Figure 1 I Radial representation of the genetic code in mRNA format. The primary importance of the second codon position in determining the type of amino acid is emphasized. The first position determines the specific amino acid and the third (wobble) position demonstrates the degeneracy of the genetic code. The natural expansion of the genetic code at opal (SeC) and amber (Pyl) is illustrated in pink. ... 3 Figure 2 I Common Trp orientations. a) Edge-to-face orientation in a β-hairpin peptide (1LE0) b)
Parallel-displaced orientation in a parallel β-sheet (2KI0) c) Cation-π interaction between Arg and Trp (2B2U). ... 6 Figure 3 I Overview of some biological processes with SAM participation. a) SAM biosynthesis. b)
Donation of the amino group for biotin biosynthesis. c) Donation of ribosyl group for tRNA modification. d) Donation of aminoalkyl group for tRNA modification. e) Methyl group donation in a range of biological reactions involving DNA, RNA, proteins, and natural products. f) Aminoalkyl group used in polyamine synthesis. g) Donation of the aminoalkyl group in the synthesis of the quorum-sensing molecule N-acylhomoserine lactone. h) SAM aminoalkyl group utilized in 1-aminocyclopropane-1-carboxylic acid (ACC) synthesis (precursor of the plant hormone ethylene). i) SAM as a source of methylene groups in cyclopropane fatty acid (CFA) synthesis. ... 9 Figure 4 I Schematic overview of the two main techniques for ncAA incorporation: selective
pressure incorporation (SPI, top) and stop-codon suppression (SCS, bottom). ... 13 Figure 5 I Biosynthesis of pyrrolysine. The three enzymes PylB, PylC, and PylD catalyze the
biosynthesis of pyrrolysine from two lysines. ... 15 Figure 6 I Active site of PylRS. The enzyme is shown in pink, bound Pyl-AMP is shown in sticks and
Y384 is depicted in blue sticks (PDB entry: 2ZIM) ... 15 Figure 7 I Tyrosyl-tRNA synthetase and tRNATyr from Methanocaldococcus jannaschii. a) Tyrosyl
tRNA secondary structures from M. jannaschii and E. coli, identity elements are shown in red.
An orthogonal Mj𝑡𝑅𝑁𝐴𝐶𝑈𝐴𝑇𝑦𝑟 with mutated bases illustrated in red is also shown. b) Crystal structure of wildtype M. jannaschii TyrRS with bound tRNA. The N-terminal domain is shown in orange, the Rossmann fold in red, the CP1 domain in green, the KMSKS loop in yellow, and the C-terminal domain in blue. ... 17 Figure 8 I Schematic overview of double-sieve selection. During positive selections, all functional
library members are selected, while negative selections sieve out variants that are capable of charging cAAs. Typically, selection cycles are repeated around 2-3 times. ... 19 Figure 9 I Reaction scheme of the reaction catalyzed by the tryptophan synthase TrpBA. In a
simple metabolic conversion, indole (or its analog) reacts with serine to form tryptophan (or its analog [3,2]Tpa). ... 22 Figure 10 I Chemical structures of methionine and its analogs used in this study. ... 24 Figure 11 I Schematic overview of the strategy to generate a biocontained organism. The E. coli
trpS gene is to be replaced with an orthogonal aaRS/tRNA pair capable of discriminating between tryptophan and its analogs, rendering the strain-dependent (“addicted”) on these unnatural substrates. The strain would not be able to survive outside the laboratory, as the ncAAs 4-F-Trp, 5-F-Trp, and [3,2]Tpa do not occur in a natural environment. ... 25
Figure 12 I L-methionine de novo biosynthesis pathway in E. coli. The responsible genes and their products are denoted in dark purple. The genes that were deleted in this study to achieve full methionine-auxotrophy are highlighted by the purple box. ... 28 Figure 13 I Genomic and phenotypic verification of Met-auxotrophy for the strain MG1655
∆metEH::FRT. a) Agarose gel of a colony-PCR of ∆metEH::FRT. On the right is a schematic illustration of where the primers bind and which fragment lengths are expected. dKO: double knockout, wt: wildtype. b) Sequencing analysis of the metE and metH loci. The red bars annotate the respective up- and downstream regions of the targeted genes, the blue bars denote FRT sequences. c) Optical densities of cultures cultivated for 48 h in the absence (-M) and presence (+M) of 1 mM Met. d) Cells plated on agar without any Met (left) and supplemented with 1 mM Met (right). ... 29 Figure 14 I Comparison of the structures of trifluoromethionine (TfMet), methionine (Met), and
ethionine (Eth). Structures are represented as ball and stick models and Connolly molecular surfaces are shown in transparent blue. ... 31 Figure 15 I Schematic overview of processes involved in formylation and deformylation of Met. .. 32 Figure 16 I Schematic overview of feedback inhibition and repression of methionine biosynthesis
