Supplementary Online Material
1 Creation of the transformants: detailed information
1.1 Synthetic constructs
The PhaC1 protein sequence from Pseudomonas resinovorans (ENA accession number
AAD26365.2) was back translated using the averaged codon usage of the genes of the
sophorolipid pathway (Genbank Accession numbers HM440974.1 (UGTB1), HQ670751
(AT), HM440973 (UGTA1), EU552419 (CYP52M1) and unpublished results) which was
determined using an online tool (Stothard, 2000). An SKL (TCTAAGCTG) peroxisomal
target sequence (PTS) found in at least one peroxisomal protein of Candida bombicola (Van
Bogaert et al., 2009a) was added to the 3’ terminus of the codon-optimized PHAC1co
sequence. Additionally the up- and downstream regulatory regions of the CYP52M1 gene
were added to the 5’ (488 bp) and 3’ (190 bp) side of the PHAC1co sequence respectively.
The 5’ UTR region, was extended beyond the promotor sequences to 1098 bp to obtain 1000
bp of homology needed for homologous recombination at the CYP52M1 locus. Codon usage
of the U. maydis glucosyltransferase gene UGT1 was optimized for C. bombicola in a
comparable way and XbaI restriction sites were added at both termini of the gene. The two
constructs were ordered at GenScript (Piscataway, USA) and obtained cloned into a pUC
vector.
1.2 Creation of the PHAC1co expression cassette
The PHAC1co construct was amplified from the obtained vector using primers
P55_FOR_upCYP_extNheI and P58_REV_PHAC1tCYP_extEcoRI (Table SI) yielding a
fragment of 2986 bp. The primers respectively contained NheI and EcoRI extensions allowing
subsequent digestion with said restriction enzymes for further subcloning. Creation of the
cassette occurred in three steps. All primers used for generation of the cassette can be found in
Table SI. First the URA3gene with its5’ and 3’ regulatory sequences (1970bp) was amplified
from genomic DNA of C. bombicola ATCC 22214, using primers P1_FOR_URA3v and
P2_REV_URA3v. This amplicon was subsequently cloned into the pGEM-T® vector
(Promega) yielding ‘pGEM-T_URA3’. Secondly the region for homologous recombination at
the 3’ end of the CYP52M1 locus (downcyp) was amplified from genomic DNA of C.
bombicola using primers P53_FOR_downCYP_extSpeI and P54_REV_downCYP_extNotI.
The resulting amplicon was digested with the unique cutters SpeI and NotI as was the pGEMT_URA3 vector and the restriction fragments were ligated after purification, yielding pGEMT_URA3_downcyp. A third and last step consisted of introduction of the synthetic construct
into the expression cassette. pGEM-T_URA3_downcyp was digested using the unique cutters
NheI and EcoRI. This double restriction digest yielded two fragments (5644 bp and 358 bp) of
which the largest one was gel purified and subsequently ligated with the digested and purified
synthetic construct. The PHAC1coexpression cassette was amplified from the resulting vector
(pGEM-T_cassette_PHAC1co)
using
primers
P63_FOR_cassPHAC1co
and
P64_REV_cassPHAC1co. The resulting linear PCR fragment was purified and used for
transformation of the C. bombicola PT36 strain (Figure S1). Transformants were selected on
SD plates. Correct integration of the PHAC1co expression cassette into the genome was
controlled by performing PCR on genomic DNA with primers further up- and downstream
than the primers used for amplification of the expression cassette, UDPGTA1Rev and
A21TotRev respectively. The resulting PCR product was purified and sent to LGC genomics
(Germany) for sequence analysis.
