Synthetic Biology Journal ›› 2022, Vol. 3 ›› Issue (3): 587-601.DOI: 10.12211/2096-8280.2021-053
• Research Article • Previous Articles Next Articles
Chaofan YANG, Yuchao JIANG, Moli SANG, Shengying LI, Wei ZHANG
Received:
2021-04-30
Revised:
2021-06-08
Online:
2022-07-13
Published:
2022-06-30
Contact:
Wei ZHANG
杨超凡, 姜玉超, 桑茉莉, 李盛英, 张伟
通讯作者:
张伟
作者简介:
基金资助:
CLC Number:
Chaofan YANG, Yuchao JIANG, Moli SANG, Shengying LI, Wei ZHANG. Studies on the functional modulating effect of redox partners on the cytochrome P450 enzyme MycG[J]. Synthetic Biology Journal, 2022, 3(3): 587-601.
杨超凡, 姜玉超, 桑茉莉, 李盛英, 张伟. 还原伴侣对细胞色素P450酶MycG功能调控的研究[J]. 合成生物学, 2022, 3(3): 587-601.
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URL: https://synbioj.cip.com.cn/EN/10.12211/2096-8280.2021-053
Fig. 3 Constructed P450 systems based on the recombination and reorganization of MycG and different redox partners(0978 and 1499 represent the ferredoxin reductase SelFdR0978 and ferredoxin SelFdx1499 from Synechococcus elongates PCC 7942, respectively; Black linker: L21, HQVAMLRDGDSFGGGPRHGAG; Gray linker: L9, GERREIRVD)
菌株 | 用途 | 来源 |
---|---|---|
E. coli DH5α | 用于本研究重组质粒克隆 | 擎科生物公司 |
E. coli BL21(DE3) | 用于本研究重组蛋白表达 | 擎科生物公司 |
BL21/pACYC-MycG | 用于表达质粒pACYC-MycG | 本实验室 |
BL21/pET28b-RhFRED | 用于表达质粒pET28b-RhFRED | 本实验室 |
BL21/pET28b-MycG-RhFRED | 用于表达质粒pET28b-MycG-RhFRED | 本实验室 |
BL21/pET28b-SelFdR0978 | 用于表达质粒pET28b-SelFdR0978 | 本实验室 |
BL21/pET28b-SelFdx1499 | 用于表达质粒pET28b-SelFdx1499 | 本实验室 |
DH5α/BL21/pET28b-FMN | 用于克隆/表达质粒pET28b-FMN | 本研究 |
DH5α/BL21/pET28b-Fe2S2 | 用于克隆/表达质粒pET28b-Fe2S2 | 本研究 |
DH5α/BL21/pET28b-Fe2S2-FMN | 用于克隆/表达质粒pET28b-Fe2S2-FMN | 本研究 |
DH5α/BL21/pET28b-MycG-FMN | 用于克隆/表达质粒pET28b-MycG-FMN | 本研究 |
DH5α/BL21/pET28b-MycG-Fe2S2 | 用于克隆/表达质粒pET28b-MycG-Fe2S2 | 本研究 |
DH5α/BL21/pET28b-FMN-MycG | 用于克隆/表达质粒pET28b-FMN-MycG | 本研究 |
DH5α/BL21/pET28b-Fe2S2-MycG | 用于克隆/表达质粒pET28b-Fe2S2-MycG | 本研究 |
DH5α/BL21/pET28b-MycG-Fe2S2-FMN | 用于克隆/表达质粒pET28b-MycG-Fe2S2-FMN | 本研究 |
DH5α/BL21/pET28b-Fe2S2-FMN-MycG | 用于克隆/表达质粒pET28b-Fe2S2-FMN-MycG | 本研究 |
DH5α/BL21/pET28b-FMN-Fe2S2-MycG | 用于克隆/表达质粒pET28b-FMN-Fe2S2-MycG | 本研究 |
DH5α/BL21/pET28b-Fe2S2-MycG-FMN | 用于克隆/表达质粒pET28b-Fe2S2-MycG-FMN | 本研究 |
DH5α/BL21/pET28b-FMN-MycG-Fe2S2 | 用于克隆/表达质粒pET28b-FMN-MycG-Fe2S2 | 本研究 |
Tab. 1 Strains and plasmids used in this study
