Fermentative production of ophthalmic acid

Tomokazu ITO

Department of Applied Molecular Biosciences, Graduate School of
Bioagricultural Sciences, Nagoya University

Research aims
Escherichia coli YggS is a highly conserved pyridoxal phosphate (PLP)-binding
protein whose biochemical function is unknown. We found that a yggS-deficient E. colis t r a i n ( Δ y g g S ) a c c u m u l a t e s a n u n u s u a l me t a b o l i t e , o p h t h a l mi c a c i d(γ-L-glutamyl-L-2-aminobutyryl-glycine, Fig. 1)1). This ∆yggS mutant is the firstmicroorganism that accumulates high levels of ophthalmic acid. Ophthalmic acid is
known to be an oxidative stress biomarker and a potent agonist of a calcium-sensing
receptor that elicits the kokumi taste. Although ophthalmic acid has potential uses in
research and industrial applications, it is currently very expensive, and no fermentative
production method has been reported. In the present study, we tried to develop the firstk n o w n f e r me n t a t i v e me t h o d f o r t h e p r o d u c t i o n o f o p h t h a l mi c a c i d .
Methods
Intracellularophthalmic

acid

concentrations for ΔyggS strains
overexpressing threonine dehydratase(IlvA),

alanine-valinetransaminase(avtA), γ-glutamyltransferase (GshA), or
glutathionesynthase(GshB)

determined.

We

constructed

wereaγ-glutamyltransferase(Ggt)-deficientstrain, ΔyggS/ggt, using the ΔyggS strain.

Fig. 1

Putative metabolic pathway of ophthalmic acidA mutant strain (ΔyggS/ggt/yliA-B) lacking a glutathione importer (ΔyliA-B) was thengenerated, using the ΔyggS/ggt strain. Plasmids that express gshA and gshB, and that
express mutated gshA and gshB, were constructed and then introduced into theΔyggS/ggt strain. These E. coli cells were cultivated in a synthetic M9-glucose medium,in the presence or absence of amino acids (0–1.0 mM of L-Glu, L-2-AB, and Gly).
Total amounts of ophthalmic acid were quantified by HPLC.
Results
Ophthalmic acid is probably synthesized via sequential reactions catalyzed by GshA
and GshB in E. coli. IlvA and AvtA are expected to participate in L-2-aminobutyrate(L-2-AB) biosynthesis (Fig. 1). IlvA catalyzes the dehydration of L-Thr to generate
2-ketobutyrate (2-KB) and ammonia. AvtA converts 2-KB to L-2-AB, and vice versa.
Overexpression of these enzymes is potentially capable of elevating intracellular
ophthalmic acid levels. As expected, expression of plasmid-borne gshA and gshB in theΔyggS

strainfurtherincreasedacid levels; however, the expressionof plasmid-borne ilvA and avtA didnot.
In the ΔyggS strain, ophthalmicacidlevels

increased

duringAmount of ophthalmic acid（µmol/L)(~20-fold) intracellular ophthalmic
1.4
1.2⊿ yggS

10.8⊿ yggS⊿ggt

0.6

WT

0.4
0.2
0025

5075

100

Cultivation time (h)cultivation, and reached maximal

Fig. 2 Effect of ggt mutation on the cultivation time-dependent

concentrations

ophthalmic acid accumulation

when

theculture

reached the early stationary phase.
Ophthalmic acid levels then dropped to below the detection limit during the latestationary phase (Fig. 2), indicating the presence of some ophthalmic acid-metabolizing
enzyme(s). We speculated that Ggt is the primary enzyme responsible for thedegradation of ophthalmic acid. Therefore, we constructed a double-mutant of yggS andggt (ΔyggS/ggt) and analyzed its cultivation time-dependent production of ophthalmic
acid. As shown in Fig. 2, the ggt mutation completely suppressed the cultivation
time-dependent decreases in ophthalmic acid levels.
In E. coli, a directed fermentation method for producing glutathione was reported thatused an E. coli strain overexpressing gshA and gshB, and lacking ggt and glutathione
importers (yliA-B). Therefore, we constructed a triple-mutant lacking yggS, ggt, andyliA-B. However, no significant increases in ophthalmic acid productivity wereobserved for the strain containing the glutathione importer mutation (data not shown).
We then constructed a gshA and gshB co-expression vector. In addition, to confer
resistance to product inhibition, a point mutation was introduced into gshA, and aplasmids that express the mutated gshA (gshA[S495F]) and gshB were constructed.
These plasmids were introduced into ΔyggS/ggt, and this transfected strain wascultivated in M9 medium in the presence or absence of amino acids (0–1.0 mM ofL-Glu, L-2-AB, and Gly). In the M9 medium (without amino acids), co-expression ofgshA and gshB slightly increased ophthalmic acid productivity, but supplementation
with amino acids significantlyincreased

ophthalmic

acidproductivity. In the presence of
1 mM of L-Glu, L-2-AB, andGly, the E. coli ΔyggS/ggt
strain overexpressing gshA andgshB produced 114 μmol/L ofophthalmic acid. In the sameconditions, the E. coli strain
overexpressing gshA(S495F)
and gshB produced 186 μmol/L

Fig. 3 Effect of ggt mutation and gshA/gshB overexpression on theophthalmic acid productionof ophthalmic acid (Fig. 3).
Conclusion
In this study, we developed the first known fermentative method for producing
ophthalmic acid, using with a yggS-deficient E. coli strain. We found thatoverexpression of gshA and gshB was effective in increasing ophthalmic acidproduction. The ggt mutation completely suppressed the cultivation time-dependent
decrease of ophthalmic acid, demonstrating that Ggt is the primary enzyme responsiblefor the degradation of ophthalmic acid. We also found that the E. coli ΔyggS/ggt strain
overexpressing gshA(S495F) and gshB produced ~50 mg/L of ophthalmic acid in an M9
medium that was supplemented with 1 mM of L-Glu, L-2-AB, and Gly. Our results
reveal the method for further fermentative production of ophthalmic acid.

References
1) Ito T., Yamauchi A., Hemmi H., Yoshimura T. (2016) Ophthalmic acidaccumulation in an Escherichia coli mutant lacking the conserved pyridoxal
5′-phosphate-binding protein YggS. Journal of Bioscience and Bioengineering. Inpress.