1.3 Creation of the UGT1co expression cassette
All primers used for generation of the cassette are given in Table SI. The codon optimized
UGT1co
gene
was
amplified
from
the
pUC-based
construct
using
primers
KIUGT1_UGT1For and KIUGT1_UGT1Rev which both contained extensions for fusion
PCR. The UGT1co gene was fused to the C. bombicola UGTB1 5’ and 3’ regulatory
sequences (Saerens et al., 2011b) by means of fusion PCR (Fang et al., 2002) with PfuUltra
DNA polymerase. Amplification of the UGTB1 5’ (P) and 3’ (T) regulatory sequences
occurred
with
primer
pairs
UGTB1-989Rev/KIUGT1_UGTB1PRev
and
KIUGT1_UGTB1TFor/UGTB1+1015Rev respectively from genomic DNA of C. bombicola
wild type. The obtained 3741 bp fusion product was ligated into pGEM-T® yielding
pG_PUGT1T from which the 2967 bpPUGT1T fragment was amplified using primers
UGTB1 -989Rev and PUGT1TRev_MluI. Additionally the 3’ end of the UGTB1coding
sequence followed by the URA3 selectable marker was amplified from plasmid pGKO_ugtB1
(Saerens et al., 2011b) with primers URA3-677For_MluI and GTII +1296Rev. The purified
PUGT1T fragment and selection marker were cut overnight with the single cutter MluI and
both fragments were ligated after purification. The ligation product (5777 bp) was amplified
from the ligation mix using Phusion High Fidelity polymerase (Finnzymes) with primers
UGTB1 -989Rev and GTII+1296Rev. After purification, the obtained expression cassette
PUGT1T_URA3GT2 was cloned into the pJET® cloning vector (Clontech) and sent for
sequencing. A linear expression cassette was created from the latter using primers
UGT1URA3 For and GTII +1296Rev and transformed into the Candida bombicola G9 strain.
Transformants were selected on SD medium. Correct integration of the UGT1co expression
cassette at the UGTB1 locus was controlled by two yeast colony PCR’s. Primers KIUGT1
CtrlFor2 and KIUGT1CtrlRev were used to check 3’ cross-over while primers Ura3endFor
and GTII+239Rev were used to check cross-over at the 5’ site of the UGTB1 locus. Two
transformants arising from double cross-over events were randomly selected for investigation
of their glycolipid production.
Table SI. Primers used for creation of the PHAC1co and UGT1co expression cassettes and
genotype control of the C. bombicola transformants. All primers were ordered at Sigma
Genosys.
Primername
Sequence ‘5-3’
P55_FOR_upCYP_extNheI
GTTGCTAGCTCTCGGCAGATTTCCTTG
P58_REV_PHAC1tCYP_extEcoRI
AGAATTCGTCGGTTAAACGCACTCCTTCA
KIUGT1 UGT1For
CATAAAACAATAAAGTCTCCTATATGGCGACCGAACATATTC
KIUGT1 UGT1Rev
CATATCTAGAAAAAGAGCCAGGTCAAAAGAGGCGGACTTCTGCC
P1_FOR_URA3v
AGAACAAGGCCGAGTATGTC
P2_REV_URA3v
TGCCAGCAGATCATCATCAC
P53_FOR_downCYP_extSpeI
TTACTAGTGTTTCTTAGCCTCCCATGGAAGAAACG
AATTGGCCTTGCGGCCGCGGTGTCGACTCGCCAAATTC
P54_REV_downCYP_extNotI
CATC
P63_FOR_cassPHAC1
CTCTCGGCAGATTTCCTTGTG
P64_REV_cassPHAC1
GGTGTCGACTCGCCAAATTC
UDPGTA1Rev
CCTACCTCTCTTCCCTGATCT
A21TotRev
GCTCTTGTTCGGTACTCTTATTG
UGTB1 -989Rev
CCTCGCCACCACCTAGTTTG
KIUGT1_UGTB1P Rev
GAATATGTTCGGTCGCCATATAGGAGACTTTATTGTTTTATGC
KIUGT1_UGTB1T For
CAGAAGTCCGCCTCTTTTGACCTGGCTCTTTTTCTAGATATG
UGTB1 +1015Rev
ATTGGCTGCTTAGTGGACTC
PUGT1TRev_MluI
CATAACGCGTTTCTGCTCTCAACACCGAG
URA-677For_MluI
GAGAACGCGTGATAGTACAGGCTTTGC
GTII+1296Rev
AGAAGCTAATTCACTAATTGCCGAC
UGT1URA For
TCTGGCGAATAACAGCACTC
KIUGT1CtrlFor2
GCGCCAGTTCCCATTGTGATTACC
KIUGT1 CtrlRev
ATAGGGCGTGCATGACCAAC
URAend For
TAAAGAAACGAAGGGCCCAGCAGTC
GTII +239Rev
CTGCTCTCAACACCGAGTGTAG
Figure S1. Schematic representation of the created PHAC1co and UGT1co expression
cassettes and their integration in the genome of a C. bombicola ∆ura3 mutant. The sites used
for homologous recombination are indicated by lines.
Figure S2. Mass spectra taken at the peak maxima of the new compounds in the
chromatogram of C. bombicola UGT1co transformant (UGT1) and corresponding spectrum of
C. bombicola ∆ugtB1. (a) compound eluting at 26.68 min (b) compound eluting at 28.17 min.