C. Sso1354 promotes the consumption of longer cellodextrins

1
Hyperthermoacidophiles for biomass deconvolution
Cellodextrins hydrolysis and transport in an
extremophile Sulfolobus solfataricus
Sreedevi Madhusoodhanan, Landon Peterson, Karl
Dana, Dr. Paul Blum NCESR, NUenergy and School of
Biological Sciences, University of Nebraska
Lincoln
pblum1_at_unl.edu
B. Region of duplication in SsoP2 at the genomic
environment of Sso1354
E. Construction of cellodextrins transporter
Sso3053 cassette deletion and complementation in
Sso98/2
Abstract
Extremely thermoacidophilic microbes such as
Sulfolobus solfataricus use soluble cellodextrins
as sole carbon and energy sources. In this study,
endoglucanases and transporters required for this
process were identified using a combination of
comparative genomics and genetics in assays that
coupled substrate utilization to growth. S.
solfataricus strain-specific genomic differences
indicated that strain 98/2 lacks endoglucanase
Sso1354 while two other endoglucanases are shared
includingSso1949 and Sso2534. Plasmid-based
expression of Sso1354 in strain 98/2 conferred
the ability to rapidly hydrolyze longer
oligosaccharides including cellohexaose (G6)
through cellonanaose (G9). Protein transporters
required for cellodextrin uptake were identified
through mutagenesis and complementation of an ABC
transporter cassette including a putative
oligosaccharide binding protein, Sso3053. In
addition, Sso3053 ablation compromised growth on
glucose while inactivation of the glucose
transporter, Sso2847, had no impact. These data
demonstrate that Sulfolobus solfataricus has
redundant mechanisms for soluble cellodextrin
catabolism comprised of both uptake and
extracytoplasmic hydrolytic properties.
A
A. The construction of Sso3053 cassette knockout
in Sso98/2 by circular plasmid transformation. B.
PCR validation if the Sso3053 deletion using
Sso3053 specific internal primers. 1. DNA ladder,
2. no DNA control, Sso98/2, Sso3053 knockout. C.
pKlacSSso3053 with natural promoter construct
for complementation into PBL2028 strain. D.
PBL2028 strain genotype showing deletion of genes
from Sso3017 through Sso3050 that includes lacS
as well as insertion in the Sso3053 gene. E. PCR
validation using the Sso3053 internal primers
showing an insertion in PBL2028. 1. DNA ladder,
no DNA control, Sso98/2, PBL2028.
Sequencing results showing the deletion region in
Sso98/2 Vs SsoP2
B
C
D
E
F. Sso3053 is essential for cellodextrins
transport
C. Sso1354 promotes the consumption of longer
cellodextrins
A
B
A
C
A. Lanes 1, Glucose 2, Cellodextrins mix 3,
spent media from Sso 98/2 4, spent media from
Sso P2 5, spent media from Sso 98/2 complemented
by Sso1354 6, Non inoculated media. B. pKlacS
construct with Sso1354 native promoter insert
for complementation into Sso98/2 strain. C. The
HPLC analysis results of the spents after 3
cycles of Sso98/2, Sso98/2Sso1354 and SsoP2 in
cellodextrins.
Introduction
Cellulosic ethanol is an emerging solution for
biofuels that will make strong contributions to
American domestic energy needs. This study
focuses on the development of process-compatible
enzymes and organisms to convert biomass derived
lignocellulose feedstocks into biofuels and value
added chemicals. The Crenarchaeote Sulfolobus
solfataricus (Sso) is an extreme thermoacidophile
and is known to have three GHF12 endoglucanases.
The primary focus of the research is on one of
the three endoglucanases Sso1354. Apart from
Sso1354, the other two enzymes Sso2534 and
Sso1949 that are reported as endoglucanases
turned out to be nonfunctional on longer
cellodextrins during the in vivo analysis. The
competitive functions of the intracellular
beta-glucosidase and the putative cellodextrins
transporter in the process of cellodextrins
uptake and hydrolysis is also considered in this
study.
B
C
D
D. Role of intracellular beta glucosidase,
putative cellodextrins transporter and Sso1354
A. A proposed model for the hydrolysis and
transport of cellodextrins in Sulfolobus
solfataricus
Growth in A. BSG Glucose minimal media, B. BSM
Maltose minimal media, C. BSp14CD (v/v), D.
HPLC analysis results of the spent in BSp14CD
(v/v) by the three strains.
B
A
Conclusions Sso1354 is present at a
duplication region in SsoP2 strain Sso1354 is
naturally absent in Sso98/2 strain Sso1354 helps
in the hydrolysis of longer cellodextrins Sso3053
is required for cellodextrins transport and
partially required for maldextrins transport in
Sso. Sso2847 is not required for glucose
transport in Sso.
A The longer cellodextrins consumption pattern of
various strains in PBL2092 (Endoglucanase triple
knock out) spent media B. HPLC analysis results
of the spent of various strains in PBL2092
(Endoglucanase triple knock out) spent 3.
Sso1354 hydrolyzes longer cellodextrins upto G9.
Beta glucosidase and cellodextrins transporter
assists in the consumption of cellodextrins upto
G8.