Identification, characterization of a flavin-containing monooxygenase MoA and its function in a

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1 1 Applied Microbiology and Biotechnology 2 Supplementary Material 3 4 Identification, characterization of a flavin-containing monooxygenase MoA and its function in a specific sophorolipid molecule metabolism in Starmerella bombicola Running title: Identification, characterization of a flavin-containing monooxygenase Jiashan Li 1, Hui Li 1, Weiwei Li 1, Chengqiang Xia 1, Xin Song 1, 2, * State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Shan Da Nan Road 27, Jinan, Shandong , P. R. China National Glycoengineering Research Center, Shandong University, Shan Da Nan Road 27, Jinan, Shandong , P. R. China *Corresponding author 15 songx@sdu.edu.cn 16 Tel/Fax:

2 Electronic Supplementary Material Contents 1. Primers used in the construction of deletion cassettes 2. Primers used in identification of deletion mutants 3. Primers used in the construction of overexpression cassette 4. Primers used in construction of recombinant plasmid 5. Identification of total sophorolipids from oleic acid as hydrophobic carbon source 6. Identification of total sophorolipids from linoleic acid as hydrophobic carbon source 7. Identification of sophorolipid substrates by HPLC-MS 2

3 Primers used in the construction of deletion cassettes Electronic supplementary material Table S1 Primers used in the construction of the moa and mob deletion cassettes Gene Primers Sequence (5 to 3 ) moa-5 flankingfor moa-5 flankingrev AAGCTTACGAATGTCAGGTGAA GATTACAGGAATGATACAACAGGCCTCTCGGCATGACGAC moa mob moa-3 flankingfor moa-3 flankingrev moa-gfor moa-grev mob-5 flankingfor mob-5 flankingrev mob-3 flankingfor mob-3 flankingrev mob-gfor mob-grev AGCT GATAATGATTCCGCAGCACAAGCAATATCAGGGTACTTGTG CTT GTCCCATGCTTTATGCTGTG AACAGGTGAGCCCTTCGTGGT AAGTCGCGGTGCAGCAATCA GCTGAGAATCGTACTCGCTGA GATTACAGGAATGATACAACAGGCCAGAAGGCTCTATCTG GAGTATGG GATAATGATTCCGCAGCACAAGCCGCTCGCATATCATGTGG TA TGGATGGAAGACGGCACA TTGATACTGTCATCGAAGGCATAGGA TGCCCTATCCGAGTCTCCAAAC hph hphexfor GGCCTGTTGTATCATTCCTGTA hphexrev GCTTGTGCTGCGGAATCATTA 3

4 Primers used in identification of deletion mutants Electronic supplementary material Table S2 Primers of the deleted coding sequences used in identification of deletion mutants Gene Primers Sequence (5 to 3 ) moa mob moa-cds-for moa-cds-rev mob-cds-for mob-cds-rev GAATAGCTGCAGTCAGAGCCCT TCTCTCTAGCCCCAAGCGAC CTTGTCAGCGAGCCCTATGAT CATGTGCATGTACTCAACATCCG 4

5 Primers used in the construction of overexpression cassette Electronic supplementary material Table S3 Primers used in the construction of Peno::moA overexpression cassette Primers Sequence (5 to 3 ) shph-for shph-rev Peno-for Peno-rev moa-for moa-rev Ttrpc-for Ttrpc-rev 5 flankingfor 5 flankingrev 3 flankingfor 3 flankingrev P-for T-rev OE-nest-for OE-nest-rev GTTAACTAGACGTGCATACATGCGAGTT GAGAGAGTGATTTCTAGCGGCGGCCGCTATGGTTTCCACGTGCACTTACA GCGGCCGCCGCTAGAAATCACTCTCTCGTTTC GGCCCGGGAACGGCCATTTCTAATAGATGTTT TTAGAAATGGCCGTTCCCGGGCCTAGGATGGGCCGCAGGGTTGTT TTCAGTAACGTTAAGTGGATCGGATCCCTAATTGGTGCTGAGTCTCTTCTCT GGATCCGATCCACTTAACGTTACTGAAATCA GTTTAAACAACCCAGGGGCTGGTGAC CAGAAACGGGAATAATGGACATT CTCGCATGTATGCACGTCTAGTTAACGTATGGTCCAGTGGTTTCAAAGGTT GTCACCAGCCCCTGGGTTGTTTAAACTTCTGCAGGAGCGGTTTTG AGCTGTTGAGGCTGATGATAAATC TCCATTACACCCTCGCGGCTCTCTC CAAGAGCGGATTCCTCAGTCT ATGTCACTTCACCGCACACTATTTC TTACTGAAAAATTTCTTGCACCCT 5

