A glucoamylase::GFP gene fusion to study protein secretion by individual hyphae of Aspergillus niger.
Although Aspergillus niger is used as a host for heterologous protein production, yields are generally lower than those obtained for homologous proteins. Mechanisms of protein secretion and the secretory pathway in filamentous fungi are poorly characterised, although there is evidence to suggest that secretion occurs by a mechanism similar to that in other eukaryotes, but with proteins destined for secretion being directed to the hyphal tip. We report on a method using a glucoamylase: GFP gene fusion which allows us for the first time to monitor, in vivo, protein secretion in A. niger at the single hyphal level. A synthetic green fluorescent protein (sGFP(S65T)) was fused to truncated A. niger glucoamylase (GLA:499). Southern blot analysis of transformants confirmed that the gene fusion had successfully integrated into the A. niger genome. Confocal and fluorescence microscopy revealed that the GLA::GFP fusion protein is fluorescent in A. niger and appears to be directed to the hyphal tip. In young mycelia, hyphal cell wall fluorescence is apparent and immunogold labelling of GFP confirmed that GFP was partially localised within the hyphal cell wall. Using Western blotting, extracellular GLA::GFP was detected only in culture filtrates of young mycelia grown in a soya milk medium. The actin inhibitor latrunculin B was used to disrupt the secretion process, and its effects on the distribution of GLA::GFP were monitored.
Gordon
C L
CL
1.800 Stopford Building, School of Biological Sciences, University of Manchester, M13 9PT, Manchester, UK.
Archer
D B
DB
Jeenes
D J
DJ
Doonan
J H
JH
Wells
B
B
Trinci
A P
AP
Robson
G D
GD
D000199:Actins / Q000378:metabolism; D001234:Aspergillus niger / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D015139:Blotting, Southern; D015153:Blotting, Western; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D003470:Culture Media; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics* / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D053778:Thiazolidines
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D003470:Culture Media; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D013844:Thiazoles; D053778:Thiazolidines; D049452:Green Fluorescent Proteins; D005087:Glucan 1,4-alpha-Glucosidase; C037068:latrunculin B