Bacterial strain and plasmid constructions

The E. coli DH5α strain (F’, Φ80dlacZ∆M15, ∆(lacZYA-argF), U169, deoR, recA1, endA1, hsdR17, (rK^-, mK⁺), supE44, λ-, thi-1, gyrA96, relA1; Woodcock et al., 1989) was used for plasmid generation and amplification. Plasmids used in this study are listed in Tab. 3.

Oligonucleotides used as primers for plasmid generation are listed in Tab. S1. For the generation of a plasmid encoding an ASC1-birA* fusion gene (see also Fig. 8A), the ASC1-containing high-copy number plasmid pME2624 served as backbone. This plasmid was linearized by PCR with a forward primer annealing downstream of the ASC1 ORF and a reverse primer annealing to the 3’-end of the ASC1 ORF excluding the stop codon. The reverse primer featured a large overhang containing a 36 base pair linker sequence and a sequence complementary to the first 20 base pairs of the birA* gene after the start codon. The birA* allele

30

containing the point mutation R118G was amplified from plasmid pRS313 (kindly provided by Dr. H. D. Schmitt, Max Planck Institute of Biophysical Chemistry, Göttingen) without its start codon. The reverse primer contained a sequence complementary to the plasmid backbone. The linearized plasmid backbone and the birA* fragment were fused by homologous recombination using the In-Fusion^® HD Cloning Kit (#639650, Clontech, Mountain View, California, USA).

The coding sequence of the ASC1-birA* fusion was verified by DNA sequencing and the plasmid was named pME4478. Similarly, a plasmid expressing the mere birA* was constructed (see also Fig. 8B): Plasmid pME2624 was linearized by PCR with the forward primer annealing downstream of the ASC1 ORF and a reverse primer annealing to the plasmid backbone upstream of the ASC1 ORF with a 20 bp overhang complementary to the birA* gene. The birA* allele including the ATG start codon was amplified from plasmid pRS313 and fused with the linearized plasmid backbone by homologous recombination. The coding sequence of birA* was verified by DNA sequencing and the plasmid was named pME4480. A plasmid encoding an asc1^DE-birA* fusion was generated by site directed mutagenesis (see also Fig. 8C). The mutated asc1^DE allele features two amino acid exchanges: R38D and K40E. To insert these substitutions within the ASC1-birA* allele, pME4478 bearing the ASC1-birA* fusion gene was used as template. With a complementary primer pair carrying the two mutations in its central part, the ASC1-birA* plasmid was fully amplified resulting in the asc1^DE-birA* vector. The template DNA was removed by DpnI treatment which exclusively digests methylated DNA, and thus only the parental vectors. The resulting plasmid was verified by DNA sequencing and termed pME4479. To obtain plasmid pME4481 ASC1 and its native promoter were amplified from pME4364 with oligonucleotides that generated a SacI and an XhoI site for restriction digestion and cloning into pME2789. Plasmids bearing asc1 mutant alleles with codons exchanged to the amber stop codon for the incorporation of the artificial photoreactive amino acid ρ-benzoyl-phenylalanine (Bpa) were also generated by site directed mutagenesis using pME2834 and pME2624 as templates. The asc1 alleles were confirmed by DNA sequencing and are named pME4174-pME4181, pME4183, pME4185, pME4186 and pME4482-pME4525.

Tab. 3: Plasmids used in this work.

Plasmid Description Reference

pUG72 AmpR, pUCori, loxP::URA3::loxP Gueldener et al., 2002 pME2787 MET25Prom, CYC1Term, URA3, 2 µm Mumberg et al., 1994 pME2789 GAL1Prom, CYC1Term, TRP1, CEN/ARS Mumberg et al., 1994

31

Plasmid Description Reference

pME2791 GAL1Prom, CYC1Term, URA3, CEN/ARS Mumberg et al., 1994

pME2624 pME2787 with ASC1 Our collection

pME2834 pME2787 with ASC1-Strep Our collection

pME4364 pME2791 with ASC1 with its native promoter (500 bp) Schmitt et al., 2017 pHK1249 EDC3Prom, URA3, CEN/ARS; EDC3-mCH Buchan et al., 2008 pESC Yrs ADH1Prom, TRP1, 2 µm, TyrRS/tRNACUA Chin et al., 2003 pRS313 PGK1Prom, CYC1Term, HIS3, CEN/ARS, birA^R118G

