Production of bacterial-derived enzymes in Aspergillus niger
with applications in lignocellulosic biofuel production.
Stephanie
The development biofuels from Lignocellulosic (LC)
biomass, which includes a variety of plant feedstocks
such as agricultural and forestry residues and dedicated
energy crops, is one route to clean and renewable
alternatives to petroleum-base fuel. The first step in
converting plant biomass to fuels is breaking down
cellulose, the most abundant plant cell wall polymer, into
fermentable sugars. Pretreament of LC biomass with
ionic liquids (ILs) greatly reduces the difficulty in
breaking down the plant cell wall, allowing more
efficient enzymatic hydrolysis of cellulose to sugar.
Unfortunately, currently available commercial cellulase
cocktails are not active in the presence of ILs, an
incompatibility that required the addition of expensive
and wasteful washing steps. Due to the efficacy of IL
pretreatment, finding IL-tolerant cellulolytic enzymes is
necessary. An ideal place to look for such enzymes is high
salt and high temperature environments that harbor
halophilic and thermophilic bacteria, which tend to
produce highly stable enzymes that may be IL-tolerant.
Researchers at JBEI have identified 17 novel bacterialderived cellulase enzymes which have been isolated from
enriched compost material. However, enzyme production
at a scale large enough to be commercially viable is
limited due to the inability of common laboratory
heterologous expression systems (E. coli and
Saccharomyces cerevisiae) to produce sufficient titers of
these enzymes. A frequently used commercial host,
Aspergillus niger (A. niger), which is known for large scale
heterologous/endogenous protein production, is a
promising solution to this limitation. The Fungal
Biotechnology Group at JBEI is focused on understanding
and engineering enzyme hyper production phenotypes
into A. niger for use as a high-titer expression platform
for cellulase enzymes discovered at JBEI. The Fungal
Biotechnology group has created DNA constructs for
expression of heterologous enzymes in A. niger and has
begun screening these enzymes for expression. To
determine how well these bacterial-derived enzymes
express in A. niger, bacterial genes were transformed into
A. niger and the enzymes were assayed for activity using
the model cellulose substrates Azo-CM-cellulose (CMC),
p-Nitrophenyl β-D-cellobioside (pNPC) or p-Nitrophenyl
β-D-glucaopyranoside (pNPG) assays. Here we show that
these enzymes can be functionally expressed in A. niger
with varying degrees of success. Based on our initial
results, we will perform further assays on these enzymes
and apply forward and reverse genetic approaches to
identify genetic features responsible for high protein
secretion and engineer these features into a master high
production strain.
Objectives
→Express novel bacterial derived enzymes in
A. niger
→Assay expressed enzymes for activity .
John
1,4
Gladden
BioEnergy Institute, Lawrence Berkeley National Lab, 2University of California, Berkeley,
3Pacific Northwest National Laboratory, 4Sandia National Laboratory
Approach
Procedure
DNA
Build an A. niger expression construct based on the A. niger glucoamylase gene (glaA), which is a highly
expressed secreted enzyme. Sequences covering the glaA promoter, secretion signal peptide, and propeptide
were fused in frame with the thermophilic cellulases followed by the trpC terminator sequence from A.
nidulans. The hph gene from E. coli was used as a selectable marker, endowing transformants with resistance
to hygromycin B. All these parts were cloned into the E. coli bluescript (chloramphenicol resistant) backbone
to make the shuttle vector.
Shuttle vector
transformed into
protoplasts isolated
from A. niger
Bluescript
Plasmid Backbone
Transformants
grown on agar
plates containing
hygromycin
Glucoamylase
Promoter
A.Niger/ E. coli
Shuttle Vector
Hygromycin B
Resistance Gene
Heterologous Gene
with His/Strep Tags
Colonies selected
and spores grown
on agar slants
The shuttle vector was
assembled with GFP to test
the construct functionality in
A. niger. Expression was
detected but not all the GFP
appeared to be secreted
(targeting to the hyphal tips).
Some vesicles were observed
to persist in the hyphae.
trpC Terminator
Results
Maximum values of β-glucosidase activity and
cellobiohydrolase activity
Hmm-140p inoculated
with spores and
incubated for 3 days
Maximum values for endo 1,4-β-Glucanase activity
substrate: Azo-CM-cellulose
substrate: p-nitrophenyl glucopyranoside (pNPG) and pnitrophenyl β-D-cellobioside (pNPC)
8
7
6
5
pNPC
pNPG
4
3
2
1
Activity Units*/mL ezyme
solution/min
Abstract
Jed
1,3
Lynn ,
M p-nitrophenol released/
L enzyme solution/min
1Joint
2
Sibert ,
0.3
0.25
0.2
0.15
0.1
0.05
0
0
J26
Supernatant isolated
by centrifugal filter
J30
Gene transformed into A. niger
Gene transformed into A. niger
*Activity Units determined by reference to Trichoderma sp. endocellulase
standard curve on Azo-CM-Cellulose
Conclusions
→Active bacterial enzymes secreted
successfully from A. niger
Supernatant
tested for
activity
J28
Future Work
→Increase enzyme yield using forward
and reverse genetics approaches
Acknowledgments
This work, conducted by the Joint BioEnergy
Institute was supported by the U.S. Department of
Energy, Office of Workforce Development for
Teachers and Scientists (WDTS) under the Science
Undergraduate Laboratory Internship (SULI)
program, Office of Science, Office of Biological and
Environmental Research, of the U.S. Department of
Energy under Contract No. DE-AC02-05CH11231.