in E. coli. Proteins and their genes are denoted in purple, repression is represented by dashed arrows, and feedback inhibition by double arrows. The aporepressor MetJ requires SAM as cofactor. ... 34 Figure 17 I Elucidation of optimal ALE starting conditions. a) Optical densities of ∆metEH::FRT
cultivated in minimal media with Met concentrations ranging from 5 µM to 1 mM measured after 24 h (dashed lines) and 48 h (solid lines). Inset compares phosphate-buffered minimal media (NMM) with its MOPS-buffered counterpart (MOPS). b) OD600 values of ∆metEH::FRT cultivated in MOPS-buffered minimal media with 20 µM Met and Eth concentrations ranging from 5 µM to 0.5 mM measured after 24 h (dashed lines) and 48 h (solid lines). The inset shows cultures cultivated with 5 µM-250 µM Eth without the addition of Met. c) OD600 values of
∆metEH::FRT cultivated in MOPS-buffered minimal media with 20 µM Met and TfMet concentrations ranging from 5 µM to 5 mM measured after 24 h (dashed lines) and 48 h (solid lines). The inset shows cultures cultivated without any Met. Values represent the mean of three cultures with the SD as error bars. ... 36 Figure 18 I Schematic representation of the effects of small and large passage sizes during
adaptive laboratory cultivation. Large passages are more likely to adequately represent the population, including colonies with different beneficial mutations. The larger the passage size, however, the smaller the dilution necessary to discard accumulated waste products and for supplementation of fresh nutrients. It also results in shorter lag and exponential phases, while increasing stationary phase and possibly favoring the onset of the death phase. Small passages, on the other hand, result in longer lag and exponential phases with shorter stationary phases. If the passage size is too small, beneficial mutations may be lost. ... 38 Figure 19 I Adaptive laboratory evolution towards Met analog utilization. Optical densities (OD600)
are plotted against the number of passages. a) Control experiment with 20 µM Met and no analog addition. b) Adaptation towards Eth usage. c) Adaptation towards TfMet usage. ... 39 Figure 20 I Overview of the 1st approach for the replacement of the E. coli metK with either the
M. jannaschii or S. solfataricus metK. Mj: M. jannaschii, Ss: S. solfataricus. The plasmid harboring the λ red system and the helper plasmid have temperature-sensitive origins of replication and can be removed via incubation at 42°C. The linearized pieces of the rescue plasmid are digested by endogenous restriction enzymes. ... 42
Figure 21 I Colony PCR of a representative clone from the first metK KO approach. Left: Agarose gel of the colony PCR. Bands are shown for a representative clone from the KO attempt (∆metK) as well as ∆metEH as wt ctrl. Right: Schematic overview showing where the primers bind and the fragment lengths of their PCR products. ... 43 Figure 22 I Schematic overview of processes involved in the CAGO technique. Left homo: left
homology region, R short: first 40-50 bp of the right homology region, CmR: chloramphenicol resistance cassette, Right homo: right homology region. The scissors represent Cas9-mediated DNA cleavage of the universal N20PAM sequence, DSB: double-strand break. ... 45 Figure 23 I Sequencing chromatogram of a representative colony from the CAGO attempt with
the M. jannaschii and S. solfataricus editing cassettes. The sequencing chromatograms are aligned to the E. coli control cassette with its left homology arm (dark blue), the E. coli metK gene (green), R short fragment (light blue), Cm resistance cassette (grey), N20PAM (purple), and right homology arm (dark blue). ... 46 Figure 24 I Crystal structure of the E. coli MAT with bound SAM (PDB: 1RG9). The isoleucine at
position 302 is highlighted in red and bound SAM is shown as sticks colored by elements. The distance between the methyl group from SAM and the Ile side chain is shown in yellow (3.6 Å). . 47 Figure 25 I Establishing the strain ∆metEH::FRT metK(I302V). a) Verification of the isoleucine to
valine point mutation at position 302 (ATC -> GTC) of the E. coli metK. b) LB-agar plate verifying the removal of the pCAGO plasmid. Left: ampicillin supplementation, right: without ampicillin. .. 48 Figure 26 I Comparison of optical densities of the new strain metK(I302V) and its ancestor
∆metEH::FRT in the presence of increasing Eth concentrations. a) OD600 values of ∆metEH::FRT cultivated in NMM19 supplied with 15 µM Met and Eth concentrations ranging from 0-1 mM measured over 48 h. b) OD600 values of metK(I302V) cultivated in NMM19 supplied with 15 µM Met and Eth concentrations ranging from 0-1 mM measured over 48 h. ... 48 Figure 27 I Comparison of OD600 values and CFU between ∆metEH::FRT and metK(I302V) for
