122NISR RESEARCH GRANT
2003 Young Investigator Research Grant

Functional Analysis of the Proteins Involved in the AromaticAmino Acids Metabolism in Bacteria
Takane KATAYAMA
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University
PurposeThe aromatic amino acids are compounds of great importance in industry, as they are precursors of many drugs
and food additives. But, the efficient fermentation methods
for them have not been developed yet. Our works were focused on (I) functional analysis of the transporters of aromatic amino acids and (II) functional analysis of the regulatory protein TyrR (a chief regulator of the genes involved in
the aromatic amino acid metabolism), the results of which
will significantly contribute to establishment of the basisfor aromatic amino acid production using bacteria.
1) Aromatic amino acids transportera) A novel phenylalanine (Phe) transport system in Escherichia coli 1)
It has so far been considered that, in E. coli, the active
transport of aromatic amino acids is mediated by five distinct permeases, AroP, Mtr, PheP, TnaB, and TyrP, and that
the AroP and PheP systems are responsible for the accumulation of Phe in cells. However, we found a low level of accumulation of Phe in the strain TK1170 (D aroP D pheP
D mtr D tna D tyrP). Growth of the aromatic transporternegative Phe strain (D aroP D pheP D mtr D tna D tyrP
pheA18::Tn10) in minimal medium supplemented with Phe
was severely inhibited in the presence of Ile and Leu (Fig.
1(A)). Moreover, the addition of Val and Leu severely decreased the Phe uptake activity of the cells, while the presence of Glu did not affect it (Fig. 1(B)). These results
strongly suggested that the branched-chain amino acids

transporter could function as the third Phe transporter.
Branched-chain amino acids are transported by the LIVI/LS and BrnQ systems. Whereas BrnQ is a single membrane protein, the LIV-I/LS system comprises two substrate-binding proteins (BP), LIV-BP and LS-BP, located in
the periplasm, and four inner membrane proteins LivHMGF. In order to determine whether BrnQ or LIV-I/LS carries out the uptake of Phe, a series of E. coli strains expressing individual transport systems were constructed andassayed for transport; AroP-expressing strain (D brnQ
D livHMGF D pheP), BrnQ-expressing strain (D aroP
D livHMGF D pheP), LIV-I/LS-expressing strain (D aroP
D brnQ D pheP), and PheP-expressing strain (D aroP D brnQ
D livHMGF ). As shown in Fig. 2 (A), LIV-I/LS-expressingstrain (D aroP D brnQ D pheP) was able to accumulate Phe
in its cells, demonstrating the involvement of the LIV-I/LS
system in Phe transport. Although the initial rate and thesteady-state level were lower than those in the strains expressing AroP and PheP, the LIV-I/LS-expressing cells exhibited the highest Phe uptake activity when cells weregrown in the presence of tyrosine and then assayed fortransport (Fig. 2 (B)), indicating the physiological significance of this system.
b) LIV-I/LS system as a determinant of azaserine sensitivity of E. coli 2)
Azaserine, which was discovered in a culture filtrate ofStreptomyces fragilis, is an antibiotic compound. It has
been shown that inhibitory effect of azaserine on E. coli isalleviated effectively in the presence of aromatic aminoFig. 1. Inhibition of Phe uptake by branched-chain amino acids. (A) Growth inhibition of Phe-auxotrophic E. coli strain (D aroP D mtr D pheP D tna D tyrP
pheA18::Tn10) in the presence of branched-chain amino acids. Disks were impregnated with 1 mM various amino acids. (B) Phe uptake activity of E. colistrain TK1170 (D aroP D mtr D pheP D tna D tyrP). Phe was added to the final concentration of 50 m M in either the absence or presence of 5 m M Glu, Leu, orVal.

123Fig. 2. The LIV-I/LS system as the third Phe transporter in E. coli. (A) Phe uptake was measured in various E. coli cells [BrnQ-(D aroP D livHMGF
D pheP), LIV-I/LS-(D aroP D brnQ D pheP), PheP-(D aroP D brnQ D livHMGF ), AroP-(D brnQ D livHMGF D pheP ), and None-(D aroP D brnQ D livHMGFD pheP) expressing cells] in the presence of 1 m M L-(U-14C)-Phe. (B) Accumulation of Phe in E. coli cells expressing individual Phe transport systems wasmeasured in the presence of 1 mM tyrosine.

