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Submitted for Biotechnology Letters
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Section of the journal: Microbial and Enzyme Technology
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Synthesis of Rosmarinic Acid Analogues in Escherichia coli
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Yibin Zhuang1,3,#, Jingjie Jiang1,2,#, Huiping Bi1,3,#, Hua Yin1,3, Shaowei Liu2*, Tao Liu1,3*
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1Tianjin
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2Department
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and Technology, Shanghai, 200237, China
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3Key
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Address correspondence to Shaowei Liu, swliu@ecust.edu.cn; Tao Liu, liu_t@tib.cas.cn
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#
Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
of Food science and Technology, School of Biological Engineering, East China University of Science
Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
Yibin Zhuang, Jingjie Jiang and Huiping Bi contributed equally to this article
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Supplemental NMR and MS data of products
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1. Compound 3: 1H NMR (CD3OD, 400 MHz), δ H 6.77 (d, J = 2.0 Hz, H-2), 6.71 (d, J = 8.0 Hz, H- 5),
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6.63 (dd, J = 8.0 Hz, J = 2.0 Hz, H-6), 3.02 (dd, J = 14.3 Hz, J = 8.6 Hz, H-7a), 3.12 (dd, J = 14.3 Hz, J
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= 4.2 Hz, H-7b), 5.21 (dd, J = 8.4 Hz, J = 4.2 Hz, H-8), 7.19 (d, J = 2.0 Hz, H-2'), 6.82 ( d, J = 8.2 Hz,
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H-5'), 7.08 (dd, J = 8.2 Hz, J = 2.0 Hz, H-6'), 7.62 (d, J = 15.9 Hz, H-7'), 6.37 (d, J = 15.9 Hz, H-8'),
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3.90 (s, OCH3-3'); 13C NMR (MeOD, 100 MHz), δ C 128.0 (C-1), 116.2 (C-2), 144.8 (C-3), 143.9 (C-4),
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115.1 (C-5), 120.4 (C-6), 36.6 (C-7), 73.4 (C-8), n d (C-9), 126.3 (C-1'), 110.4 (C-2'), 148.0 (C-3'), 149.3
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(C-4'), 114.9 (C-5'), 122.9 (C-4'), 146.2 (C-7'), 113.4 (C-8'), 167.0 (C-9'), 55.1 (OCH3-3'). HRESI-MS
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m/z 375.1063 [M + H]+ (calcd. for C19H19O8 375.1080).
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2. Compound 4: 1H NMR (CD3OD, 400 MHz), δ H 6.72 (d, J = 2.1 Hz, H-2), 6.69 (d, J = 8.0 Hz, H-5),
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6.56 (dd, J = 8.0 Hz, J = 2.0 Hz, H-6), 2.91 (dd, J = 14.3 Hz, J = 8.8 Hz, H-7a), 3.05 (dd, J = 14.3 Hz, J
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= 4.1 Hz, H-7b), 5.08 (dd, J = 8.8 Hz, J = 4.1 Hz, H-8), 6.62 (d, J = 2.1 Hz, H-2'), 6.65 (d, J = 8.0 Hz,
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H-5'), 6.43 (dd, J = 8.0 Hz, J = 2.1 Hz, H-6'), 2.72 (t, J = 7.4 Hz, H-7'), 2.57 (t, J = 7.4 Hz, H-8'); 13C
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NMR (MeOD, 100 MHz), δ C 128.0 (C-1), 116.1 (C-2), 144.7 (C-3), 144.8 (C-4), 114.9 (C-5), 120.4 (C-
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6), 36.5 (C-7), 73.7 (C-8), 172.5 (C-9), 132.1 (C-1'), 115.0 (C-2'), 144.8 (C-3'), 143.2 (C-4'), 115.0 (C-
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5'), 119.1 (C-6'), 29.8 (C-7'), 35.7 (C-8'), 172.8 (C-9'). HRESI-MS m/z 363.1112 [M + H]+ (calcd. for
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C18H19O8 363.1080).
