In some cases, incorporation of deuteratued tyrosine (d-tyr) is required by the design of experiments. This can be achieved in theoretically via two routes. One is by inactivating the gene specific for tyrosine biosynthesis and supplement in the culture medium exogenous d-tyr as universal protein expression building block. An alternative way is direct supplement of d-tyr at certain concentration range where feedback inhibition of Tyr biosynthesis starts to prevent host strain from adopting endogenous regular Tyr rather than exogenous d-tyr as protein expression building block.
For the first route, tyrA gene was chosen for inactivation [213]. TyrA gene product is known as chorismate mutase/prephenate dehydrogenase (CM/PDH, TyrA) which is a bifunctional enzyme occurring as a homodimer with a molecular weight of approximately 78,000 and is involved in tyrosine biosynthesis in Escherichia coli [214]. First, the common shikimate pathway intermediate chorismate undergoes rearrangement into prephenate
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catalyzed by the N-terminal CM domain of TyrA. Second, oxidative decarboxylation of prephenate into to 4-hydroxyphenylpyruvate is catalyzed by C-terminal PDH domain of TyrA. The product is eventually transaminated using glutamate as nitrogen source into L-tyrosine via TyrB (Figure 5.5.) [215]. TyrB is shared by other biosynthetic pathway like Leu, Phe and Asp as aminotransferase [216], while TyrA only specialized in the first two steps converting chorismate to tyrosine. TyrA gene was therefore chosen for inactivation since only it would discontinue tyrosine biosynthesis while leaving the other biosynthetic pathways undisturbed.
Figure 5.5. Part of shikimate pathway for biosynthesis of L-tyrosine from chorismate to tyrosine. Tyr A is a bifunctional enzyme responsible for chorismate rearrangement and prephenate decarboxylation while Tyr B serves as an aminotransferase.
Inactivation of tyrA gene in E. coli chromosome relies on homologous recombination via bacteriophage λ-Red system [213]. This strategy applies molecular cloning methods to construct short linear recombinant DNA molecules in vitro that include nucleotide sequences homologous to the regions flanking target genes on the chromosome ~30-50 basepairs (bp). The synthetic genes are used as primers to integrate with drug resistance cassette. The resulting new drug resistance cassette flanked by homologous ends is introduced into the target strain and recombinate with chromosomal target gene which is replaced by the drug marker as a result.
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The λ-Red system also possesses auxiliary components that stabilize linear DNA fragments in E. coli, and therefore, the construction of recombinant plasmids is no longer required. Instead, linear double-stranded DNA molecules for homologous recombination can be generated by two series of PCR reactions as shown in Figure 5.6. [217]. First round of PCR reactions amplify ~500bp upstream/downstream regions of the target chromosomal gene with 5’ ends overhang of UP.R and DOWN.F primers overlaping with antibiotic resistance marker. The overhangs are used in the second round of PCR to extend and amplify an antibiotic resistance marker. Two different markers sandwiched by FLP recognition tags (FRT, for later on deletion of drug cassette if needed) were designed with kanamycin resistance gene (kan) and chloramphenicol resistance gene (cat) and assembled into pKD4 and pKD3 plasmids respectively as standard PCR templates in our lab. Recombination of PCR product with genomic DNA is catalyzed by co-transformation of a helper plasmid called pKD46 which harbors three genes (γ,
β and exo) that make up the λ-Red system under the control of a tight arabinose-inducible ParaB
promoter. After the transformation of the PCR-generated linear recombinant DNA into the cells that have expressed the λ-Red enzymes and replacement of the target gene by the antibiotic cassette, the antibiotic gene can be readily eliminated by the action of FLP recombinase harbored by another helper plasmid named pCP20 (Figure 5.6). The scars left behind after the elimination of FRT-flanked resistance markers were designed to contain stop codons in all six reading frames to minimize the effects on the downstream genes. The ability to easily eliminate the resistance marker is very valuable in the construction of auxotrophs that require multiple genes to be knocked out. In addition, both pKD46 and pCP20 plasmids are equipped with temperature-sensitive replicons so that they can be readily removed after the desired recombination events.
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Figure 5.6. Schematics showing the deletion of a chromosomal gene with the λ-Red recombination system originally developed by Datsenko and Wanner [217] and modified by Lin [213]. In the first step, ~500 bp from each end of the target is amplified by 1st PCR. Please note that UP.R and DOWN.F primers have 5’ overhands that overlap with the two ends of the resistance cassette. The 2nd PCR to generate the linear recombinant DNA involves three templates: the resistance cassette and the two PCR products from the first step. Replacement of the target gene by the resistance cassette is achieved by transformation of cells that has already expressed the λ-Red system from pKD46 with the linear DNA. If desired, the resistance cassette can be removed by the action of Flp recombinase expressed from pCP20.
The alternative way of d-tyr incorporation is achieved by taking advantage of alleviated feedback inhibition by the end product (tyrosine) of tyrosine biosynthesis [218]. A collection of genes, as shown in Figure 5.7., responsible for tyrosine biosynthesis (aroF, aroL, tyrA, tyrB) and transport (aroP, trpP) belong to a group of transcription units under control of a single protein (regulon), TyrR, in E. coli [219]. Binding to specific DNA sequences known as strong/weak TyrR boxes, the TyrR protein can either activate or repress transcription at different σ70 promotors [220]. For efficient regulation to occur, the TyrR protein must interact with small ligand molecules ATP, Tyr and Phe. In the absence of cofactor or in the presence of Phe alone, TyrR
transformed into cells with λ-Red recombinase (pKD46) ~ 500 bp downstream ~ 500 bp upstream Target ~ 500 bp upstream ~ 500 bp downstream FRT Resistance cassette FRT ~ 500 bp upstream ~ 500 bp downstream FRT Resistance cassette FRT PCR PCR PCR FLP recombinase (pCP20) ~ 500 bp upstream ~ 500 bp downstream FRT ~ 500 bp upstream ~ 500 bp downstream FRT Resistance cassette FRT UP.F UP.F
UP.R DOWN.F DOWN.R
DOWN.R Step 1 Step 2 Step 3 Step 4 chromosome chromosome chromosome
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exists as a dimer, while in the presence of ATP and Tyr, TyrR self-aggregates to form a hexamer [221]. The genes in the TyrR regulon that are repressed by Tyr contain at least two TyrR boxes located adjacent to each other and differing in their respective affinities for TyrR protein (Figure 5.7.). Practically, addition of 1mM tyrosine/Phe into growth medium will be enough to switch the host strain from endogenous biosynthetic Tyr to exogenous supplemental Tyr. This is proved in MacMillan’s work on P. d. aa3 d4-tyr incorporation with 0.9 mM d4-tyr supplement at 90%
labeling efficiency. At 1mM Tyr supplement, host E. coli strain also starts to encounter growth inhibition [218] presumably by disruption of both aromatic amino acid uptake machinery and other downstream biosynthesis like vitamins (quinones, folate) and siderophores (enterobactin).
Figure 5.7. Left: Common and terminal pathways for aromatic amino acid biosynthesis and genes for aromatic amino acid transport [222]. Right: Examples of genes regulated by TyrR with SB/WB boxes and bound TyrR shown [223].