菌株 | 用途 | 来源 |
---|---|---|
E. coli DH5α | 用于本研究重组质粒克隆 | 擎科生物公司 |
E. coli BL21(DE3) | 用于本研究重组蛋白表达 | 擎科生物公司 |
BL21/pACYC-MycG | 用于表达质粒pACYC-MycG | 本实验室 |
BL21/pET28b-RhFRED | 用于表达质粒pET28b-RhFRED | 本实验室 |
BL21/pET28b-MycG-RhFRED | 用于表达质粒pET28b-MycG-RhFRED | 本实验室 |
BL21/pET28b-SelFdR0978 | 用于表达质粒pET28b-SelFdR0978 | 本实验室 |
BL21/pET28b-SelFdx1499 | 用于表达质粒pET28b-SelFdx1499 | 本实验室 |
DH5α/BL21/pET28b-FMN | 用于克隆/表达质粒pET28b-FMN | 本研究 |
DH5α/BL21/pET28b-Fe2S2 | 用于克隆/表达质粒pET28b-Fe2S2 | 本研究 |
DH5α/BL21/pET28b-Fe2S2-FMN | 用于克隆/表达质粒pET28b-Fe2S2-FMN | 本研究 |
DH5α/BL21/pET28b-MycG-FMN | 用于克隆/表达质粒pET28b-MycG-FMN | 本研究 |
DH5α/BL21/pET28b-MycG-Fe2S2 | 用于克隆/表达质粒pET28b-MycG-Fe2S2 | 本研究 |
DH5α/BL21/pET28b-FMN-MycG | 用于克隆/表达质粒pET28b-FMN-MycG | 本研究 |
DH5α/BL21/pET28b-Fe2S2-MycG | 用于克隆/表达质粒pET28b-Fe2S2-MycG | 本研究 |
DH5α/BL21/pET28b-MycG-Fe2S2-FMN | 用于克隆/表达质粒pET28b-MycG-Fe2S2-FMN | 本研究 |
DH5α/BL21/pET28b-Fe2S2-FMN-MycG | 用于克隆/表达质粒pET28b-Fe2S2-FMN-MycG | 本研究 |
DH5α/BL21/pET28b-FMN-Fe2S2-MycG | 用于克隆/表达质粒pET28b-FMN-Fe2S2-MycG | 本研究 |
DH5α/BL21/pET28b-Fe2S2-MycG-FMN | 用于克隆/表达质粒pET28b-Fe2S2-MycG-FMN | 本研究 |
DH5α/BL21/pET28b-FMN-MycG-Fe2S2 | 用于克隆/表达质粒pET28b-FMN-MycG-Fe2S2 | 本研究 |
引物名称 | 核苷酸序列(5′~3′) |
---|---|
arm-FP | catatggctgccgcgcg |
arm-RP | tagctcgagcaccaccacc |
Fe2S2-FP | gagcacgccttcgacctcga |
Fe2S2-RP | tcagagtcgcagggccagcc |
FMN-FP | gtcaccgtcgagcgcctgga |
FMN-RP | cgaggtgaagtgctcgacgt |
MycG-FMN-FP | ggcagccatatgacttc |
MycG-FMN-RP | cgaggtgaagtgctcgacgt |
Fe2S2-arm-FP | cgcgcggcagccatatggagcacgccttcgacctc |
Fe2S2-arm-RP | ggtggtggtgctcgagtcagagtcgcagggccag |
FMN-arm-FP | cgcgcggcagccatatggtcaccgtcgagcgcctg |
FMN-arm-RP | ggtggtggtgctcgagctacgaggtgaagtgctcgacg |
MycG-FMN-arm-FP | cgcgcggcagccatatgacttcagctgaacctagggc |
MycG-FMN-arm-RP | ggtggtggtgctcgagctacgaggtgaagtgctcgacg |
MycG-Fe2S2-WL-FP | ttcagctgaagtcatcatatggctgccgcg |
MycG-Fe2S2-WL-RP | gcggtgtcccgcaccgagcacgccttcgacctcgaa |
MycG-L9-FP | atgacttcagctgaacctagggc |
MycG-L9-RP | ggtgcgggacaccg |
Fe2S2-L9-FP | gcgctcgacggtgacgacgtccggatcgagcgccgcgagggagagtcgcagg |
Fe2S2-L9-RP | acgtcgagcacttcacctcgtgactcgagcaccaccacca |
FMN-WL-FP | catatggctgccgcgcggcacc |
FMN-WL-RP | gtcaccgtcgagcgcctggacc |
MycG-FP | acttcagctgaacctagggc |
MycG-FP2 | accatcggagaacccgccgcccgggcggtgtcccgcaccacttcagctgaacctaggg |
MycG-RP | ccacacgaccggcagctcga |
