Biosensor-Assisted Adaptive Laboratory Evolution for Violacein Production
Abstract
:1. Introduction
2. Results
2.1. Optimization of the Violacein Production System
2.2. Development of Tryptophan-Responsive Sensors
2.3. Tryptophan-Responsive Sensor-Driven ALE for Violacein Overproduction
2.4. Examination of the Evolved Strain
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains, Plasmids, and Reagents
4.2. Construction of Heterologous Violacein Pathway
4.3. Development of Tryptophan-Responsive Sensors
4.4. Validation of Tryptophan-Responsive Sensors
4.5. Adaptive Laboratory Evolution Procedure
4.6. Culture Conditions for Violacein Production
4.7. Analytical Methods
4.8. Whole-Genome Sequencing
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
ALE | Adaptive Laboratory Evolution |
E. coli | Escherichia coli |
C. violaceum | Chromobacterium violaceum |
E4P | Erythrose 4-phosphate |
PEP | Phosphoenolpyruvate |
DAHP | 3-deoxy-d-arabinoheptulosonate 7-phosphate |
L-TRP | L-tryptophan |
IPAI | indole-3-pyruvic acid imine dimer |
PDV | prodeoxyviolacein |
PV | proviolacein |
DV | deoxyviolacein |
5′-UTR | 5′-untranslated region |
SNP | single nucleotide polymorphism |
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Name | Sequence (5′-3′) |
---|---|
Primers used for the construction of violacein production plasmid | |
homology_pCDF_F2 | acaagcttgcggccgcataatgcttaagtc |
homology_pCDF_R | attatacgagccgatgattaattgtcaaggatccccatggtatatctccttattaaagttaaacaaaattatttc |
UTR_vioA_F | gtgagcggataacaattgaaacggacataaggaggaaattctatgaaacattcttccgatatctgcattg |
term_vioA_R | gctgttgcagcgtatcgccgcgtaacgcaaaaaaccccgcttcggcggggttttttcgcgaattcttgac |
UTR_vioB_F | tgtgtggaattgtgagcggataacaattaatcgagttcgaaggaggaaaagtcatgagcattctggatttcccgc |
term_vioB_R | gcaaactgagccgcgaggcctaacgcaaaaaaccccgcttcggcggggttttttcgcgcggccgcata |
homology_vioA_F2 | gggatccttgacaattaatcatcggctcgtataatgtgtggaattgtgagcggataacaattgaaacgga |
term_vioA_R | gctgttgcagcgtatcgccgcgtaacgcaaaaaaccccgcttcggcggggttttttcgcgaattcttgac |
homology_vioB_F2 | ttcggcggggttttttcgcgaattcttgacaattaatcatcggctcgtataatgtgtggaattgtgagcggataaca |
term_vioB_R2 | gcaaactgagccgcgaggcctaacgcaaaaaaccccgcttcggcggggttttttcgcgagctcggcgcgcctgca |
homology_vec_F | cctaggctgctgccaccgctgagcaataactagc |
homology_vec_R | cttgcggccgcataatgcttaagtcgaaca |
UTR_vioC_F | tgtgtggaattgtgagcggataacaattaaggacaacaaaaggaggagaactaatgaaacgtgcgattatcgttggtg |
term_vioC_R | ggtacaagattggtcgcgtgaattaacgcaaaaaaccccgcttcggcggggttttttcgc |
UTR_vioD_F | tgtgtggaattgtgagcggataacaattccattggagagaaggagggaaattcatgaagattctggtcattggtgctg |
term_vioD_R | tgcgttatgctttgcagcgctaacgcaaaaaaccccgcttcggcggggttttttcgcctcgagttg |
UTR_vioE_F | aatgtgtggaattgtgagcggataacaattaaaacgaaaataaggaggaaattctatggagaaccgtgagccacc |
term_vioE_R | cggttttcgcggccaagcgctaacgcaaaaaaccccgcttcggcggggttttttcgcggtaccttgacaattaatcat cggctcg |
homology_vioC_F2 | cgcttcggcggggttttttcgcctcgagttgacaattaatcatcggctcgtataatgtgtggaattgtgagcggataac |
term_vioC_R2 | cttcggcggggttttttcgccctaggctgctgccaccgctgagcaataactagc |
homology_vioD_F2 | gcggtaccttgacaattaatcatcggctcgtataatgtgtggaattgtgagcggataac |
