genetically engineered glucose/xylose co ... - Purdue University

fretfulcrunchBiotechnology

Dec 10, 2012 (4 years and 9 months ago)

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GENETIC ENGINEERING OF GLUCOSE/XYLOSE CO
GENETIC ENGINEERING OF GLUCOSE/XYLOSE CO
-
-
FERMENTING
FERMENTING
SACCHAROMYCES
SACCHAROMYCES
YEAST FOR CO
YEAST FOR CO
-
-
PRODUCTION OF ETHANOL
PRODUCTION OF ETHANOL
AND VARIOUS INDUSTRIAL ENZYMES
AND VARIOUS INDUSTRIAL ENZYMES
Miroslav Sedlak
Miroslav Sedlak
1
1
,
,
Zhengdao
Zhengdao
Chen
Chen
1
1
,
,
Yanfang
Yanfang
Pang
Pang
2
2
, Todd Applegate
, Todd Applegate
2
2
, and Nancy Y.W. Ho
, and Nancy Y.W. Ho
1
1
1
1
Laboratory of Renewable Resources Engineering (LORRE),
Laboratory of Renewable Resources Engineering (LORRE),
2
2
Department of Animal Sciences
Department of Animal Sciences
Purdue University, West Lafayette, IN, 47907
Purdue University, West Lafayette, IN, 47907

The PCR and Overlap-Extension PCR (OE-PCR) were used to construct a phytase
expression cassette (Figure 1A).
• The phytase expression cassette was cloned into pKS2µKmXK and transformed to
Saccharomyces yeasts (Figure 1B).
• The expression and secretion of phytase by Saccharomyces yeasts during growth
and ethanol fermentation was monitored by immunoblot and measuring of
enzymatic activity (Figure 2, 3 and Table 1).
• The deglycosilation of phytase produced by Saccharomyces yeast was performed by
Endoglycosidase H
f
(New England Biolabs Inc) (Figure 4).
• The isolation of phytase from the fermentation medium was carried out by ultra-
filtration using the High-performance Ultra-filtration Cell Model 2000 (Amicon).
• The effect of Saccharomyces produced phytase on the chicken’s performance, and
tibia ash was tested in male chickens (Tables 2 and 3).
Material and Methods
Material and Methods
Recent studies have proven ethanol to be an ideal liquid fuel for transportation,
and renewable cellulosic materials (lignocelluloses) to be attractive feedstock for
ethanol-fuel production by fermentation. The major fermentable sugars from the
hydrolysis of cellulosic biomass (such as rice straw, sugarcane bagasse, corn fiber,
softwood, hardwood, grasses, etc.) are D- glucose and D-xylose. The efficient
fermentation of xylose from hydrolysates of lignocelluloses will significantly improve
the economics of large-scale fuel ethanol production from these types of feed-stock. In
recent years we have developed genetically engineered yeasts that can effectively co-
ferment both glucose and xylose simultaneously to ethanol.
Our genetically engineered glucose/xylose co-fermenting Saccharomyces yeasts, such
as 424A(LNH-ST) (our super-stable engineered Saccharomyces yeast) have exceeded
all expectations in co-fermenting glucose and xylose to ethanol. Despite using such
efficient yeasts, fuel ethanol production from lignocellulosic biomass is still a costly
process. The production costs for ethanol from lignocellulosic biomass have to be very
low in order to be competitive and profitable. One way to reduce the overall cost for
the production of ethanol is to produce high valued co-products or by-products
during ethanol production. We believe that making our yeast able to co-produce
various industrial enzymes could create additional high priced products for sale and
therefore reduce the overall cost of ethanol production.
One important industrial enzyme is phytase, which is used as a supplement in animal
feed. Phosphorus is an expensive nutrient in poultry diets; therefore, there is an
incentive to minimize dietary levels of phosphorus supplements during formulation.
Recently there has been concern about phosphorus levels in manure of farm animals,
which are largely from indigestible phytic phosphorus present in animal feed. The
phytase improves phosphorus utilization from animal diet and reduces phosphorus
pollution from their excreta.
In this presentation we report successful over-expression and secretion of
Escherichia coli phytase encoded by the appA gene by Saccharomyces yeasts during
growth and ethanol fermentation. We also show that phytase produced by our
Saccharomyces yeasts is more efficient in releasing phytate-bound phosphorus in
chickens than two commercially available phytases.
Introduction
Introduction
In the present study, we demonstrated that Saccharomyces yeasts are
able to express the E. coli appA gene. We verified extracellular presence
of phytase by Western blotting analyses and by measuring phytase
activity in a cultural medium.
The Western blotting analyses demonstrated the presence of two
major protein bands of approximately 58.3 and 55.5 kDa in size (Figure
3, 4). Deglycosylation of the enzyme produced a single protein band of
approximately 50 kDa (Figure 4), which is in agreement with the results
reported by others, indicating that two bands of higher masses are due to
different degree of glycosylation. We have also shown that the E. coli
phytase can be produced in Saccharomyces yeast under the control of a
constitutive (PGI) or inducible (Gal10) Promoter (Figure 2). The levels of
phytase secretion were 12000-15000 U/L in 24 hours from both promoters
(Figure 2). This is 4-5 times more than was achieved by cloning and
expression of phytase (phyA) from Aspergillus niger in S. cerevisiae. Our
results indicated that the E. coli phytase was produced mainly during the
growth stage (Figure 3, Table 1), but the level of phytase remained
reasonably stable during ethanol fermentation (Table 1).
Phytic acid serves as storage of phosphorus in grains used as the feed
for farm animals and is relatively unavailable to most animals. However,
the enzyme phytase effectively releases phosphorus from phytate when
the enzyme is included in the diet. The phytase as a nutritional
supplement has been shown to dramatically increase hydrolysis of
phytate-phosphorus, thereby minimizing the need for inorganic
phosphorus supplementation and lowering the phosphorus content of
manure.
Because the E. coli phytase produced by Saccharomyces yeast is
glycosylated, we tested its effectiveness as a feed additive for poultry in
vivo. We tested bird performance (Table 2) and bone characteristics of
male chickens (Table 3) that were fed sub-optimal levels of phosphorus
with and without 750 U phytase/kg. We also compared efficiency of
phytase produced by our Saccharomyces yeast to two commercially
available phytases (Tables 2 and 3). Our results indicated that the
phytase produced by our recombinant yeast is nearly twice as efficient as
commercially available phytases (Tables 2 and 3). The increase in
efficiency of our yeast-produced phytase could be explained by the
differences in biochemical properties between phytases of different
origins. However, the glycosylation of our cloned enzyme might also
contribute to higher stability of the enzyme and make it more resistant to
gastric and pancreatic proteases.
The results presented in this study confirmed the feasibility of
producing important industrial enzymes such as phytase as the co-
products of ethanol fermentation. High valued co-products or by-
products like phytase could make production of ethanol more cost-
effective and profitable.
Result and Conclusion
Result and Conclusion
6753 bps
XK
Km
Ori
Amp
pKS2µKmXK

