Genetic Engineering of S. cerevisiae for Pentose Utilization - EERE

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11 Δεκ 2012 (πριν από 4 χρόνια και 11 μήνες)

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Oral presentation 5
-
02


Genetic Engineering of
S. cerevisiae

for Pentose Utilization


Peter Richard
*, Ritva Verho, John Londesborough and Merja Penttilä


VTT Biotechnology, PO Box 1500, 02044 VTT, Finland

Phone: +358 9 456 7190 email: Peter.Richard@vtt
.fi


The two most widespread pentose sugars in our biosphere are D
-
xylose and L
-
arabinose. The pentose
catabolic pathways are relevant for microorganisms living on decaying plant material and also in
biotechnology when cheap raw materials such as plant hyd
rolysates are fermented to ethanol.
In fungi,
i.e. in yeast and mold, L
-
arabinose is sequentially converted to L
-
arabinitol, L
-
xylulose, xylitol and D
-
xylulose and enters the pentose phosphate pathway as D
-
xylulose 5
-
phosphate. In molds the reductions
are

NADPH
-
linked and the oxidations are NAD
+
-
linked.

We recently identified the two missing genes in
this pathway. The functional overexpression of all the genes of the pathway in
S. cerevisiae

led to growth
on L
-
arabinose and ethanol production under anaerob
ic conditions however at very low rates.
In this
communication we show that in a yeast species the L
-
arabinose pathway is similar, i.e. it has the same
two reduction and two oxidation reactions, but the reduction by L
-
xylulose reductase is not performed by

a strictly NADPH
-
dependent enzyme as in molds but by a strictly NADH
-
dependent enzyme. To our
knowledge this is the first report of an NADH
-
linked L
-
xylulose reductase. D
-
xylose fermentation to
ethanol with recombinant
S. cerevisiae

is often slow and has
a low yield. One reason is that the
catabolism of these pentoses through the corresponding fungal pathways creates an imbalance of redox
cofactors. The process, although redox neutral, requires NADPH which must be regenerated in a separate
process. To faci
litate the NADPH regeneration, the recently discovered gene
GDP1

coding for a fungal
NADP GAPDH was expressed in a
S. cerevisiae

strain with the D
-
xylose pathway. Glucose 6
-
phosphate
dehydrogenase is the main path for NADPH regeneration, however it causes
futile CO
2

production and
creates a redox imbalance on the pathway for anaerobic fermentation to ethanol. The deletion of the
corresponding gene,
zwf1
, in combination with overexpression of
GDP1

could stimulate D
-
xylose
fermentation with respect to rate an
d yield; i.e. less CO
2

and xylitol were produced. Through redox
engineering a yeast strain, which was mainly producing xylitol and CO
2

from D
-
xylose, was converted to
a strain producing mainly ethanol.