PMID- 16021486
OWN - NLM
STAT- MEDLINE
DCOM- 20060605
LR  - 20061115
IS  - 0175-7598 (Print)
IS  - 0175-7598 (Linking)
VI  - 69
IP  - 6
DP  - 2006 Feb
TI  - A new method for screening and isolation of hypersecretion mutants in Aspergillus 
      niger.
PG  - 711-7
AB  - Although filamentous fungi have a unique property of secreting a large amount of 
      homologous extracellular proteins, the use of filamentous fungi as hosts for the 
      production of heterologous proteins is limited because of the low production 
      levels that are generally reached. Here, we report a general screening method for 
      the isolation of mutants with increased protein production levels. The screening 
      method makes use of an Aspergillus niger strain that lacks the two major 
      amylolytic enzymes, glucoamylase (GlaA) and acid amylase (AamA). The 
      double-mutant strain grows poorly on starch and its growth is restored after 
      reintroducing the catalytic part of the glucoamylase gene (GlaA512). We show that 
      the fusion of a heterologous protein, a laccase from Pleurotus ostreatus (Pox2), 
      to the catalytic part of glucoamylase (GlaA512-Pox2) severely hampers efficient 
      production of the glucoamylase protein, resulting in a slow-growth phenotype on 
      starch. Laccase-hypersecreting mutants were obtained by isolating mutants that 
      displayed improved growth on starch plates. The mutant with the highest growth 
      rate on starch displayed the highest laccase activity, indicating that increased 
      glucoamylase protein levels are correlated with higher laccase production levels. 
      In principle, our method can be applied to any low-produced heterologous protein 
      that is secreted as a fusion with the glucoamylase protein.
FAU - Weenink, Xavier O
AU  - Weenink XO
AD  - Clusius Laboratory, Fungal Genetics Research Group, Institute of Biology, Leiden 
      University, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands.
FAU - Punt, Peter J
AU  - Punt PJ
FAU - van den Hondel, Cees A M J J
AU  - van den Hondel CA
FAU - Ram, Arthur F J
AU  - Ram AF
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20050714
PL  - Germany
TA  - Appl Microbiol Biotechnol
JT  - Applied microbiology and biotechnology
JID - 8406612
RN  - 0 (Recombinant Proteins)
RN  - 9005-25-8 (Starch)
RN  - EC 1.10.3.2 (Laccase)
RN  - EC 3.2.1.3 (Glucan 1,4-alpha-Glucosidase)
SB  - IM
MH  - Aspergillus niger/*genetics/growth & development/isolation & 
      purification/*metabolism
MH  - Biomass
MH  - Glucan 1,4-alpha-Glucosidase/genetics
MH  - Laccase/genetics/*metabolism
MH  - Mutation
MH  - Pleurotus/enzymology
MH  - Protein Transport/*genetics
MH  - Recombinant Proteins/genetics/*metabolism
MH  - Starch/metabolism
EDAT- 2005/07/16 09:00
MHDA- 2006/06/06 09:00
CRDT- 2005/07/16 09:00
PHST- 2005/03/08 00:00 [received]
PHST- 2005/04/23 00:00 [accepted]
PHST- 2005/04/21 00:00 [revised]
PHST- 2005/07/16 09:00 [pubmed]
PHST- 2006/06/06 09:00 [medline]
PHST- 2005/07/16 09:00 [entrez]
AID - 10.1007/s00253-005-0013-y [doi]
PST - ppublish
SO  - Appl Microbiol Biotechnol. 2006 Feb;69(6):711-7. doi: 10.1007/s00253-005-0013-y. 
      Epub 2005 Jul 14.