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3. Compound 5: 1H NMR (CD3OD, 400 MHz), δ H 6.58 (d, J = 2.0 Hz, H-2), 6.56 (d, J = 8.0 Hz, H-5),
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6.35 (dd, J = 8.0 Hz, J = 2.0 Hz, H-6), 2.89 (dd, J = 14.3 Hz, J = 8.6 Hz, H-7a), 2.99 (dd, J = 14.3 Hz, J
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= 4.1 Hz, H-7b), 5.09 (dd, J = 8.6 Hz, J = 4.1 Hz, H-8), 7.01 (d, J = 8.5 Hz, H-2', 6'), 6.66 ( d, J = 8.5
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Hz, H-3', 5'), 2.77 (t, J = 7.6 Hz, H-7'), 2.59 (t, J = 7.6 Hz, H-8'); 13C NMR (MeOD, 100 MHz), δ C 127.0
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(C-1), 116.1 (C-2), 145.5 (C-3), 143.9 (C-4), 114.8 (C-5), 119.2 (C-6), 36.3 (C-7), 73.6 (C-8), 171.2 (C2
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9), 131.6 (C-1'), 130.7 (C-2', 6'), 115.5 (C-3', 5'), 156.5 (C-4'), 30.0 (C-7'), 35.7 (C-8'), 172.8 (C-9').
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HRESI-MS m/z 347.1120 [M + H]+ (calcd. for C18H19O7 347.1131).
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4. Compound 7: 1H NMR (CD3OD, 400 MHz), δ H 7.13 (d, J = 8.4 Hz, H-2, 6), 6.74 (d, J = 8.4 Hz, H-
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3, 5), 3.08 (dd, J = 14.3 Hz, J = 8.2 Hz, H-7a), 3.17 (dd, J = 14.3 Hz, J = 4.4 Hz, H-7b), 5.22 (dd, J = 8.2
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Hz, J = 4.4 Hz, H-8), 7.45 (d, J = 8.6 Hz, H-2', 6'), 6.81 ( d, J = 8.6 Hz, H-3', 5'), 7.62 (d, J = 15.9 Hz,
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H-7'), 6.33 (d, J = 15.9 Hz, H-8'); 13C NMR (MeOD, 100 MHz), δ C 127.2 (C-1), 130.1 (C-2, 6), 114.8
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(C-3, 5), 155.9 (C-4), 36.3 (C-7), 73.3 (C-8), 172.2 (C-9), 125.7 (C-1'), 129.9 (C-2', 6'), 115.4 (C-3', 5'),
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160.0 (C-4'), 145.9 (C-7'), 113.1 (C-8'), 167.1 (C-9'). HRESI-MS m/z 329.0998 [M + H]+ (calcd. for
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C18H17O6 329.1025).
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5. Compound 8: 1H NMR (CD3OD, 400 MHz), δ H 7.14 (d, J = 8.5 Hz, H-2, 6), 6.74 (d, J = 8.5 Hz, H-
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3, 5), 3.08 (dd, J = 14.3 Hz, J = 8.4 Hz, H-7a), 3.17 (dd, J = 14.3 Hz, J = 4.3 Hz, H-7b), 5.22 (dd, J = 8.4
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Hz, J = 4.3 Hz, H-8), 7.19 (d, J = 1.9 Hz, H-2'), 6.82 ( d, J = 8.2 Hz, H-5'), 7.07 (dd, J = 8.2 Hz, J = 1.9
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Hz, H-6'), 7.61 (d, J = 15.9 Hz, H-7'), 6.37 (d, J = 15.9 Hz, H-8'), 3.90 (s, OCH3-3') ; 13C NMR (MeOD,
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100 MHz), δ C 127.3 (C-1), 130.0 (C-2), 114.8 (C-3), 156.0 (C-4), 114.8 (C-5), 130.0 (C-6), 36.4 (C-7),
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73.4 (C-8), n d (C-9), 126.2 (C-1'), 110.3 (C-2'), 148.0 (C-3'), 149.4 (C-4'), 115.0 (C-5'), 122.9 (C-4'),
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146.1 (C-7'), 113.4 (C-8'), 167.0 (C-9'), 55.0 (OCH3-3'). HRESI-MS m/z 359.1238 [M + H]+ (calcd. for
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C19H19O7 359.1131).
