Synthetic Biology Journal ›› 2021, Vol. 2 ›› Issue (6): 1046-1060.DOI: 10.12211/2096-8280.2021-098
Hutao GAO, Jia WANG, Xinxiao SUN, Xiaolin SHEN, Qipeng YUAN
Received:
2021-10-21
Revised:
2021-11-29
Online:
2022-01-21
Published:
2021-12-31
Contact:
Xiaolin SHEN, Qipeng YUAN
高虎涛, 王佳, 孙新晓, 申晓林, 袁其朋
通讯作者:
申晓林,袁其朋
作者简介:
基金资助:
CLC Number:
Hutao GAO, Jia WANG, Xinxiao SUN, Xiaolin SHEN, Qipeng YUAN. De novo biosynthesis of 3-phenylpropanol in E. coli[J]. Synthetic Biology Journal, 2021, 2(6): 1046-1060.
高虎涛, 王佳, 孙新晓, 申晓林, 袁其朋. 在大肠杆菌中从头生物合成3-苯丙醇[J]. 合成生物学, 2021, 2(6): 1046-1060.
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URL: https://synbioj.cip.com.cn/EN/10.12211/2096-8280.2021-098
Strains | Genotype | Source |
---|---|---|
Trans 5α | Lab Stock | |
BW25113(F′) | Lab Stock | |
BW25113(F′) ∆pykA ∆pykF | This study | |
G01 | BW25113(F′) harboring pZE-CCR-4CL1 | This study |
G02 | BW25113(F′) harboring pCS-ER | This study |
G03 | BW25113(F′) harboring pZE-CCR-4CL1 and pCS-ER | This study |
G04 | BW25113(F′) harboring pZE-ER | This study |
G05 | BW25113(F′) harboring pCS-Carsfp | This study |
G06 | BW25113(F′) harboring pZE-ER and pCS-Carsfp | This study |
G07 | BW25113(F′) harboring pZE-RgTAL-ER and pCS-Carsfp | This study |
G08 | BW25113(F′) harboring pZE-tac-RgTAL-ER-Carsfp | This study |
G09 | BW25113(F′)∆pykA∆pykF harboring pZE-tac-RgTAL-ER-Carsfp | This study |
G10 | BW25113(F′) harboring pCS-lac-APTA and pZE-tac-RgTAL-ER-Carsfp | This study |
G11 | BW25113(F′) harboring pCS-tac-APTA and pZE-tac-RgTAL-ER-Carsfp | This study |
G12 | BW25113(F′)∆pykA∆pykF harboring pCS-tac-APTA and pZE-tac-RgTAL-ER-Carsfp | This study |
Tab. 1 Strains used in this study
Strains | Genotype | Source |
---|---|---|
Trans 5α | Lab Stock | |
BW25113(F′) | Lab Stock | |
BW25113(F′) ∆pykA ∆pykF | This study | |
G01 | BW25113(F′) harboring pZE-CCR-4CL1 | This study |
G02 | BW25113(F′) harboring pCS-ER | This study |
G03 | BW25113(F′) harboring pZE-CCR-4CL1 and pCS-ER | This study |
G04 | BW25113(F′) harboring pZE-ER | This study |
G05 | BW25113(F′) harboring pCS-Carsfp | This study |
G06 | BW25113(F′) harboring pZE-ER and pCS-Carsfp | This study |
G07 | BW25113(F′) harboring pZE-RgTAL-ER and pCS-Carsfp | This study |
G08 | BW25113(F′) harboring pZE-tac-RgTAL-ER-Carsfp | This study |
G09 | BW25113(F′)∆pykA∆pykF harboring pZE-tac-RgTAL-ER-Carsfp | This study |
G10 | BW25113(F′) harboring pCS-lac-APTA and pZE-tac-RgTAL-ER-Carsfp | This study |
G11 | BW25113(F′) harboring pCS-tac-APTA and pZE-tac-RgTAL-ER-Carsfp | This study |
