yea_1914_sm_materials_and_methods

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<Supporting information>
+A: Supplementary materials and methods
+B: Disruption of genes encoding DAG acyltransferase homologues in Y. lipolytica
90812
Gene disruptions in strain 90812 were done by double homologous recombination with
linear disrupting cassettes, as previously described (Yadav et al., 2007). Briefly, the
DGAT2 disrupting cassette consisted of hygromycin resistance gene flanked by 640 bp
of 5 DGAT2 sequence at one end and 453 bp of 3 DGAT2 sequence at the other. The
PDAT disrupting cassette was Y. lipolytica URA3 gene (GenBank AJ306421) and E.
coli vector flanked by 494 bp of 5 PDAT sequence at one end and 498 bp of 3 PDAT
sequence at the other. The DGAT1 disrupting cassette was Y. lipolytica LEU gene
(GenBank AAA35244) flanked by 598 bp of 5 DGAT1 sequence at one end and and
658 bp of 3 DGAT1 sequence at the other. Strain 90812 DAG acyltransferase mutants
are shown in Table 3.
+B: Disruption of DAG acyltransferase genes in Y. lipolytica 20362
For DGAT1 and DGAT2 gene disruptions, linear disruption cassettes contained Y.
lipolytica URA3 gene (GenBank AJ306421) flanked by 34 bp Lox sites (‘floxed’ URA3),
such that URA3 can be excised to result in uracil auxotrophy by the transient expression of
Cre site-specific recombinase from an autonomously replicating plasmid, followed by
curing of the Cre plasmid, as previously described (Damude et al., 2006). The ‘floxed’
URA3 was flanked by 735 bp 5 DGAT2 sequence at one end and 720 bp 3 DGAT2
sequence at the other in the DGAT2 disruption cassette, and by 598 bp 5 DGAT1 sequence
at one end and 658 bp 3 DGAT1 sequence at the other in the DGAT1 disruption cassette.
The PDAT disrupting cassette was either the linear DNA fragment used in strain 90812 or
a 7121 bp circular plasmid, pY185. Linear DAG acyltransferase disruption cassettes were
transformed into Y2224. Uracil prototrophs were screened by PCR (see below) to identify
two dgat1 mutants (one designated L190) out of 30 transformants, one dgat2 mutant
(L110) out of 10 transformants, and one pdat mutant (L192) out of 24 transformants. An
uracil auxotroph of dgat1 mutant (L113) was used to obtain a dgat1/dgat2 double mutant
(L171).
+B: Disruption of PDAT in dgat1/dgat2 double mutant in Y. lipolytica 20362
When a uracil auxotroph (L183) of dgat1/dgat2 double mutant (L171) was transformed
with the linear PDAT disruption cassette, no pdat mutant was identified in 170
transformants by PCR using primers 37/38 (data not shown). Disruption of PDAT in
L171was then attempted by an alternative method that uses a plasmid and a two-step
homologous recombination event (Leon et al., 2002). For this strain, L183 was
transformed with plasmid pY185, which consists of an E. coli plasmid carrying Y.
lipolytica URA3 gene and 1006 bp 5 PDAT sequence and 1005 bp of 3 PDAT sequence,
separated by 889 bp of ‘stuffer’, a non functional DNA fragment derived from the Y.
lipolytica LEU gene. Plasmid pY185 was linearized with XbaI site present in the 5 PDAT
homology to facilitate integration of the entire circular plasmid (Leon et al., 2002)
(manuscript Figure 1). Strain L183 was transformed by linearized pY185 and the resultant
uracil prototrophs screened by PCR, using PCR primers 910/911 to identify four out of 46
transformants as first crossover events by the presence of 1173 bp PCR product. These
represent a ‘conditional’ pdat mutant, in which both the inserted pY185 (containing the
disrupted copy of PDAT and URA3 marker) as well as the native wild-type PDAT gene
are present (Figure 3). All four first crossover events were plated on FOA plates and
nine resulting uracil auxotrophs of each were screened by PCR, using primers 912/913.
