National Laboratory
  2. Technical University of Munich
  3. Chinese Academy of Sciences
  4. Tianjin University
  5. Technische Universitat Munchen
  6. Johann Wolfgang Goethe-Universitat Frankfurt
  7. Universitat fur Bodenkultur Wien
  8. University of Minnesota
  9. University of California, Berkeley
  10. Private author
  11. BATTELLE (PACIFIC NW LAB)
  12. Joint BioEnergy Institute
  13. Technische Universitat Braunschweig
+ Show Author Affiliations

Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein–encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain ?cre-1, while glucose repression was still mostly functional in ?exo-1. Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in ?exo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in ?exo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.

Research Organization:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Organization:
USDOE
DOE Contract Number:
AC05-76RL01830
OSTI ID:
1810707
Report Number(s):
PNNL-SA-163826
Journal Information:
Proceedings of the National Academy of Sciences of the United States of America, Vol. 118, Issue 26
Country of Publication:
United States
Language:
English

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