(based on van Werven and Timmers, 2006)

H. D. Schmitt (MPI-BPC) pME4478 MET25Prom, CYC1Term, URA3, 2 µM, ASC1-birA* This work pME4479 MET25Prom, CYC1Term, URA3, 2 µM, asc1^DE-birA* This work pME4480 MET25Prom, CYC1Term, URA3, 2 µM, birA* This work pME4481 pME2789 with ASC1 with its native promoter (500 bp) This work pME4174 pME2787 with asc1^P30Amber-Strep This work pME4175 pME2787 with asc1^D51Amber-Strep This work pME4176 pME2787 with asc1^F54Amber-Strep This work pME4177 pME2787 with asc1^A75Amber-Strep This work pME4178 pME2787 with asc1^A95Amber-Strep This work pME4179 pME2787 with asc1^K118Amber-Strep This work pME4180 pME2787 with asc1^K137Amber-Strep This work pME4181 pME2787 with asc1^Q139Amber-Strep This work pME4183 pME2787 with asc1^T209Amber-Strep This work pME4185 pME2787 with asc1^P267Amber-Strep This work pME4186 pME2787 with asc1^Q299Amber-Strep This work pME4482 pME2787 with asc1^N17Amber-Strep This work pME4483 pME2787 with asc1^K62Amber-Strep This work pME4484 pME2787 with asc1^K107Amber-Strep This work pME4485 pME2787 with asc1^K129Amber-Strep This work pME4486 pME2787 with asc1^N148Amber-Strep This work pME4487 pME2787 with asc1^N174Amber-Strep This work pME4488 pME2787 with asc1^N196Amber-Strep This work pME4489 pME2787 with asc1^Q237Amber-Strep This work pME4490 pME2787 with asc1^R275Amber-Strep This work

32 Tab. 3: Continued.

Plasmid Description Reference

pME4491 pME2787 with asc1^F278Amber-Strep This work pME4492 pME2787 with asc1^Y281Amber-Strep This work pME4493 pME2787 with asc1^H288Amber-Strep This work

pME4494 pME2787 with asc1^N17Amber This work

pME4495 pME2787 with asc1^P30Amber This work

pME4496 pME2787 with asc1^F54Amber This work

pME4497 pME2787 with asc1^K62Amber This work

pME4498 pME2787 with asc1^I67Amber This work

pME4499 pME2787 with asc1^A75Amber This work

pME4500 pME2787 with asc1^K87Amber This work

pME4501 pME2787 with asc1^A95Amber This work

pME4502 pME2787 with asc1^K107Amber This work

pME4503 pME2787 with asc1^K118Amber This work

pME4504 pME2787 with asc1^K129Amber This work

pME4505 pME2787 with asc1^K137Amber This work

pME4506 pME2787 with asc1^Q139Amber This work

pME4507 pME2787 with asc1^N148Amber This work

pME4508 pME2787 with asc1^K161Amber This work

pME4509 pME2787 with asc1^D165Amber This work

pME4510 pME2787 with asc1^N174Amber This work

pME4511 pME2787 with asc1^F186Amber This work

pME4512 pME2787 with asc1^N196Amber This work

pME4513 pME2787 with asc1^T209Amber This work

pME4514 pME2787 with asc1^A226Amber This work

pME4515 pME2787 with asc1^K228Amber This work

pME4516 pME2787 with asc1^Q237Amber This work

pME4517 pME2787 with asc1^E239Amber This work

pME4518 pME2787 with asc1^N248Amber This work

pME4519 pME2787 with asc1^T258Amber This work

pME4520 pME2787 with asc1^P267Amber This work

pME4521 pME2787 with asc1^R275Amber This work

pME4522 pME2787 with asc1^F278Amber This work

33

Plasmid Description Reference

pME4523 pME2787 with asc1^Y281Amber This work

pME4524 pME2787 with asc1^H288Amber This work

pME4525 pME2787 with asc1^Q299Amber This work

2.3 Cultivation of microorganisms