All 36 showed ca. 2504 bp PCR products, confirming them to be second crossover
events with the loss of URA3 and indicative of the presence of only the native PDAT
gene and not the 2134 bp product indicative of the presence of only the pdat mutant
gene (data not shown). This is in contrast to the expected frequency of 50% of each.
One first crossover line, designated L280, was also transformed with a DNA fragment
carrying a Y. lipolytica mutant acetolactate synthase gene that confers resistance to
sulphonylurea (Damude et al., 2006) and a chimeric gene comprised of Y. lipolytica YAT1
promoter (Damude et al., 2006) linked to Y. lipolytica DGAT2 ORF. One resulting
sulphonylurea-resistant transformant was designated L290. After overnight growth in
YPD, L280 and L290 were plated on FOA plates and 30 normal-sized colonies each of
L280 and L290 were picked after 5 days and 36 small L280 colonies were picked after
8 days. All were confirmed second crossover events by uracil auxotrophy and by PCR,
using primers 912/144. Of the 30 L290 second crossover events, 25 showed the
presence of only the wild-type PDAT gene and five of only the pdat mutant gene,
whereas all 30 L280 second crossover events showed the presence only of the wild-type
PDAT gene. However, of the 36 small L280 second crossover colonies, colony PCR
showed the presence of the wild-type PDAT gene in 18 (one designated L296), pdat
mutant gene (two of which were designated L294 and L297) in seven, and no PCR
product was seen in 11. PCR using primers 921/922 also confirmed that L294 and L297
are dgat1/dgat2/pdat triple mutants (TMs), while L296 is dgat1/dgat2 double mutant
(DM). Strain 20362 DAG acyltransferase mutants are shown in Table 3.
+B: PCR screening for DAG acyltransferase mutants
Targeted disruptions were identified by PCR, using two sets of PCR primers. One set
was designed to amplify a junction fragment using ‘integration-specific’ primers, one
primer specific to the genomic sequence absent in the disrupting cassette, and the other
specific to the disrupting cassette outside the target gene homology regions. The second
set was designed to amplify the native fragment using ‘native’ primers specific to 5
and 3 genomic regions of the target gene outside the two homology regions in the
disrupting cassette. Disruptions were confirmed when transformants were positive for
the junction fragment and negative for the native fragment expected from the wild-type
gene. PCR primers used for screening DAG acyltransferase mutants in strain 20362 are
shown in Table 1. PCR was done either by colony PCR or on extracted genomic DNA.
Colony PCR was performed using MasterAmp kit (Epicentre, Madison, WI, USA)
and the manufacturer’s instructions. A single colony was resuspended in 25 l
containing 0.2 M of each primer, 0.25 mM each dNTP and 0.25 l (1.25 u) of
MasterAmp Taq DNA polymerase (Epicentre, Cat. No. Q82110) in the manufacturer’s
reaction buffer. Genomic DNA was extracted using YeaStar Genomic DNA kit (Zymo
Research, Cat. No. D2002) and the manufacturer’s instructions. PCR on extracted DNA
was performed in 25 l containing 1 l template DNA, 0.2 M each primer, 0.25 mM
each dNTP and 0.5 l (2.5 u) TaKaRa Taq (Takara Bio Inc., Cat. No. RR002M) in the
manufacturer’s reaction buffer. In both methods PCR amplification was carried out in a
MasterCycler Gradient PCR machine (Eppendorf) as follows: initial denaturation at
95°C 4 min, 30 cycles (40 cycles for colony PCR) of denaturation (95°C, 30 s),
annealing (55°C, 1 min) and elongation (72°C, 1 min) and a final elongation (72°C, 10
min). 15 l of each PCR reaction was loaded on 1% agarose gel and subjected to
electrophoresis. 1 kb DNA Ladder (Novagen, Cat. No, 70537) was used as size marker.
The expected PCR product sizes (Table 2) were used to confirm gene disruption.
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