Structural and functional analyses of bacterial organic acidexporters for the efficient fermentative production of organicacids
Kei NANATANI
Graduate School of Agricultural Science, Tohoku University

Research aims
To date, organic acids, the raw materials used to produce many polymers, are mainly derived
from fossil fuels. However, with the popularization of green biotechnology, many studies
worldwide are seeking to produce organic acids through microbial fermentation. However,
harvesting biologically produced organic acids is hindered by their strong polarity, which
prevents them from crossing the cell membrane. Therefore, membrane transporters arenecessary to mediate organic acid efflux. In this study, we investigated the organic acidexport activity of bacterial membrane transporters, focusing on the following three subjects.
Subject 1: Expression and purification of aspartate:alanine exchanger (AAEx)
transporters
Methods: In order to crystallize AAEx membrane transporters, we first needed to identify
proteins having a good in vitro stability. To this end, we cloned the genes encoding bacterial
homologs of AAEx transporters and constructed expression vectors. Next, we optimized theconditions for expressing and purifying these AAEx homologs.
Results: We have successfully expressed and purified AAEx transporters and are now
optimizing the buffer conditions required to improve the purification step and crystallize
AAEx transporters.
Subject 2: Establishment of in vivo methods for the functional analysis of AAEx
transporters
Methods: To study the substrate transport mechanism of AAEx transporters, we selected a
representative member of the AAEx transporter family, the aspartate:alanine antiporter(AspT) from Tetragenococcus halophilus[1]. To assess the export function of AspT, we
exploited an Escherichia coli strain containing a deletion of the L-alanine exporter ygaW
(MLA301ΔygaW)[2]. This strain is unable to export L-alanine. Upon addition of L-Ala-L-Ala
to the medium, the cells accumulate L-alanine, which inhibits cell growth. We hypothesizedthat the expression of a functional AspT in the MLA301ΔygaW E. coli strain would restoreboth the L-alanine export function and cell growth.
Results: First, we grew the E. coli MLA301ΔygaW strain in a medium containing
to optimize the growth conditions, Isopropyl β-D-1-thiogalactopyranosideconcentration, and growth time. In cells transformed with an empty vector, we observed
growth inhibition. However, as anticipated, the expression of AspT restored L-Ala-L-Ala

L-Ala-L-Ala

resistance and promoted cell growth (Fig. 1). We believe that this method will prove usefulfor analyzing the function of AAEx transporters.
Subject 3: Establishment of amethod
toprepare
stableproteoliposomes forfunctional analyses

invitroMethods: The preparation of stable
proteoliposomes is a prerequisite
for in vitro functional analyses oftransporters. Due to a lack ofstability of the proteoliposomes, ourprevious method only allowed us tomeasure the transport activity for 1 h.
We hypothesized that traces ofdetergents that were used tosolubilize the transporters, might

Fig. 1. AspT expression restores L-Ala-L-Ala
resistance in L-alanine export-deficient cells
The L-Ala-L-Ala-sensitive MLA301ΔygaW E. coli
strain was transformed with either the pTrc_aspT
expression vector (□) or the empty pTrc99A vector(○) vector. Cell growth was monitored using
medium containing 0.5 mM L-Ala-L-Ala.

have remained in the preparation and destabilized the proteoliposomes. To eliminate these
detergent traces, we employed Bio-Beads resin, which has a high affinity for detergents, inour liposome preparation protocol.
Results: Using Bio-Beads resin to optimize our proteoliposomes preparation method, wewere able to prepare stable liposomes with a high transport activity. Moreover, the liposomes
prepared using this method allowed us to measure the transport activity for more than 3hours. We believe that this method will help us perform detailed functional analyses ofAAEx transporters.
Conclusion
To facilitate the production of organic acids through microbial fermentation further, weaimed to solve the structure and investigate the function of AAEx transporters. We havemade significant progress towards achieving these aims by successfully purifying AAEx
transporters and establishing functional transport assays.

References
1) Sasahara, A., Nanatani, K., Enomoto, M., Kuwahara, S., and Abe, K. (2011) Substratespecificity of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus inreconstituted liposomes. J. Biol. Chem. 286: 29044-29052.
2) Hori, H., Yoneyama, H., Tobe, R., Ando, T., Isogai, E., and Katsumata, R. (2011)
Inducible L-alanine exporter encoded by the novel gene ygaW (alaE) in Escherichia coli.
Appl. Environ. Microbiol. 77: 4027-4034.