G12 | BW25113(F′)∆pykA∆pykF harboring pCS-tac-APTA and pZE-tac-RgTAL-ER-Carsfp | This study |
Plasmids | Description | Source |
---|---|---|
pZE12-luc | pLlacO-1; luc; ColE1 ori; Ampr | Lab Stock |
pCS27 | pLlacO-1, P15A ori, Kanr | Lab Stock |
pZE-CCR-4CL1 | pZE-luc carrying CCR from Leucaena leucocephala, and 4CL1 from Arabidopsis thaliana | This study |
pZE-ER | pZE-luc carrying ER from Clostridium acetobutylicum | This study |
pCS-ER | pCS27 carrying ER from C. acetobutylicum | This study |
pCS-carsfp | pCS27 carrying Car from Mycobacterium marinum and Sfp from Bacillus subtilis | This study |
pZE-RgTAL-ER | pZE-luc carrying TAL from Rhodobacter glutinis and ER from C.acetobutylicum | This study |
Tab. 2 Plasmids used in this study
Plasmids | Description | Source |
---|---|---|
pZE12-luc | pLlacO-1; luc; ColE1 ori; Ampr | Lab Stock |
pCS27 | pLlacO-1, P15A ori, Kanr | Lab Stock |
pZE-CCR-4CL1 | pZE-luc carrying CCR from Leucaena leucocephala, and 4CL1 from Arabidopsis thaliana | This study |
pZE-ER | pZE-luc carrying ER from Clostridium acetobutylicum | This study |
pCS-ER | pCS27 carrying ER from C. acetobutylicum | This study |
pCS-carsfp | pCS27 carrying Car from Mycobacterium marinum and Sfp from Bacillus subtilis | This study |
pZE-RgTAL-ER | pZE-luc carrying TAL from Rhodobacter glutinis and ER from C.acetobutylicum | This study |
Primer | Sequence 5′-3′ |
---|---|
CCR-4CL1-1-F-KpnI | gggaaaGGTACCatgcctgctgcggctccagc |
CCR-4CL1-1-R-BamHI | gggaaaGGATCCttatttggtcggcagcggcaggtg |
CCR-4CL1-2-F-BamHI | gggaaaGGATCCaggagatataccatggcgccacaagaacaagcagt |
CCR-4CL1-2-R-XbaI | gggaaaTCTAGAttacaatccatttgctagttttgccctc |
ER-KpnI-F | gggaaaGGTACCatgaacaaatacaagaaattatttgaaccaatcaaaattgg |
ER-XbaI-F | gggaaaTCTAGAttatatatggtttgcaacttcaaaagcatccc |
ER框-SpeI-F | gggaaaACTAGTaattgtgagcggataacaattgacattgtga |
ER框-SacI-R | gggaaaGAGCTCacaacagataaaacgaaaggcccagtc |
TAL框-SpeI-F | gggaaaACTAGTctcgagaattgtgagcggataacaattga |
TAL框-SacI-R | gggaaaGAGCTCcgacaaacaacagataaaacgaaaggcc |
Car-KpnI-F | gggaaaGGTACCatgtcacctatcacccgcgagg |
Car-BamHI-R | gggaaaGGATCCtcacagcaagcccagcagac |
sfp-BamHI-F | gggaaaGGATCCaggagatataccatgaagatttacggaatttatatgg |
sfp-XbaI-R | GGGAAAtctagattataaaagctcttcgtacgagactattgtgat |
AP-NheI-R | gggaaaGCTAGCttatttcttcagttcagccaggcttaacc |
TA-NheI-F | gggaaaGCTAGCaggagatataccatgtcctcacgtaaagagcttg |
APTA-XbaI-F | gggaaaTCTAGAatgacacaacctctttttctgatcggg |
APTA-BamHI-R | gggaaaGGATCCttacccgcgacgcgctttta |
pCS-tac-反-BamHI-F | gggaaaGGATCCgtcgccaatcacgcgtgaagagc |
pCS-tac-反-XbaI-R | gggaaaCATATGttataaaagctcttcgtacgagacta |
tac-Car框-F-SpeI | gggaaaACTAGTctcgagttgacaattaatcatcggctcg |
