Table 2.
Primers used
| Primers | Sequences (5′-3′) | Applications |
|---|---|---|
| The overexpression of mutated DAHP synthase ARO4 and chorismate mutase ARO7 to avoid feedback inhibition | ||
| 1 | CTGCACAACCCACATAATTC | Amplify ARO4 upstream homologous region for ARO4fbr overexpression from S. cerevisiae genome, forward primer |
| 2 | CGACCTGCAGCGTACGAAGCTTCAGCTGGAGAGACTCTTCTAGTTATTCTTAATTAC | Amplify ARO4 upstream homologous region for ARO4fbr overexpression from S. cerevisiae genome, reverse primer |
| 3 | GTAATTAAGAATAACTAGAAGAGTCTCTCCAGCTGAAGCTTCGTACGCTGCAGGTCG | Amplify URA3 selection marker for ARO4fbr overexpression from pUG72, forward primer |
| 4 | CATCTTTGTAAAACTTCGATACCACACACGCGGCCGCATAGGCCACTAGTGGATCTG | Amplify URA3 selection marker for ARO4fbr overexpression from pUG72, reverse primer, reverse primer |
| 5 | CAGATCCACTAGTGGCCTATGCGGCCGCGTGTGTGGTATCGAAGTTTTACAAAGATG | Amplify PTEF1-ARO4fbr-TADH1 DNA fragment for ARO4fbr overexpression from pLC-m1, forward primer |
| 6 | CATCCACGAAATCAGTGAAAGGTCAGCGCACACACCATAGCTTCAAAATGTTTCTAC | Amplify PTEF1-ARO4fbr-TADH1 DNA fragment for ARO4fbr overexpression from pLC-m1, reverse primer |
| 7 | GTAGAAACATTTTGAAGCTATGGTGTGTGCGCTGACCTTTCACTGATTTCGTGGATG | Amplify ARO4 downstream homologous region for ARO4fbr overexpression from S. cerevisiae genome, forward primer |
| 8 | CCTATTTCTGGTACTGGCTTTTC | Amplify ARO4 downstream homologous region for ARO4fbr overexpression from S. cerevisiae genome, reverse primer |
| 9 | GATCGTATATGACACAACGAGG | Amplify ARO7 upstream homologous region for ARO7fbr overexpression from S. cerevisiae genome, forward primer |
| 10 | CGACCTGCAGCGTACGAAGCTTCAGCTGATCTTATACCAATTTTATGCAGGATGCTG | Amplify ARO7 upstream homologous region for ARO7fbr overexpression from S. cerevisiae genome, reverse primer |
| 11 | CAGCATCCTGCATAAAATTGGTATAAGATCAGCTGAAGCTTCGTACGCTGCAGGTCG | Amplify URA3 selection marker for ARO7fbr overexpression from pUG72, forward primer |
| 12 | GATAAGACCCCATTCTTTGAAGGTACTTCCCGGCCGCATAGGCCACTAGTGGATCTG | Amplify URA3 selection marker for ARO7fbr overexpression from pUG72, reverse primer, reverse primer |
| 13 | CAGATCCACTAGTGGCCTATGCGGCCGGGAAGTACCTTCAAAGAATGGGGTCTTATC | Amplify PPGK1-ARO7fbr-TCYC1 DNA fragment for ARO7fbr overexpression from pLC-m1, forward primer |
| 14 | GTGGAATTGTTACCGTGATAGCCTTCATGCCTTCGAGCGTCCCAAAACCTTCTCAAGC | Amplify PPGK1-ARO7fbr-TCYC1 DNA fragment for ARO7fbr overexpression from pLC-m1, reverse primer |
| 15 | GCTTGAGAAGGTTTTGGGACGCTCGAAGGCATGAAGGCTATCACGGTAACAATTCCAC | Amplify ARO7 downstream homologous region for ARO7fbr overexpression from S. cerevisiae genome, forward primer |
| 16 | GTCATCGACTTTGTCATTGCC | Amplify ARO7 downstream homologous region for ARO7fbr overexpression from S. cerevisiae genome, reverse primer |
| The knockout of ARO10 (phenylpyruvate decarboxylase) and PDC5 (pyruvate decarboxylase) to avoid production of aromatic alcohols and direct the pathway flux to aromatic amino acids | ||
| 17 | CCTCTTCTTCTTGTGTGTTTAAGC | Amplify ARO10 upstream homologous region for ARO10 knockout from S. cerevisiae genome, forward primer |