increasing Eth concentrations. a) Values for the strain ∆metEH::FRT with Eth concentrations increasing from 0-100 µM from top to bottom. b) Values for the strain metK(I302V) with Eth concentrations increasing from 0-100 µM from top to bottom. Values represent the mean of two (50 µM Eth, 100 µM Eth) and three (0 µM Eth, 15 µM Eth) experiments with the standard deviation as error bars. ... 50 Figure 28 I Second adaptive laboratory evolution towards Met analog utilization. Optical densities
(OD600) are plotted against the number of passages. a) Control experiment with 10 µM Met and no analog addition. b) Adaptation towards Eth usage... 51 Figure 29 I Comparison of OD600 values and CFU between the adapted populations 2 (top) and 7
(bottom) and the corresponding control populations cultivated in media supplemented with Eth. a) Populations cultivated in Eth-supplemented media for 31 passages. b) Control populations cultivated in NMM19 lacking Eth for 31 passages and then cultivated in Eth-supplemented media for this experiment. ... 52 Figure 30 I Overview of OD600 values (top) and CFU (bottom) of all the adapted populations and
the control populations cultivated in media supplemented with Eth. a) Populations cultivated in Eth-supplemented media for 31 passages. b) Control populations cultivated in NMM19 lacking Eth for 31 passages and then cultivated in Eth-supplemented media for this experiment. For better visibility, CFU/mL values are plotted as lines in these summary graphs. ... 53 Figure 31 I Overview of the biocontainment approach. Step 1: Double-sieve selection of an aaRS
library, consisting of consecutive rounds of positive and negative selections. During positive
selections, the target ncAA is supplied and functional library members are selected. Negative selections take place in the absence of the ncAA to sort out library members incorporating cAAs.
Step 2: Screening of the selected variants for promising candidates by comparing cell growth and fluorescence in the absence and presence of the ncAA. These steps require an amber anticodon. Step 3: Replacement of the endogenous trpS gene in the adapted strain with a selected aaRS/tRNA pair for suppression of Trp codons. The tRNA anticodon needs to be mutated from amber to Trp. ... 56 Figure 32 I M. mazei PylRS library. a) Crystal structure of M. mazei PylRS active site with bound ATP
and modeled in [3,2]Tpa. ATP (pink) and [3,2]Tpa (blue) are shown as sticks. Residues chosen for randomization are colored in yellow and also shown as sticks. PDB: 3VQV b) Chromatograph of the whole library sequencing. c) Sequencing results of seven randomly picked library members. 58 Figure 33 I Number of colonies on positive selection plates supplemented with 0.1 mM [3,2]Tp
compared to control plates lacking [3,2]Tp. The colonies were counted using ImageJ, where every colony larger than 5 pixels was counted. The colony numbers per plate were normalized to the culture volume for better comparison. P1-4: positive selection 1-4, ctrl: control plates. P1 and P2 represent the mean of 4 and 3 plates, respectively, with the SD represented as an error bar. ... 60 Figure 34 I Screening for promising M. mazei PylRS library members. After the fourth positive
selection, 30 colonies were resuspended in 50 µL sterile ddH2O and 2 µL of the suspension were spotted on NMM19 –Trp plates with and without 0.1 mM [3,2]Tp and with increasing Cm concentrations. The cells were incubated overnight at 37 °C. ... 61 Figure 35 I Fluorescence assay of sfGFP(R2TAG) with the PylRS HLLNQ mutant in media
supplemented with and without Trp or [3,2]Tp. a) Absorbance at 600 nm. b) Fluorescence after 15 h of incubation. ... 63 Figure 36 I Comparison of the structures of pyrrolysine, tryptophan, and its analog [3,2]Tpa... 64 Figure 37 I The tryptophanyl-tRNA synthetase. a) Structure of the B. stearothermophilus TrpRS
dimer with bound tryptophan (shown in pink). PDB: 1MB2 b) Active site of the B.
stearothermophilus TrpRS with bound tryptophan. Highlighted in red and shown as sticks are the residues chosen for randomization in the TrpRS library, bound Trp is shown in green. c) Residues that interact with bound Trp-Amp in the B. stearothermophilus crystal structure (reproduced from Doublié et al94). ... 65 Figure 38 I Sequencing results of the E. coli TrpRS library. a) Sequencing chromatogram of the
library mixture. b) Sequencing results of randomly picked colonies. ... 66 Figure 39 I Overview of the processes involved in selection with a genetic replacement system.