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6. Compound 9: 1H NMR (CD3OD, 400 MHz), δ H 7.05 (d, J = 8.6 Hz, H-2, 6), 6.71 (d, J = 8.4 Hz, H-
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3, 5), 2.98 (dd, J = 14.3 Hz, J = 8.6 Hz, H-7a), 3.09 (dd, J = 14.3 Hz, J = 4.1 Hz, H-7b), 5.09 (dd, J = 8.6
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Hz, J = 4.1 Hz, H-8), 6.62 (d, J = 2.1 Hz, H-2'), 6.65 ( d, J = 8.2 Hz, H-5'), 6.43 ( dd, J = 8.2 Hz, J = 4.1
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Hz, H-6'), 2.72 (t, J = 7.4 Hz, H-7'), 2.57 (t, J = 7.4 Hz, H-8'); 13C NMR (MeOD, 100 MHz), δ C 127.3
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(C-1), 130.0 (C-2, 6), 114.8 (C-3, 5), 155.9 (C-4), 36.2 (C-7), 73.6 (C-8), n d (C-9), 132.1 (C-1'), 114.9
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(C-2'), 144.8 (C-3'), 143.2 (C-4'), 114.9 (C-5'), 119.1 (C-6'), 29.8 (C-7'), 35.7 (C-8'), 172.8 (C-9'). HRESI-
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MS m/z 347.1137 [M + H]+ (calcd. for C18H19O7 347.1131).
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7. Compound 10: 1H NMR (CD3OD, 400 MHz), δ H 7.06 (d, J = 8.5 Hz, H-2, 6), 6.72 (d, J = 8.5 Hz,
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H-3, 5), 2.97 (dd, J = 14.3 Hz, J = 8.6 Hz, H-7a), 3.10 (dd, J = 14.3 Hz, J = 4.0 Hz, H-7b), 5.08 (dd, J =
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8.9 Hz, J = 4.0 Hz, H-8), 6.92 (d, J = 8.5 Hz, H-2', 6'), 6.67 ( d, J = 8.5 Hz, H-3', 5'), 2.77 (t, J = 7.6 Hz,
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H-7'), 2.59 (t, J = 7.6 Hz, H-8'); 13C NMR (MeOD, 100 MHz), δ C 127.4 (C-1), 130.0 (C-2,6), 114.8 (C-
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3, 5), 155.9 (C-4), 36.3 (C-7), 73.8 (C-8), n d (C-9), 131.2 (C-1'), 128.8 (C-2', 6'), 114.7 (C-3', 5'), 155.3
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(C-4'), 29.6 (C-7'), 35.8 (C-8'), 172.8 (C-9'). HRESI-MS m/z 331.1183 [M + H]+ (calcd. for C18H19O6
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331.1182).