MycG-Fe2S2-FMN-WL-FP | aggttcagctgaagtcatatggctgccgcgcgg |
MycG-Fe2S2-FMN-WL-RP | gcggtgtcccgcaccgagcacgccttcgacc |
Fe2S2-FMN-G-WL-FP | gggttctccgatggtgaccggttgatggcggtgcagcaccgaggtgaagtgctcgacg |
Fe2S2-FMN-G-WL-RP | ctgccggtcgtgtggtgactcgagcaccaccac |
FMN-L9-FP | gtcaccgtcgagcgcctgga |
FMN-L9-RP | gacgtccggatcgagcgccg |
FMN-L21-FP | gtcaccgtcgagcgcctgga |
FMN-L21-RP | taggttcagctgaagtggtgc |
Fe2S2-L21-FP | gagcacgccttcgacctcga |
Fe2S2-L21-RP | taggttcagctgaagtggtg |
FMN-Fe2S2-G-WL-FP | ctcgatccggacgtcgagcacgccttcgac |
FMN-Fe2S2-G-WL-RP | gcgctcgacggtgaccatatggctgccgcgcg |
FMN-G-Fe2S2-WL-FP | gcgctcgacggtgaccatatggctgccgcgc |
FMN-G-Fe2S2-WL-RP | acttcagctgaacctagggc |
Fe2S2-G-FMN-WL-FP | aggtcgaaggcgtgctccatatggctgccgcg |
Fe2S2-G-FMN-WL-RP | acttcagctgaacctagggc |
FMN-MycG-WL-FP | ctgccggtcgtgtggtagctcgagcaccaccac |
FMN-MycG-WL-RP | gggttctccgatggtgaccggttgatggcggtgcagcaccgaggtgaagtgctcgacg |
Fe2S2-MycG-WL-FP | ctgccggtcgtgtggtagctcgagcaccaccac |
Fe2S2-MycG-WL-RP | tgatggcggtgcagcacgagtcgcagggccagcc |
Tab. 2 Oligos used in this study
引物名称 | 核苷酸序列(5′~3′) |
---|---|
arm-FP | catatggctgccgcgcg |
arm-RP | tagctcgagcaccaccacc |
Fe2S2-FP | gagcacgccttcgacctcga |
Fe2S2-RP | tcagagtcgcagggccagcc |
FMN-FP | gtcaccgtcgagcgcctgga |
FMN-RP | cgaggtgaagtgctcgacgt |
MycG-FMN-FP | ggcagccatatgacttc |
MycG-FMN-RP | cgaggtgaagtgctcgacgt |
Fe2S2-arm-FP | cgcgcggcagccatatggagcacgccttcgacctc |
Fe2S2-arm-RP | ggtggtggtgctcgagtcagagtcgcagggccag |
FMN-arm-FP | cgcgcggcagccatatggtcaccgtcgagcgcctg |
FMN-arm-RP | ggtggtggtgctcgagctacgaggtgaagtgctcgacg |
MycG-FMN-arm-FP | cgcgcggcagccatatgacttcagctgaacctagggc |
MycG-FMN-arm-RP | ggtggtggtgctcgagctacgaggtgaagtgctcgacg |
MycG-Fe2S2-WL-FP | ttcagctgaagtcatcatatggctgccgcg |
MycG-Fe2S2-WL-RP | gcggtgtcccgcaccgagcacgccttcgacctcgaa |
MycG-L9-FP | atgacttcagctgaacctagggc |
MycG-L9-RP | ggtgcgggacaccg |
Fe2S2-L9-FP | gcgctcgacggtgacgacgtccggatcgagcgccgcgagggagagtcgcagg |
Fe2S2-L9-RP | acgtcgagcacttcacctcgtgactcgagcaccaccacca |
FMN-WL-FP | catatggctgccgcgcggcacc |
FMN-WL-RP | gtcaccgtcgagcgcctggacc |
MycG-FP | acttcagctgaacctagggc |
MycG-FP2 | accatcggagaacccgccgcccgggcggtgtcccgcaccacttcagctgaacctaggg |
MycG-RP | ccacacgaccggcagctcga |
MycG-Fe2S2-FMN-WL-FP | aggttcagctgaagtcatatggctgccgcgcgg |
MycG-Fe2S2-FMN-WL-RP | gcggtgtcccgcaccgagcacgccttcgacc |
Fe2S2-FMN-G-WL-FP | gggttctccgatggtgaccggttgatggcggtgcagcaccgaggtgaagtgctcgacg |