| 18 | CGACCTGCAGCGTACGAAGCTTCAGCTGGCTTAAGGGAGTTTCTTTGTTATCTTG | Amplify ARO10 upstream homologous region for ARO10 knockout from S. cerevisiae genome, reverse primer |
| 19 | CAAGATAACAAAGAAACTCCCTTAAGCCAGCTGAAGCTTCGTACGCTGCAGGTCG | Amplify URA3 selection marker for ARO10 knockout from pUG72, forward primer |
| 20 | CACACGATAGGAATGACAGAAAAAAGCGGCCGCATAGGCCACTAGTGGATCTG | Amplify URA3 selection marker for ARO10 knockout from pUG72, reverse primer |
| 21 | CAGATCCACTAGTGGCCTATGCGGCCGCTTTTTTCTGTCATTCCTATCGTGTG | Amplify ARO10 downstream homologous region for ARO10 knockout from S. cerevisiae genome, forward primer |
| 22 | CGAAGTATTTCGGACTCTTTCTTC | Amplify ARO10 downstream homologous region for ARO10 knockout from S. cerevisiae genome, reverse primer |
| 23 | GTTGAAAATGACGACGAGCCTG | Amplify PDC5 upstream homologous region for PDC5 knockout from S. cerevisiae genome, forward primer |
| 24 | CGACCTGCAGCGTACGAAGCTTCAGCTGGTGTAATAAGAAAGAGAGGAAAGGAC | Amplify PDC5 upstream homologous region for PDC5 knockout from S. cerevisiae genome, reverse primer |
| 25 | GTCCTTTCCTCTCTTTCTTATTACACCAGCTGAAGCTTCGTACGCTGCAGGTCG | Amplify URA3 selection marker for PDC5 knockout from pUG72, forward primer |
| 26 | CCTAAACATCTATAACCTTCAAAAGCGGCCGCATAGGCCACTAGTGGATCTG | Amplify URA3 selection marker for PDC5 knockout from pUG72, reverse primer |
| 27 | CAGATCCACTAGTGGCCTATGCGGCCGCTTTTGAAGGTTATAGATGTTTAGG | Amplify PDC5 downstream homologous region for PDC5 knockout from S. cerevisiae genome, forward primer |
| 28 | CTCTTGCTTGTTCGGAGTTC | Amplify PDC5 downstream homologous region for PDC5 knockout from S. cerevisiae genome, reverse primer |
| Overexpression of l-tyrosine pathway genes ARO2 and TYR1 | ||
| 29 | CGTGAACACGTACACTATACG | Amplify ARO2 upstream homologous region for ARO2 overexpression from S. cerevisiae genome, forward primer |
| 30 | CGACCTGCAGCGTACGAAGCTTCAGCTGCGATGAGAATAACGCTTAGATGATGCCG | Amplify ARO2 upstream homologous region for ARO2 overexpression from S. cerevisiae genome, reverse primer |
| 31 | CGGCATCATCTAAGCGTTATTCTCATCGCAGCTGAAGCTTCGTACGCTGCAGGTCG | Amplify URA3 selection marker for ARO2 overexpression from pUG72, forward primer |
| 32 | CATCTTTGTAAAACTTCGATACCACACACGCGGCCGCATAGGCCACTAGTGGATCTG | Amplify URA3 selection marker for ARO2 overexpression from pUG72, reverse primer, reverse primer |
| 33 | CAGATCCACTAGTGGCCTATGCGGCCGCGTGTGTGGTATCGAAGTTTTACAAAGATG | Amplify PTEF1-ARO2-TADH1 DNA fragment for ARO2 overexpression from pLC-m2, forward primer |
| 34 | CATAACTCTTGAGGGGTTTTGTTTCTATCGCACACACCATAGCTTCAAAATGTTTCTAC | Amplify PTEF1-ARO2-TADH1 DNA fragment for ARO2 overexpression from pLC-m2, reverse primer |
| 35 | GTAGAAACATTTTGAAGCTATGGTGTGTGCGATAGAAACAAAACCCCTCAAGAGTTATG | Amplify ARO2 downstream homologous region for ARO2 overexpression from S. cerevisiae genome, forward primer |
| 36 | CATTGATGTTACCTTCACCAAGG | Amplify ARO2 downstream homologous region for ARO2 overexpression from S. cerevisiae genome, reverse primer |
| 37 | CCCATCTTTGAAAACTCTAACG | Amplify TYR1 upstream homologous region for TYR1 overexpression from S. cerevisiae genome, forward primer |
