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Title: Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger

Abstract

Plant biomass, once reduced to its composite sugars, can be converted to fuel substitutes. One means of overcoming the recalcitrance of lignocellulose is pretreatment followed by enzymatic hydrolysis. However, currently available commercial enzyme cocktails are inhibited in the presence of residual pretreatment chemicals. Recent studies have identified a number of cellulolytic enzymes from bacteria that are tolerant to pretreatment chemicals such as ionic liquids. The challenge now is generation of these enzymes in copious amounts, an arena where fungal organisms such as Aspergillus niger have proven efficient. Fungal host strains still need to be engineered to increase production titers of heterologous protein over native enzymes, which has been a difficult task. Here, we developed a forward genetics screen coupled with whole-genome resequencing to identify specific lesions responsible for a protein hyper-production phenotype in A. niger. As a result, this strategy successfully identified novel targets, including a low-affinity glucose transporter, MstC, whose deletion significantly improved secretion of recombinant proteins driven by a glucoamylase promoter.

Authors:
 [1];  [1];  [2];  [3];  [4];  [1]; ORCiD logo [1]
  1. Joint BioEnergy Institute, Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
  2. Joint BioEnergy Institute, Emeryville, CA (United States); Wright-Patterson Air Force Base, Dayton, OH (United States)
  3. Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  4. Joint BioEnergy Institute, Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1416031
Alternate Identifier(s):
OSTI ID: 1420441; OSTI ID: 1431407; OSTI ID: 1433118
Report Number(s):
PNNL-SA-132880
Journal ID: ISSN 0175-7598; PII: 8717
Grant/Contract Number:  
AC02-05CH11231; AC05-76RL01830
Resource Type:
Journal Article: Published Article
Journal Name:
Applied Microbiology and Biotechnology
Additional Journal Information:
Journal Volume: 102; Journal Issue: 4; Journal ID: ISSN 0175-7598
Publisher:
Springer
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Aspergillus niger; Heterologous protein expression; Forward mutagenesis; Whole-genome resequencing; MstC

Citation Formats

Reilly, Morgann C., Kim, Joonhoon, Lynn, Jed, Simmons, Blake A., Gladden, John M., Magnuson, Jon K., and Baker, Scott E. Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger. United States: N. p., 2018. Web. doi:10.1007/S00253-017-8717-3.
Reilly, Morgann C., Kim, Joonhoon, Lynn, Jed, Simmons, Blake A., Gladden, John M., Magnuson, Jon K., & Baker, Scott E. Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger. United States. doi:10.1007/S00253-017-8717-3.
Reilly, Morgann C., Kim, Joonhoon, Lynn, Jed, Simmons, Blake A., Gladden, John M., Magnuson, Jon K., and Baker, Scott E. Sat . "Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger". United States. doi:10.1007/S00253-017-8717-3.
@article{osti_1416031,
title = {Forward genetics screen coupled with whole-genome resequencing identifies novel gene targets for improving heterologous enzyme production in Aspergillus niger},
author = {Reilly, Morgann C. and Kim, Joonhoon and Lynn, Jed and Simmons, Blake A. and Gladden, John M. and Magnuson, Jon K. and Baker, Scott E.},
abstractNote = {Plant biomass, once reduced to its composite sugars, can be converted to fuel substitutes. One means of overcoming the recalcitrance of lignocellulose is pretreatment followed by enzymatic hydrolysis. However, currently available commercial enzyme cocktails are inhibited in the presence of residual pretreatment chemicals. Recent studies have identified a number of cellulolytic enzymes from bacteria that are tolerant to pretreatment chemicals such as ionic liquids. The challenge now is generation of these enzymes in copious amounts, an arena where fungal organisms such as Aspergillus niger have proven efficient. Fungal host strains still need to be engineered to increase production titers of heterologous protein over native enzymes, which has been a difficult task. Here, we developed a forward genetics screen coupled with whole-genome resequencing to identify specific lesions responsible for a protein hyper-production phenotype in A. niger. As a result, this strategy successfully identified novel targets, including a low-affinity glucose transporter, MstC, whose deletion significantly improved secretion of recombinant proteins driven by a glucoamylase promoter.},
doi = {10.1007/S00253-017-8717-3},
journal = {Applied Microbiology and Biotechnology},
issn = {0175-7598},
number = 4,
volume = 102,
place = {United States},
year = {2018},
month = {1}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1007/S00253-017-8717-3

Citation Metrics:
Cited by: 5 works
Citation information provided by
Web of Science

Figures / Tables:

Table 1 Table 1: Strains used in this study

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Works referenced in this record:

On the safety of Aspergillus niger - a review
journal, January 2002

  • Schuster, E.; Dunn-Coleman, N.; Frisvad, J.
  • Applied Microbiology and Biotechnology, Vol. 59, Issue 4-5, p. 426-435
  • DOI: 10.1007/s00253-002-1032-6

A Thermophilic Ionic Liquid-Tolerant Cellulase Cocktail for the Production of Cellulosic Biofuels
journal, May 2012


Glycoside Hydrolase Activities of Thermophilic Bacterial Consortia Adapted to Switchgrass
journal, July 2011

  • Gladden, John M.; Allgaier, Martin; Miller, Christopher S.
  • Applied and Environmental Microbiology, Vol. 77, Issue 16, p. 5804-5812
  • DOI: 10.1128/AEM.00032-11

    Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.