skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

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

Save / Share:

Works referenced in this record:

Aspergillus niger membrane-associated proteome analysis for the identification of glucose transporters
journal, September 2015


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


Recombinant microbial systems for improved β-galactosidase production and biotechnological applications
journal, November 2011


DELLY: structural variant discovery by integrated paired-end and split-read analysis
journal, September 2012


Expression of naturally ionic liquid-tolerant thermophilic cellulases in Aspergillus niger
journal, December 2017


Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression.
journal, October 1996

  • Ozcan, S.; Dover, J.; Rosenwald, A. G.
  • Proceedings of the National Academy of Sciences, Vol. 93, Issue 22
  • DOI: 10.1073/pnas.93.22.12428

A broader role for AmyR in Aspergillus niger: regulation of the utilisation of d-glucose or d-galactose containing oligo- and polysaccharides
journal, August 2011

  • vanKuyk, Patricia A.; Benen, Jaques A. E.; Wösten, Han A. B.
  • Applied Microbiology and Biotechnology, Vol. 93, Issue 1
  • DOI: 10.1007/s00253-011-3550-6

Expression Profile of Amylolytic Genes in Aspergillus nidulans
journal, October 2006

  • Nakamura, Takashi; Maeda, Yuki; Tanoue, Nami
  • Bioscience, Biotechnology, and Biochemistry, Vol. 70, Issue 10
  • DOI: 10.1271/bbb.50694

Ionic liquid-tolerant microorganisms and microbial communities for lignocellulose conversion to bioproducts
journal, November 2016

  • Yu, Chaowei; Simmons, Blake A.; Singer, Steven W.
  • Applied Microbiology and Biotechnology, Vol. 100, Issue 24
  • DOI: 10.1007/s00253-016-7955-0

BreakDancer: an algorithm for high-resolution mapping of genomic structural variation
journal, August 2009

  • Chen, Ken; Wallis, John W.; McLellan, Michael D.
  • Nature Methods, Vol. 6, Issue 9
  • DOI: 10.1038/nmeth.1363

The low affinity glucose transporter HxtB is also involved in glucose signalling and metabolism in Aspergillus nidulans
journal, March 2017

  • dos Reis, Thaila Fernanda; Nitsche, Benjamin M.; de Lima, Pollyne Borborema Almeida
  • Scientific Reports, Vol. 7, Issue 1
  • DOI: 10.1038/srep45073

LUMPY: a probabilistic framework for structural variant discovery
journal, January 2014


Regulation of the glaA gene of Aspergillus niger
journal, January 1990

  • Fowler, Timothy; Berka, Randy M.; Ward, Michael
  • Current Genetics, Vol. 18, Issue 6
  • DOI: 10.1007/BF00327025

Identification of the mstE Gene Encoding a Glucose-inducible, Low Affinity Glucose Transporter in Aspergillus nidulans
journal, January 2006

  • Forment, Josep V.; Flipphi, Michel; Ramón, Daniel
  • Journal of Biological Chemistry, Vol. 281, Issue 13
  • DOI: 10.1074/jbc.M508198200

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

The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data
journal, July 2010


On the safety of a new generation of DSM Aspergillus niger enzyme production strains
journal, August 2003

  • van Dijck, Piet W. M.; Selten, Gerard C. M.; Hempenius, Rixta A.
  • Regulatory Toxicology and Pharmacology, Vol. 38, Issue 1
  • DOI: 10.1016/s0273-2300(03)00049-7

Discovery and characterization of ionic liquid-tolerant thermophilic cellulases from a switchgrass-adapted microbial community
journal, January 2014

  • Gladden, John M.; Park, Joshua I.; Bergmann, Jessica
  • Biotechnology for Biofuels, Vol. 7, Issue 1
  • DOI: 10.1186/1754-6834-7-15

VariantAnnotation: a Bioconductor package for exploration and annotation of genetic variants
journal, March 2014


Deletion of glucose oxidase changes the pattern of organic acid production in Aspergillus carbonarius
journal, August 2014


Pyrithiamine Resistance Gene ( ptrA ) of Aspergillus oryzae : Cloning, Characterization and Application as a Dominant Selectable Marker for Transformation
journal, January 2000

  • Kubodera, Takafumi; Yamashita, Nobuo; Nishimura, Akira
  • Bioscience, Biotechnology, and Biochemistry, Vol. 64, Issue 7
  • DOI: 10.1271/bbb.64.1416

Transceptors as a functional link of transporters and receptors
journal, March 2017


Carbon repression in aspergilli
journal, June 1997


Toward a Systems Approach to Understanding Plant Cell Walls
journal, December 2004


The Sequence Alignment/Map format and SAMtools
journal, June 2009


Ypt/Rab GTPases: Principles learned from yeast
journal, November 2014

  • Lipatova, Zhanna; Hain, Adelaide U.; Nazarko, Volodymyr Y.
  • Critical Reviews in Biochemistry and Molecular Biology, Vol. 50, Issue 3
  • DOI: 10.3109/10409238.2015.1014023

Current Pretreatment Technologies for the Development of Cellulosic Ethanol and Biorefineries
journal, September 2015

  • Silveira, Marcos Henrique Luciano; Morais, Ana Rita C.; da Costa Lopes, Andre M.
  • ChemSusChem, Vol. 8, Issue 20
  • DOI: 10.1002/cssc.201500282

Characterization of a polyketide synthase in Aspergillus niger whose product is a precursor for both dihydroxynaphthalene (DHN) melanin and naphtho-γ-pyrone
journal, April 2011

  • Chiang, Yi-Ming; Meyer, Kristen M.; Praseuth, Michael
  • Fungal Genetics and Biology, Vol. 48, Issue 4
  • DOI: 10.1016/j.fgb.2010.12.001

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