The gene of interest is replaced with an antibiotic resistance cassette while the rescue plasmid complements for the chromosomal loss. Subsequent elimination of the rescue plasmid followed by transformation of the library yield functional library members, which can complement for the loss of the wild type gene of interest. ... 67 Figure 40 I Assessment of the optimal time point of library transformation for the genetic
replacement system. G2748 ∆trpS was cultivated in LB media at 30 °C and I-SceI mega-nuclease expression was induced at the indicated time points. Samples were taken at different time points, OD600 was measured and CFU were assessed by plating 2 µL of a dilution series of the samples on LB agar plates. ... 69 Figure 41 I Colony forming units of the samples taken during the final optimization experiment
with the recombination system. a) Colony forming units of the samples taken 1 h and 2 h after
induction of I-SceI mega-nuclease expression. b) Colony forming units of the transformations with three control plasmids; wildtype ecTrpRS on two different backbones and ecTrpRS with an amber stop codon at position 3 for assessment of false positives due to recombination events. . 70 Figure 42 I Active site of the M. jannaschii TyrRS with bound tyrosine (green). Residues chosen for
mutation are shown as sticks and highlighted in red. PDB: 1J1U ... 71 Figure 43 I Indole analog tolerance of NEB10-beta cells under selection conditions. After
transformation with the MjTyrRS library, a dilution series was prepared from recovered NEB10-beta cells and 2 µL of each dilution were plated on LB agar plates containing 37 µg/mL Cm and increasing concentrations of 4-F-indole or [3,2]Tp. ... 72 Figure 44 I Number of colonies on positive selection plates supplemented with the indicated
indole analog compared to control plates lacking the analog. The colonies were counted using ImageJ, where every colony larger than 5 pixels was counted. P1-3: positive selection 1-3, ctrl:
control plates. P1 represents the mean of 3 ([3,2]Tp) and 5 (4-F-indole) agar plates with the SD represented as the error bar. a) Double-sieve selection with [3,2]Tp. The colony numbers for P1 were adjusted to the volume of recovered transformations spread on P2 and P3 plates (450 µL / plate). b) Selection experiments with 4-F-indole. ... 73 Figure 45 I Serial dilutions of the two promising MjTyrRS variants 33 and 39 on increasing Cm
concentrations with and without [3,2]Tp. ... 74 Figure 46 I Normalized fluorescence of MjTyrRS variant 33 and 39 after 15 h of incubation. Six
biological replicates were cultivated in ZYP 5052 media supplied with the appropriate antibiotics, as well as either 0.5 mM [3,2]Tp, 0.1 mM 4-F-indole, or no ncAA at all. Fluorescence was normalized to absorption at 600 nm. ... 75 Figure 47 I Identification of the amino acid incorporated in response to the amber codon in
sfGFP(R2TAG). The reporter protein sfGFP(R2TAG) was co-expressed with MjTyrRS variants 33 or 39 in the presence of [3,2]Tp. The ESI-MS spectra were deconvoluted using the Agilent Mass Hunter BioConfirm software for masses between 27 kDa and 30 kDa. The highest peak was normalized to 100. a) Deconvoluted spectrum of sfGFP(R2TAG) co-expressed with variant 33.
