Biofuels in Perspective

(a)
Flo1p
FSProROL
FLProROL
Signal sequence
(b)
1
Amp r
Proposed flocculation functional domain
ori
2µm
term-ICL
ProROL
gene
SalI/XhoI
45aa x 18 repeats
1099
Pro region of ROL
TRP1
pWIFSProROL
(pWIFLProROL)
BglII/BamHI
UPR-ICL
Enzymatic Production of Biodiesel 145
FS- (FL-) anchor gene
1536
Membrane anchor
BglII/BamHI
Mature region of ROL
1447
Figure 8.15 (a) Structural features of Flo1p (76), FLProROL, and FSProROL. Arrowheads indicate possible
N-glycosylation sites. Open arrow-head indicates possible GPI attachment site (amino acid 1514). Numbers
written horizontally indicate length of proteins. The 45-amino-acid repeats comprise the proposed
flocculation functional domain. (b) Yeast expression plasmids pWIFSProROL and pWIFLProROL. (From Ref.
75, with permission of American Society for Microbiology.)
flocculation ability. This result leads to the conclusion that the hydrophobic C-terminal
region of Flo1p (1448–1536 amino acids) is not necessary for flocculation, but that the
C-terminal region of the FL anchor consisting of the 1100–1447 amino acids is.
8.5.3 Methanolysis Reaction Using Yeast Cells Displaying ProROL
In methanolysis reaction of plant oil, reaction substrates such as methanol and triglyceride
can easily access surface-displayed lipase. Matsumoto et al. 75 therefore used yeast cells
displaying FSProROL and FLProROL for biodesel production without permeabilizing
treatment. Figure 8.17 shows the time course of ME content in methanolysis reaction. In
yeast cells displaying FSProROL and FLProROL, ME content after 72-h reaction reached
78.6 % and 73.5 %, respectively, with two-step addition of methanol. Further, the initial
reaction rate, defined as ME production rate, reached 3.2 g-ME/1εmin when 150 mg of
lyophilized FSProROL-displaying cells were used (data not shown), a level as high as that
for ROL in enzyme solution. 22