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8. Compound 12: 1H NMR (CD3OD, 400 MHz), δ H 7.30 (m, H-2, 6), 7.31 (m, H-3, 5), 7.28 (m, H-4),
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3.18 (dd, J = 14.2 Hz, J = 8.4 Hz, H-7a), 3.20 (dd, J = 14.2 Hz, J = 4.3 Hz, H-7b), 5.29 (dd, J = 8.4 Hz,
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J = 4.3 Hz, H-8), 7.45 (d, J = 8.6 Hz, H-2', 6'), 6.81 ( d, J = 8.6 Hz, H-3', 5'), 7.61 (d, J = 15.9 Hz, H-7'),
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6.32 (d, J = 15.9 Hz, H-8'); 13C NMR (CD3OD, 100 MHz), δ C 136.4 (C-1), 129.1 (C-2, 6), 128.1 (C-3,
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5), 126.5 (C-4), 37.0 (C-7), 72.9 (C-8), 171.8 (C-9), 125.6 (C-1'), 129.9 (C-2', 6'), 115.4 (C-3', 5'), 160.0
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(C-4'), 146.0 (C-7'), 112.9 (C-8'), 166.9 (C-9'). HRESI-MS m/z 313.1127 [M + H]+ (calcd. for C18H17O5
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313.1076)
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9. Compound 13: 1H NMR (CD3OD, 400 MHz), δ H 7.30 (m, H-2, 6), 7.31 (m, H-3, 5), 7.23 (m, H-4),
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3.10 (dd, J = 14.3 Hz, J = 8.4 Hz, H-7a), 3.21 (dd, J = 14.3 Hz, J = 4.3 Hz, H-7b), 5.19 (dd, J = 8.4 Hz,
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J = 4.3 Hz, H-8), 7.33 (d, J = 1.5 Hz, H-2'), 6.80 ( d, J = 8.2 Hz, H-5'), 7.10 (dd, J = 8.2 Hz, J = 1.5 Hz,
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H-6'), 7.61 (d, J = 15.9 Hz, H-7'), 6.37 (d, J = 15.9 Hz, H-8'), 3.82 (s, OCH3-3') ; 13C NMR (MeOD, 100
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MHz), δ C 136.6 (C-1), 128.2 (C-2,6), 129.2 (C-3,5), 126.6 (C-4), 36.5 (C-7), 72.4 (C-8), 170.7 (C-9),
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125.3 (C-1'), 111.1 (C-2'), 147.9 (C-3'), 149.5 (C-4'), 115.4 (C-5'), 123.4 (C-4'), 145.9 (C-7'), 113.5 (C4
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8'), 165.9 (C-9'), 55.6 (OCH3-3'). HRESI-MS m/z 343.1203 [M + H]+ (calcd. for C19H19O6 343.1182).
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10. Compound 14: 1H NMR (CD3OD, 400 MHz), δ H 7.23 (m, H-2, 6), 7.27 (m, H-3, 5), 7.21 (m, H-
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4), 3.07 (dd, J = 14.3 Hz, J = 8.6 Hz, H-7a), 3.19 (dd, J = 14.3 Hz, J = 4.2 Hz, H-7b), 5.18 (dd, J = 8.6
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Hz, J = 4.3 Hz, H-8), 6.61 (d, J = 2.1 Hz, H-2'), 6.65 ( d, J = 8.2 Hz, H-5'), 6.43 ( dd, J = 8.2 Hz, J = 4.1
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Hz, H-6'), 2.72 (t, J = 7.4 Hz, H-7'), 2.57 (t, J = 7.4 Hz, H-8'); 13C NMR (MeOD, 100 MHz), δ C 136.3
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(C-1), 129.0 (C-2, 6), 128.0 (C-3, 5), 126.5 (C-4), 36.9 (C-7), 72.9 (C-8), 171.7 (C-9), 132.0 (C-1'), 115.0
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(C-2'), 144.8 (C-3'), 143.2 (C-4'), 115.0 (C-5'), 119.1 (C-6'), 29.8 (C-7'), 35.6 (C-8'), 172.6 (C-9'). HRESI-
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MS m/z 331.1141 [M + H]+ (calcd. for C18H19O7 331.1182)
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11. Compound 15: 1H NMR (CD3OD, 400 MHz), δ H 7.24 (m, H-2, 6), 7.28 (m, H-3, 5), 7.23 (m, H-4),
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3.07 (dd, J = 14.3 Hz, J = 8.8 Hz, H-7a), 3.20 (dd, J = 14.3 Hz, J = 4.1 Hz, H-7b), 5.17 (dd, J = 8.8 Hz,
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J = 4.1 Hz, H-8), 6.91 (d, J = 8.5 Hz, H-2', 6'), 6.66 ( d, J = 8.5 Hz, H-3', 5'), 2.76 (t, J = 7.6 Hz, H-7'),
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2.57 (t, J = 7.6 Hz, H-8'); 13C NMR (MeOD, 100 MHz), δ C 136.4 (C-1), 128.8 (C-2, 6), 128.0 (C-3, 5),
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126.5 (C-4), 36.9 (C-7), 72.9 (C-8), 171.6 (C-9), 131.1 (C-1'), 129.0 (C-2', 6'), 114.8 (C-3',5'), 155.4 (C-
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4'), 29.5 (C-7'), 35.6 (C-8'), 172.6 (C-9'). HRESI-MS m/z 315.1219 [M +H]+ (calcd. for C18H18O5 H
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315.1232).