6 Primers used in construction of recombinant plasmid Electronic supplementary material Table S4 Primers used in construction of recombinant plasmid pmal-c2x-moa Gene Primers Sequence (5 to 3 ) moa pmal-moa-for TATGGATCCATGGGCCGCAGGGTTGTT pmal-moa-rev TATTCTAGACTAATTGGTGCTGAGTCTCTTCTCT 6

7 Identification of total sophorolipids from oleic acid as hydrophobic carbon source Electronic supplementary material Table S5 Identification of total sophorolipids from oleic acid as hydrophobic carbon source based on m/z peaks of protonated molecular ion No RT (min) MW Sophorolipid analogs Non-acetylated acidic SL with a C 18:2 fatty acid Mono-acetylated acidic SL with a C 18:1 fatty acid Di-acetylated acidic SL with a C 18:2 fatty acid Di-acetylated lactonic SL with a C 18:2 fatty acid Di-acetylated lactonic SL with a C 18:1 fatty acid 7

8 6. Identification of total sophorolipids from linoleic acid as hydrophobic carbon source Electronic supplementary material Table S6 Identification of total sophorolipids from linoleic acid as hydrophobic carbon source based on m/z peaks of protonated molecular ion No RT (min) MW Sophorolipid analogs * Di-acetylated acidic SL with a C 18:2 fatty acid* Mono-acetylated acidic SL with a C 18:0 fatty acid Mono-acetylated acidic SL with a C 18:2 fatty acid Di-acetylated acidic SL with a C 18:2 fatty acid Di-acetylated lactonic SL with a C 18:2 fatty acid Di-acetylated lactonic SL with a C 18:1 fatty acid 8

9 7. Identification of sophorolipid substrates by HPLC-MS Electronic supplementary material Table S7 Identification of sophorolipid substrates by HPLC-MS SL structure M+H-H 2 O M+H M+NH 4 M+Na M+K C18:1 DASL C18:2 DASL C18:1 DLSL

10 Electronic supplementary material Fig. S1 Construction of monooxygenase genes knock-out mutants moa and mob. (a) schematic illustration of the construction of moa/mob gene deletion cassette using double-joint PCR method, (b) schematic illustration of the construction of moa mutant by homologous recombination, (c) schematic illustration of the construction of mob mutant by homologous recombination 10

11 Electronic supplementary material Fig. S2 Construction of monooxygenase A overexpressed mutant (a) schematic illustration of the construction of overexpression cassette through three times of double-joint PCR, (b) the non-coding region was replaced by the overexpression cassette containing hygromycin (hyg) resistance gene sequence homologously 11

12 Electronic supplementary material Fig. S3 The specific MS chromatogram of purified peak 1 and peak 4 from the crude sophorolipids produced by moa. (a) the MS and MS/MS chromatogram of peak 1 [M+H] + = (b) the MS and MS/MS chromatogram of peak 4, [M+H] + = , [M+NH 4 ] + = , [M+K] + = The MS/MS fragments of [M+H] + = 705 were the same between that from peak 1 and peak 4 12

13 Electronic supplementary material Fig. S4 Identification of sophorolipid substrates by HPLC-MS (a) C18:1 DASL, (b) C18:2 DASL, (c) C18:1 DLSL 13

14 Electronic supplementary material Fig. S5 Identification of the products catalyzed by MoA when C18:2 DASL was used as substrates by MS. (a) specific chromatogram of fragments from sophorose with no acetyl [M+H] + = , [M-H 2 O+H] + = ; sophorose with one acetyl [M+H] + = , [M-H 2 O+H] + = ; sophorose with two acetyls [M-H 2 O+H] + = , [M-2H 2 O+H] + = , [M-3H 2 O+H] + = (b) specific chromatogram of fragments from hydroxylated linoleic acid [M+H] + = , [M-H 2 O+H] + =