term_vioD_R | tgcgttatgctttgcagcgctaacgcaaaaaaccccgcttcggcggggttttttcgcctcgagttg |
homology_vioE_F2 | cttgcggccgcataatgcttaagtcgaacattgacaattaatcatcggctcgtataatgtgtggaattgtgagcggataac |
term_vioE_R | cggttttcgcggccaagcgctaacgcaaaaaaccccgcttcggcggggttttttcgcggtaccttgacaattaatcatcg gctcg |
Primers used for the construction of tryptophan-responsive sensor | |
T7_term_F | ctagcataaccccttggggc |
pET23b_vec_R | ccgagatctcgatcccgcgaaat |
tnaC_homo_F | caacgctgcccgagatctcgatcccgcgaaatgcatgcccgcgcttacgaagccgcattc |
tnaA_homo_w_tetA | ccgaggatgacgatgagcgcattgttagatttcatcggttcagggagatgtttaaagttttccattac |
tetA_F | atgaaatctaacaatgcgctcatcgtcatc |
sfgfp_homo_R | gcatggacgagctgtacaagtaaaccgctgagcaataactagcataaccccttggggc |
D21T_blunt_F | acacaccgcccttgatttgccc |
D21T_blunt_R | gacaattttgttgtcaatattgaacc |
Gene | Function | Position | Mutation | Amino Acid Change | |
---|---|---|---|---|---|
EPWSV | EPWSV2 | ||||
galR | DNA-binding transcriptional dual regulator [42] | 2975313 | G | C | R20P |
ptrA | Protease 3 [43] | 2956475 | A | T | S356T |
rnr | RNase R [44] | 4413454 | A | C | E707D |
rpoC | RNA polymerase subunit β’ [45] | 3450328 | T | A | Q335L |
tcyP | Cystine/sulfocystein:cation symporter [46] | 1812711 | A | C | T22P |
Name | Relevant Characteristics 1 | Source |
---|---|---|
Strains | ||
Mach1-T1R | F− φ80(lacZ)ΔM15 ΔlacX74 hsdR(rK−mK+) ΔrecA1398 endA1 tonA | Invitrogen |
E. coli W3110 | F− λ− rph-1 INV(rrnD, rrnE) | ATCC39936 |
ATCC31743 | E. coli W3110-ΔtrpABCDE-ΔtrpR-ΔtnaA/pSC101-trpABCDE | ATCC31743 |
EPW | ATCC31743, KanR | This study |
EPWS | EPW/TrpSENmut | This study |
EPWSV | EPWS/pVio | This study |
EPWSV1 | Evolved strain derived from EPWSV | This study |
EPWSV2 | Evolved strain derived from EPWSV | This study |
EPWSV3 | Evolved strain derived from EPWSV | This study |
EPWSV4 | Evolved strain derived from EPWSV | This study |
Plasmids | ||
pCDFDuet-1 | Expression vector, SmR, CloDF13 ori | Novagen |
pET-23b | Expression vector, AmpR, pBR322 ori | Novagen |
pCPA | pCDF-Ptac::synUTRvioA-vioA::Ter- Ptac::synUTRvioB-vioB::Ter | This study |
pVio | pCDF-Ptac::synUTRvioA-vioA::Ter- Ptac::synUTRvioB-vioB::Ter-Ptac::synUTRvioE-vioE::T-Ptac::synUTRvioD-vioD::Ter-Ptac::synUTRvioC-vioC::Ter | This study |
TrpSEN | pET-23b-PtnaC::tnaC-tetA-sfgfp::Ter | This study |
TrpSENmut | pET-23b-PtnaC::tnaC(D21T)-tetA-sfgfp::Ter | This study |
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Gwon, D.-a.; Seok, J.Y.; Jung, G.Y.; Lee, J.W. Biosensor-Assisted Adaptive Laboratory Evolution for Violacein Production. Int. J. Mol. Sci. 2021, 22, 6594. https://doi.org/10.3390/ijms22126594
Gwon D-a, Seok JY, Jung GY, Lee JW. Biosensor-Assisted Adaptive Laboratory Evolution for Violacein Production. International Journal of Molecular Sciences. 2021; 22(12):6594. https://doi.org/10.3390/ijms22126594
Chicago/Turabian StyleGwon, Da-ae, Joo Yeon Seok, Gyoo Yeol Jung, and Jeong Wook Lee. 2021. "Biosensor-Assisted Adaptive Laboratory Evolution for Violacein Production" International Journal of Molecular Sciences 22, no. 12: 6594. https://doi.org/10.3390/ijms22126594