acids, especially Phe, and partially in the presence ofbranched-chain amino acids. The alleviating effect of aromatic amino acids could be explained by competition for
the uptake of substrates by AroP (a general aromatic permease) because all azaserine-resistant mutants isolated so
far have lesions in the aroP gene. However, why Phe is themost effective among the aromatic amino acids and how
branched-chain amino acids affect azaserine sensitivity
have not been elucidated. Recently, we found that the LIVI/LS system, initially assumed to be a branched-chainamino acid-specific transporter, is the third Phe transporter.
This finding led us to envisage the possible linkage between
the alleviating effects of Phe and branched-chain aminoacids on azaserine toxicity.
As shown in Fig. 3 (A), the disruption of liv was effective for increased azaserine resistance of E. coli cells only
when D aroP co-existed. Whereas the D aroP strain was resistant to 0.1 mM azaserine at maximum, the strain carrying
D aroP D liv exhibited resistance to 0.2 mM azaserine, but
no significant resistance was observed for the D liv strain.
Fig. 3 (B) shows the results of complementation analysis.
Introduction of the liv genes into the D aroP D liv strain
made the cells more sensitive to azaserine as compared tothe D aroP strain, maybe due to the increased gene dosage.
The Ki value for azaserine in the inhibition of Phe uptake
was estimated to be 0.6 mM (data not shown). These results
revealed that the LIV-I/LS system is one of the major determinants of azaserine sensitivity of E. coli.
2) TyrR, a chief regulator of the genes involved in thearomatic amino acid metabolism
3,4-Dihydroxyphenyl-L-alanine (L-DOPA) is an aromatic amino acid used in the treatment of Parkinson’s disease, and can be produced by the reverse reaction of tyrosine phenol-lyase (Tpl). In order to construct a high-Tpl-expressing strain, we tried to elucidate the mechanism underlying expression of Tpl. First, we identified TyrR as a positive regulator of tpl, and revealed its action on the tpl promoter where TyrR changes its conformation from a dimer
to a hexamer upon the binding of L-tyrosine so that it interacts with RNA polymerase. TyrR is known to be a chieftranscriptional regulator of the genes involved in the aro-Fig. 3. The LIV-I/LS system as a determinant of azaserine sensitivity.
(A) MG1655, its D aroP, D livJ-yhhK-livKHMGF, and D aroP D livJ-yhhKlivKHMGF derivatives were streaked on M63-glucose minimal solid
medium containing azaserine (AS) at a final concentration of 0–0.2 mM.
(B) The LIV-I and LS systems are both involved in azaserine sensitivity, asrevealed by complementation analysis.

matic amino acid metabolism and regulate expression ofthe genes positively and/or negatively by altering its conformation. Thus, elucidation of the mechanism underlying thedimer-hexamer conformational change of TyrR is of great
importance not only for creating a high-Tpl-expressing
strain but also for figuring out the metabolic flow of thearomatic amino acids in cells.
a) Identification of an amino acid residue involved inhexamer formation of TyrR
Wilson et al. reported that E275Q amino acid substitution severely impairs the ability of TyrR to form a hexamer.
Indeed, a mutant TyrRE275Q was incapable of activating tpl
even in the presence of tyrosine (data not shown). Using thetpl-lac reporter system, we obtained several revertants ofthis mutant. Sequence analysis showed that all revertants
carry the common amino acid replacements in the central
domain of TyrR. We are now trying to determine the properties of these mutant TyrRs using the purified proteins.
b) Efficient production of L-DOPA using cells carrying amutant TyrR3)
Using the lac reporter system, we obtained several mu-124

tant TyrR proteins with enhanced ability to activate tpl. Themutations dispersed in the N-terminal (NT), central (Cen),and C-terminal (CT) domains of TyrR were accumulated bygenetic manipulations, and these tyrR alleles were introduced into the tyrR-deficient cells, and then the recombinant strains were evaluated as to L-DOPA productivity. Thehighest value (33 g l1 h1) was obtained for the cells carrying the tyrRNT Cen CT allele, and was 18-fold higher than theheretofore achieved level (1.8 g l1 h1) with the use ofwild-type cells. Our work could be a good example demonstrating that the exploitation of a transcriptional regulator isalso one of the promising ways for improving the quality
and quantity of a final product in microbial biotechnology.

References1)

Koyanagi, T., Katayama, T., Suzuki, H., and Kumagai, H. (2004).
Identification of the LIV-I/LS system as the third phenylalaninetransporter in Escherichia coli K-12. Journal of Bacteriology 186(2):343–350.
2) Koyanagi, T., Katayama, T., Suzuki, H., and Kumagai, H. (2004).
The LIV-I/LS system as a determinant of azaserine sensitivity of Escherichia coli K-12. FEMS Microbiology Letters. 237(1): 73–77.
3) Koyanagi, T., Katayama, T., Suzuki, H., and Kumagai, H. (2004). Effective production of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) withErwinia herbicola cells carrying a mutant transcriptional regulator
TyrR. Journal of Biotechnology. in press.