1
2
3
4
5
Whole-cell biotransformation systems for reduction of prochiral
6
carbonyl compounds to chiral alcohol in Escherichia coli
7
8
9
10
Bingjuan Li1, Yuxia Li1, Dongmei Bai1, Xin Zhang1, Huiying Yang2, Jie Wang2,
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Gang Liu1, Juejie Yue1, Yan Ling1, Dongsheng Zhou2, Huipeng Chen1
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13
14
15
16
17
18
Table S1 Oligonucleotide primers used in this study
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Oligonucleotide
ADHF
ADHR
FDHF
FDHR
ADH-LINR1
ADH-LINR2
LIN-FDHF1
LIN-FDHF2
FDH-LINR1
FDH-LINR2
LIN-ADHF1
LIN-ADHF2
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Sequence (5,→3,)
GACCATGGGCTCTAACCGTCTGGAC
GTCTCGAGTTACTGAGCGGTGTAAC
GACCATGGGCAAAATCGTTCTGGTTCTG
GTTGCGGCCGCTTATTTTTTGTCGT
GCCGCCCCCGCTGCCGCCGCCCCCTGCGGCCGCCTGAG
TTTGGCCGCTGCTTCTTTGGCCGCTGCTTCTGCGGCCGCCTGAG
CGGCAGCGGGGGCGGCGGCAGCAAAATCGTTCTGGTTCTG
GCAGCGGCCAAAGAAGCAGCGGCCAAAAAAATCGTTCTGG
CGCCCCCGCTGCCGCCGCCCCCTTTTTTGTCGTGTT
TTTGGCCGCTGCTTCTTTGGCCGCTGCTTCTTTTTTGTCGTGTT
CGGCAGCGGGGGCGGCGGCAGCTCTAACCGTCTGGAC
GCAGCGGCCAAAGAAGCAGCGGCCAAATCTAACCGTCTGGAC
Linker sequences are underlined. The stop codons are italicized.
1
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22
23
24
25
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Figure S1 SDS-PAGE analysis of over-expressed recombinant proteins.
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The lysates of E. coli cells were centrifuged to collect any insoluble material and the
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supernatants were separated on a 12% polyacrylamide gel. Lanes 1 and 2, soluble
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and insoluble protein fraction of Rosetta (DE3)-pETDuet (negative control),
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respectively; lanes 3 and 4, soluble and insoluble protein fraction of Rosetta
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(DE3)-pETDuet-adh-lin1-fdh, respectively; lanes 5 and Lane 6, soluble and
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insoluble protein fraction of Rosetta (DE3)-pETDuet-adh-lin2-fdh, respectively;
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lanes 7 and 8, soluble and insoluble protein fraction of Rosetta
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(DE3)-pETDuet-fdh-lin1-adh, respectively; lanes 9 and 10, soluble and insoluble
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protein fraction of Rosetta (DE3)-pETDuet-fdh-lin2-adh, respectively; lanes 11 and
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12, soluble and insoluble protein fraction of Rosetta (DE3)-pETDuet-adh/fdh,
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respectively; M, molecular-mass marker.
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41
42
43
44
45
46
47
2
48
49
50
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Table S2 Peptide mass data of the four fusion proteins
Protein tested
Intens
Measured peptide
masses (Da)
Theoretical
masses (Da)a
Coverage
(%)
ADH-LIN1-FDH
12130
10186
8286
4394
2698
2184.274
1521.915
2787.681
2402.362
2624.3
2184.1335
1521.8423
2787.4999
2402.2251
2624.1535
44.27
ADH-LIN2-FDH
9029
8558
6045
2677
1612
13006
6662
4883
2204
1860
1601
1521.837
2184.147
2787.535
2402.232
2624.165
1521.961
2184.314
2787.74
1650.058
2402.422
987.579
1521.8423
2184.1335
2787.4999
2402.2251
2624.1535
1521.8423
2184.1335
2787.4999
1648.7489
2402.2251
987.537
46.50
1521.8423
2184.1335
2787.4999
2402.2251
1648.7489
987.537
41.60
FDH-LIN1-ADH
FDH-LIN2-ADH
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14049
1521.935
7524
2184.28
5670
2787.704
2094
2402.382
1864
1650.034
1653
987.553
a Predicted mass calculated from amino acid sequences.
46.40
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Out of total trypsin-digestion, the mass spectrum revealed several protonated
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ions [M+H]+ of each peptide fragment. The main peptides were matched with the
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fusion proteins after theoretical trypsin digestion (web. Expasy.org/peptide_mass/).
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The main matched peptides are listed in Table S2. The coverage sequence of each
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fusion protein was rather high after the matching calculations.
3