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Supplementary Figures
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Supplementary Fig. 1 HPLC-MS analysis of metabolite profile of the recombinant E. coli strain
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BLRA1 supplied with ferulic acid and other substrates. (a) and (b) Feeding with ferulic acid
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dihydroxyphenyllactic acid (DHPL); (c) and (d) Feeding with ferulic acid
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acid (HPL); (e) and (f) Feeding with ferulic acid and phenyllactic acid (PL). (a), (c) and (e) Recombinant
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and 3,4-
and p-hydroxyphenyllactic
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E. coli strain BLRA0 supplied with ferulic acid in combination with DHPL，HPL or PL used as negative
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controls. (g) MS analysis of compound 3 in (b); (h) MS analysis of compound 8 in (d); (I) MS analysis
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of compound 13 in (f).
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Supplementary Fig. 2 HPLC-MS analysis of metabolite profile of the recombinant E. coli strain
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BLRA1 supplied with p-hydroxyphenyl propionic acid (HPPA) and other substrates. (a) and (b) Feeding
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with HPPA and 3,4-dihydroxyphenyllactic acid (DHPL); (c) and (d) Feeding with HPPA and p-
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hydroxyphenyllactic acid (HPL); (e) and (f) Feeding with HPPA and phenyllactic acid (PL). (a), (c) and
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(e) Recombinant E. coli strain BLRA0 supplied with HPPA in combination with DHPL，HPL or PL as
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negative controls. (g) MS analysis of compound 5 in (b); (h) MS analysis of compound 10 in (d); (i) MS
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analysis of compound 15 in (f).
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Supplementary Fig. 3 HPLC-MS analysis of metabolites profile of recombinant E. coli strain BLRA1
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supplied with mandelic acid and other substrates. (a) and (b) Feeding with mandelic acid and caffeic acid;
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(c) and (d) Feeding with mandelic acid and p-courmaric acid; (e) and (f) Feeding with mandelic acid and
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p-hydroxyphenyl propionic acid (HPPA). (a), (c) and (e) Recombinant E. coli strain BLRA0 supplied
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with mandelic acid in combination with caffeic acid，p-courmaric acid or HPPA used as negative controls.
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(g) MS analysis of compound 16 in (b); (h) MS analysis of compound 17 in (d); (i) MS analysis of
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compound 18 in (f).
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Supplementary Fig. 4 HPLC-MS analyses of fermentation broth of recombinant E. coli strain BLRA1
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fed with tyrosol and other substrates. (a) and (b) Feeding with tyrosol and caffeic acid; (c) and (d) Feeding
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with tyrosol
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acid (HPPA). (a), (c) and (e) were recombinant E. coli strain BLRA0 used as control. (g) MS analysis of
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compound 19 in (b); (h) MS analysis of compound 20 in (d); (i) MS analysis of compound 21 in (f).
and p-courmaric acid; (e) and (f) Feeding with tyrosol
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and p-hydroxyphenyl propionic