tac-Car框-R-SacI | gggaaaGAGCTCcgacaaacaacagataaaacgaaaggcc |
Tab. 3 Primers used in this study
Primer | Sequence 5′-3′ |
---|---|
CCR-4CL1-1-F-KpnI | gggaaaGGTACCatgcctgctgcggctccagc |
CCR-4CL1-1-R-BamHI | gggaaaGGATCCttatttggtcggcagcggcaggtg |
CCR-4CL1-2-F-BamHI | gggaaaGGATCCaggagatataccatggcgccacaagaacaagcagt |
CCR-4CL1-2-R-XbaI | gggaaaTCTAGAttacaatccatttgctagttttgccctc |
ER-KpnI-F | gggaaaGGTACCatgaacaaatacaagaaattatttgaaccaatcaaaattgg |
ER-XbaI-F | gggaaaTCTAGAttatatatggtttgcaacttcaaaagcatccc |
ER框-SpeI-F | gggaaaACTAGTaattgtgagcggataacaattgacattgtga |
ER框-SacI-R | gggaaaGAGCTCacaacagataaaacgaaaggcccagtc |
TAL框-SpeI-F | gggaaaACTAGTctcgagaattgtgagcggataacaattga |
TAL框-SacI-R | gggaaaGAGCTCcgacaaacaacagataaaacgaaaggcc |
Car-KpnI-F | gggaaaGGTACCatgtcacctatcacccgcgagg |
Car-BamHI-R | gggaaaGGATCCtcacagcaagcccagcagac |
sfp-BamHI-F | gggaaaGGATCCaggagatataccatgaagatttacggaatttatatgg |
sfp-XbaI-R | GGGAAAtctagattataaaagctcttcgtacgagactattgtgat |
AP-NheI-R | gggaaaGCTAGCttatttcttcagttcagccaggcttaacc |
TA-NheI-F | gggaaaGCTAGCaggagatataccatgtcctcacgtaaagagcttg |
APTA-XbaI-F | gggaaaTCTAGAatgacacaacctctttttctgatcggg |
APTA-BamHI-R | gggaaaGGATCCttacccgcgacgcgctttta |
pCS-tac-反-BamHI-F | gggaaaGGATCCgtcgccaatcacgcgtgaagagc |
pCS-tac-反-XbaI-R | gggaaaCATATGttataaaagctcttcgtacgagacta |
tac-Car框-F-SpeI | gggaaaACTAGTctcgagttgacaattaatcatcggctcg |
tac-Car框-R-SacI | gggaaaGAGCTCcgacaaacaacagataaaacgaaaggcc |
Fig. 1 Design of synthetic routes of 3-phenylpropanolRgTAL—phenylalanine ammonia lyase from Rhodobacter glutinis; 4CL1—from Arabidopsis thaliana coumarate-CoA ligase; CCR— cinnamoyl-CoA reductase from Leucaena leucocephala; ER—enoic acid reduction from Clostridium acetobutylicum Enzyme; Car—carboxylic acid reductase from Marine Mycobacterium; sfp—phosphoubiquitin transferase from Bacillus subtilis
Fig. 5 Production of 3-phenylpropanol in pykA/F knockout strainDHAP—dihydroxyacetone phosphate; PYR—pyruvate; PEP—phosphoenolpyruvate; E4P—D-erythrose-4-phosphate; DAHP—3-deoxy-D-arabinoheptanoate heptaphosphate; TktA—Transketolase; PykA/F—pyruvate kinase; PpsA—phosphoenolpyruvate synthase; AroF/AroGfbr/AroH—3-deoxy-D-arabinoheptanoate heptaphosphate synthase
Fig. 6 Strengthening of shikimate pathway on production of 3-phenylpropanolDHQ—3-dehydroquinic acid; DHS—3-dehydroshikimate; SHK—shikimic acid; CHOR—chorismate; AroB—dehydroquinic acid synthase; AroD—dehydroquinic acid dehydratase, AroE—Shikimate dehydrogenase; AroK/AroL/AroA/AroC—dehydroshikimate dehydratase; PheA—prephenate dehydrogenase
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