| 38 | CGACCTGCAGCGTACGAAGCTTCAGCTGGTTTATCAAGTGGATATGCTGTCCTCTTTC | Amplify TYR1 upstream homologous region for TYR1 overexpression from S. cerevisiae genome, reverse primer |
| 39 | GAAAGAGGACAGCATATCCACTTGATAAACCAGCTGAAGCTTCGTACGCTGCAGGTCG | Amplify URA3 selection marker for TYR1 overexpression from pUG72, forward primer |
| 40 | CATCTTTGTAAAACTTCGATACCACACACGCGGCCGCATAGGCCACTAGTGGATCTG | Amplify URA3 selection marker for TYR1 overexpression from pUG72, reverse primer, reverse primer |
| 41 | CAGATCCACTAGTGGCCTATGCGGCCGCGTGTGTGGTATCGAAGTTTTACAAAGATG | Amplify PTEF1-TYR1-TADH1 DNA fragment for TYR1 overexpression from pLC-m3, forward primer |
| 42 | CGATAATCGCGGTAGTGAGCATTACACGCACACACCATAGCTTCAAAATGTTTCTAC | Amplify PTEF1-TYR1-TADH1 DNA fragment for TYR1 overexpression from pLC-m3, reverse primer |
| 43 | GTAGAAACATTTTGAAGCTATGGTGTGTGCGTGTAATGCTCACTACCGCGATTATCG | Amplify TYR1 downstream homologous region for TYR1 overexpression from S. cerevisiae genome, forward primer |
| 44 | CGACCAGGATCCATATATC | Amplify TYR1 downstream homologous region for TYR1 overexpression from S. cerevisiae genome, reverse primer |
| Quantitative real-time PCR | ||
| 45 | GTGAACAATTACACAGCTCCTATAG | Amplify partial cDNA of S. cerevisiae ALG9 gene for real-time PCR, forward primer |
| 46 | CCTATGATTATCTGGCAGCAGGAAAG | Amplify partial cDNA of S. cerevisiae ALG9 gene for real-time PCR, reverse primer |
| 47 | CTGGTGATTTCGGCTCTATTGCCAAG | Amplify partial cDNA of S. cerevisiae TAL1 gene for real-time PCR, forward primer |
| 48 | GGTGGTCTTACCATGCTTCTTACCG | Amplify partial cDNA of S. cerevisiae TAL1 gene for real-time PCR, reverse primer |
| 49 | GACTTGTCTAAGTCCAAGACCTCTCC | Amplify partial cDNA of S. cerevisiae ENO2 gene for real-time PCR, forward primer |
| 50 | CATGGCTTCAGCGAAGGTCTTAGC | Amplify partial cDNA of S. cerevisiae ENO2 gene for real-time PCR, reverse primer |
| 51 | CCTGGAATTCAAGGCTTCTTTGCCAG | Amplify partial cDNA of S. cerevisiae ARO1 gene for real-time PCR, forward primer |
| 52 | CGCTACCATAACCGTATCACGAGTAC | Amplify partial cDNA of S. cerevisiae ARO1 gene for real-time PCR, reverse primer |
| 53 | CGCTAACGGTGAAAACGCCATTACC | Amplify partial cDNA of S. cerevisiae ARO4 gene for real-time PCR, forward primer |
| 54 | CGTCTTCAGTAGTTTCCCAACCTATAC | Amplify partial cDNA of S. cerevisiae ARO4 gene for real-time PCR, reverse primer |
| 55 | GAGAGGTCGCATTTCGCCACATG | Amplify partial cDNA of S. cerevisiae ARO7 gene for real-time PCR, forward primer |
| 56 | CAGGTGATTCGAATCTTCTGATGCG | Amplify partial cDNA of S. cerevisiae ARO7 gene for real-time PCR, reverse primer |
| 57 | GGTCATTTGGTATCCAGGCAAAGCG | Amplify partial cDNA of S. cerevisiae TYR1 gene for real-time PCR, forward primer |
| 58 | CTCGATTTCCGGCAGCTTACAACTC | Amplify partial cDNA of S. cerevisiae TYR1 gene for real-time PCR, reverse primer |
| 59 | GCTTCAGGTGCCATTGCTGAGAAG | Amplify partial cDNA of S. cerevisiae ARO2 gene for real-time PCR, forward primer |
| 60 | CCTGGTGATGGTGTTCAACAGATGC | Amplify partial cDNA of S. cerevisiae ARO2 gene for real-time PCR, reverse primer |