Fe2S2-FMN-G-WL-RP | ctgccggtcgtgtggtgactcgagcaccaccac |
FMN-L9-FP | gtcaccgtcgagcgcctgga |
FMN-L9-RP | gacgtccggatcgagcgccg |
FMN-L21-FP | gtcaccgtcgagcgcctgga |
FMN-L21-RP | taggttcagctgaagtggtgc |
Fe2S2-L21-FP | gagcacgccttcgacctcga |
Fe2S2-L21-RP | taggttcagctgaagtggtg |
FMN-Fe2S2-G-WL-FP | ctcgatccggacgtcgagcacgccttcgac |
FMN-Fe2S2-G-WL-RP | gcgctcgacggtgaccatatggctgccgcgcg |
FMN-G-Fe2S2-WL-FP | gcgctcgacggtgaccatatggctgccgcgc |
FMN-G-Fe2S2-WL-RP | acttcagctgaacctagggc |
Fe2S2-G-FMN-WL-FP | aggtcgaaggcgtgctccatatggctgccgcg |
Fe2S2-G-FMN-WL-RP | acttcagctgaacctagggc |
FMN-MycG-WL-FP | ctgccggtcgtgtggtagctcgagcaccaccac |
FMN-MycG-WL-RP | gggttctccgatggtgaccggttgatggcggtgcagcaccgaggtgaagtgctcgacg |
Fe2S2-MycG-WL-FP | ctgccggtcgtgtggtagctcgagcaccaccac |
Fe2S2-MycG-WL-RP | tgatggcggtgcagcacgagtcgcagggccagcc |
组合 | 铁氰化钾/[μmol/(L·min)) | 细胞色素C/[μmol/(L·min)) | |
---|---|---|---|
FMN-Fe2S2 | 34.38±0.05 | 13.57±0.01 | |
Fe2S2-FMN | 24.37±0.02 | 10.21±0.01 | |
MycG-FMN-Fe2S2 | 21.82±0.02 | 10.45±0.01 | |
FMN-MycG-Fe2S2 | 6.92±0.003 | 8.42±0.01 | |
Fe2S2-MycG-FMN | 6.64±0.02 | 3.23±0.01 | |
FMN-Fe2S2-MycG | 26.07±0.03 | 10.22±0.01 | |
MycG-Fe2S2-FMN | 5.85±0.006 | 5.04±0.009 | |
Fe2S2-FMN-MycG | 25.36±0.02 | 6.0±0.01 | |
MycG-FMN | 33.97±0.03 | Fe2S2:3.94±0.06 | SelFdx1499:7.04±0.03 |
FMN-MycG | 19.81±0.03 | Fe2S2:10.37±0.02 | SelFdx1499:9.56±0.02 |
SelFdR0978 | 24.23±0.04 | Fe2S2:1.63±0.02 | SelFdx1499:3.75±0.01 |
FMN | 5.04±0.07 | Fe2S2:0.46±0.03 | SelFdx1499:1.86±0.02 |
Tab. 3 Electron transfer efficiencies of different recombinant proteins
组合 | 铁氰化钾/[μmol/(L·min)) | 细胞色素C/[μmol/(L·min)) | |
---|---|---|---|
FMN-Fe2S2 | 34.38±0.05 | 13.57±0.01 | |
Fe2S2-FMN | 24.37±0.02 | 10.21±0.01 | |
MycG-FMN-Fe2S2 | 21.82±0.02 | 10.45±0.01 | |
FMN-MycG-Fe2S2 | 6.92±0.003 | 8.42±0.01 | |
Fe2S2-MycG-FMN | 6.64±0.02 | 3.23±0.01 | |
FMN-Fe2S2-MycG | 26.07±0.03 | 10.22±0.01 | |
MycG-Fe2S2-FMN | 5.85±0.006 | 5.04±0.009 | |
Fe2S2-FMN-MycG | 25.36±0.02 | 6.0±0.01 | |
MycG-FMN | 33.97±0.03 | Fe2S2:3.94±0.06 | SelFdx1499:7.04±0.03 |
FMN-MycG | 19.81±0.03 | Fe2S2:10.37±0.02 | SelFdx1499:9.56±0.02 |
SelFdR0978 | 24.23±0.04 | Fe2S2:1.63±0.02 | SelFdx1499:3.75±0.01 |
FMN | 5.04±0.07 | Fe2S2:0.46±0.03 | SelFdx1499:1.86±0.02 |
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