XhoI
ApaI
Gal10 (PGI)
LS
ATG
PHY
6xHis
PGI-LS-PHY6xHis
(1857 bps)
ApaI
XhoI
PGI
LS
ATG
PHY
6xHis
Gal10-LS-PHY6xHis
(2190 bps)
ApaI
XhoI
Gal10
LS
ATG
PHY
6xHis
Figure 1A
.
. Phytase expression cassettes
Figure 1B. Plasmid pKS2µKmXK bearing phytase expression cassette
Hours
0 20 40 60 80
U/mL
0
5
10
15
20
25
Gal10, 50 ml
Gal10, 100 ml
PGI, 50ml
PGI, 100ml
Figure 2. Production of cloned phytase under the control of
Gal10 or PGI promoter by Saccharomyces yeast
Table 1. Activity of cloned phytase secreted by Saccharomyces yeast into
medium during growth and fermentation




Time Activity (U/L)

T
0
12000

T
1
10046

T
2
9628





T
0
– 24 hours growth, T
1
– 16 hours fermentation, T
2
– 48 hours fermentation

Phytase Source Tibia ash
U/kg (%) (% nPP spared)

0 - 43.08 -0.020

750 Ronozyme 46.42 0.066

750 Nathuphos 45.88 0.052

750 LORRE 50.01 0.158

SEM 0.51 0.0125

Prob. of source effect
0.0001 0.0001


Table 3. Effect of
E. coli
phytase produced by
Saccharomyces
yeast and two
commercial phytase sources on tibia ash in male chickens at 22 d of age
Table 2. Effect of
E. coli
phytase produced by
Saccharomyces
yeast and two
commercial phytase sources on performance in male chickens

Phytase Source 8 d BW 21 d BW Gain Feed

consumed
(U/kg) g/bird

0
-
618.3 3069.7 408.6 714.0

750
Ronozyme
616.0 3225.8 435.0 672.6

750
Nathuphos
618.5 3302.3 447.3 663.7

750 LORRE 620.2 3609.7 498.3 636.7

SEM 3.24 94.42 15.59 13.76

Prob. of source effect
0.8165 0.0041 0.0039 0.0059


Figure 4. Western blot analyses of extracellular
phytase protein expressed by
Saccharomyces
yeast bearing cloned
E. coli
phytase before and
after deglycosylation. Line: N1, N2 – glycosylated
phytase; D1, D2 – deglycosylated phytase.
Figure 3. Western blot analyses of extracellular
phytase protein expressed by
Saccharomyces
yeast bearing cloned
E. coli
phytase during
growth and fermentation. Line: 1, 4 – T
0
;
2, 5 – T
1
; 3, 6 – T
2
.
A – Stained by Coomassie blue; B – Immunodetection with Anti-His HRP conjugate
M1, M2 –molecular weight markers