The highest peak corresponds to phenylalanine incorporation. The smaller peaks are each approximately 22 Da apart and likely correspond to sodium adducts (Na = 22.99 Da). b) Deconvoluted spectrum of sfGFP(R2TAG) co-expressed with variant 39. The highest peak corresponds to glutamine incorporation. The smaller peaks are each approximately 22 Da apart and likely correspond to sodium adducts (Na = 22.99 Da). ... 76 Figure 48 I Number of colonies on positive selection plates supplemented [3,2]Tp compared to
control plates lacking the analog during selections with the epPCR MjTyrRS 33 library. The colonies were counted using ImageJ, where every colony larger than 5 pixels was counted. P1-3: positive selection 1-3, ctrl: control plates. The numbers for the selection plates represent the mean of 5 (P1), 3 (P2), and 2 (P3) agar plates with the SD represented as error bars. The colony numbers were normalized to the culture volume. ... 78 Figure 49 I Serial dilutions of promising variants on increasing Cm concentrations with and
without [3,2]Tp after the second round of positive selection with the epPCR MjTyrRS 33 library. ... 79 Figure 50 I Representative serial dilutions of promising variants on increasing Cm concentrations
with and without [3,2]Tp after the third round of positive selection with the epPCR MjTyrRS 33 library. ... 80
Figure 51 I Fluorescence assay of sfGFP(R2TAG) with different MjTyrRS variants in the absence and presence of ncAAs. a) Variants picked after the second and third round of positive selection with the epPCR library based on variant 33. P2_x denotes variants picked after the second positive selection, with x designating the number of the randomly picked colony. P3_x denotes variants picked after the third round. sfGFP(R2TAG): control transformed with sfGFP, but no MjTyrRS. b) Test of the efficacy of 5-F-indole compared to 5-F-Trp in the fluorescence assay with variant 14.2. Analog concentrations used were: 0.03 mM 5-F-indole, 0.1 mM 5-F-indole/Trp, 0.5 mM 5-F-indole/Trp, 1 mM 5-F-indole/Trp, and 5 mM 5-F-indole/Trp. ... 81 Figure 52 I Screening of the epPCR library based on MjTyrRS variant 14.2. a) Number of colonies
on positive selection plates supplemented with 5-F-Trp compared to control plates lacking the analog. The colonies were counted using ImageJ, where every colony larger than 5 pixels was counted. P1-2: positive selection 1-2, ctrl: control plates. The numbers for the selection plates represent the mean of 2 agar plates with the SD represented as error bars. The colony numbers were normalized to the culture volume. b) Representative serial dilutions of variants on increasing Cm concentrations with and without 5-F-Trp after the first and second round of positive selections with the epPCR MjTyrRS 14.2 library. ... 82 Figure 53 I Fluorescence assay of MjTyrRS variants in the presence of the non-canonical amino
acid [3,2]Tpa. sfGFP(R2TAG): control transformed with sfGFP, but no MjTyrRS. Columns with error bars represent the mean of two or three values with the SD as error bars. ... 83 Figure 54 I Selection with the amino acid [3,2]Tpa on minimal media (NMM19 -Trp). a) Number of
colonies on positive selection plates supplemented with [3,2]Tpa compared to control plates lacking the amino acid. The colonies were counted using ImageJ, where every colony larger than 5 pixels was counted. The numbers for the selection plates represent the mean of 2 agar plates with the SD represented as error bars. The colony numbers were normalized to the culture volume. Between both positive selections, a negative selection was performed. b) Single colonies from the first and second round of positive selections with the MjTyrRS library on increasing Cm concentrations with and without the amino acid [3,2]Tpa. ... 84 Figure 55 I Selection with the amino acid [3,2]Tpa on LB media. a) Overview of the two different
approaches employed during selection experiments with 0.5mM [3,2]Tpa. b) Number of colonies on positive selection plates supplemented with [3,2]Tpa compared to control plates lacking the amino acid. The colonies were counted using ImageJ, where every colony larger than 5 pixels was counted. The numbers for the selection plates represent the mean of 2 agar plates with the SD represented as error bars. The colony numbers were normalized to the culture volume. c) Single colonies from the second and third rounds of positive selections with the MjTyrRS library on increasing Cm concentrations with and without the amino acid [3,2]Tpa. ... 85 Figure 56 I Schematic overview of processes involved in the CRISPR/Cas9 response. Figure
supplied by © Johan Jarnestad / The Royal Swedish Academy of Sciences. ... 115 Figure 57 I Nucleotide and amino acid sequence of the M. jannaschii methionine adenosyl
transferase. The sequence was codon-optimized for expression in E. coli by GeneArt (Thermo Fisher Scientific). ... 156 Figure 58 I Nucleotide and amino acid sequence of the S. solfataricus methionine adenosyl
transferase. The sequence was codon-optimized for expression in E. coli by GeneArt (Thermo Fisher Scientific). ... 157 Figure 59 I SDS PAGE of EcMAT overexpression after IMAC purification. EE4 = eluate fraction
1-4. ... 157
Figure 60 I SDS PAGE of heterologous MjMAT (left) and SsMAT (right) expression after IMAC purification. E1-E2 = eluate fraction 1-2, pre E = small peak eluted prior to the main peak. ... 158 Figure 61 I SDS PAGE of MjMAT (left) and EcMAT (right) after IEX purification. ... 158
Figure 60 I SDS PAGE of heterologous MjMAT (left) and SsMAT (right) expression after IMAC purification. E1-E2 = eluate fraction 1-2, pre E = small peak eluted prior to the main peak. ... 158 Figure 61 I SDS PAGE of MjMAT (left) and EcMAT (right) after IEX purification. ... 158