Development of Indole-3-Acetic Acid-Producing Escherichia coli by Functional Expression of IpdC, AspC, and Iad1

Abstract

Biosynthesis of indole-3-acetic acid (IAA) via the indole-3-pyruvic acid pathway involves three kinds of enzymes; aminotransferase encoded by aspC, indole-3-pyruvic acid decarboxylase encoded by ipdC, and indole-3-acetic acid dehydrogenase encoded by iad1. The ipdC from Enterobacter cloacae ATCC 13047, aspC from Escherichia coli, and iad1 from Ustilago maydis were cloned and expressed under the control of the tac and sod promoters in E. coli. According to SDS-PAGE and enzyme activity, IpdC and Iad1 showed good expression under the control of $P_{tac}$, whereas AspC was efficiently expressed by $P_{sod}$ originating from Corynebacterium glutamicum. The activities of IpdC, AspC, and Iad1 from the crude extracts of recombinant E. coli Top 10 were 215.6, 5.7, and 272.1 nmol/min/mg-protein, respectively. The recombinant E. coli $DH5{\alpha}$ expressing IpdC, AspC, and Iad1 produced about 1.1 g/l of IAA and 0.13 g/l of tryptophol (TOL) after 48 h of cultivation in LB medium with 2 g/l tryptophan. To improve IAA production, a tnaA gene mediating indole formation from tryptophan was deleted. As a result, E. coli IAA68 with expression of the three genes produced 1.8 g/l of IAA, which is a 1.6-fold increase compared with wild-type $DH5{\alpha}$ harboring the same plasmids. Moreover, the complete conversion of tryptophan to IAA was achieved by E. coli IAA68. Finally, E. coli IAA68 produced 3.0 g/l of IAA after 24 h cultivation in LB medium supplemented with 4 g/l of tryptophan.

Introduction

Indole-3-acetic acid (IAA) is the most abundant naturally occurring plant hormone with auxin activity, which controls many physiological processes in plants such as cell enlargement and division, tissue differentiation, and phototropic and gravitropic responses [41, 43, 48]. Several plant-associated bacteria, fungi, and yeasts, including Agrobacterium tumefaciens [14], Azospirillum brasilense [4, 17, 44], Bradyrhizobium spp. [25], Enterobacter cloacae [18], Erwinia herbicola [7], Pantoea agglomerans [1], Pseudomonas syringe subsp. savastanoi [26], Pseudomonas chlororaphis [11, 16], Saccharomyces cerevisiae [34], Streptomyces spp. [24], and Ustilago maydis [35], are known to be able to synthesize IAA in the presence of tryptophan. IAA produced by these microbes in the rhizosphere has been reported to enhance the development of the plant root system, improve mineral and water uptake by the roots, inhibit the growth of several plant pathogenic microbes such as Fusarium oxysporum and U. maydis, and be used as a communication system with the host plant [30, 34, 41, 43].

In bacteria, five different IAA-biosynthetic pathways via indole-3-acetamide (IAM), indole-3-pyruvic acid (IPA), tryptamine, indole-3-acetaldoxime, and indole-3-acetaldehyde derived from tryptophan were reported (Fig. 1) [7, 12, 41]. Of these, various genes and enzymes regarding IAM- and IPA-related pathways were well identified and characterized [41]. Plant-pathogenic bacteria causing hyperplasia such as A. tumefaciens, P. syringae subsp. savastanoi, and E. herbicola pv. gypsophilae, usually synthesize IAA via the IAM pathway [7]. The first step in the IAM pathway is catalyzed by tryptophan 2-monooxygenase (IaaM), which converts tryptophan to IAM, and is followed by the second step in which IAM is hydrolyzed to IAA by IAM hydrolase (IaaH) [6, 23, 47]. The IPA pathway has been described in a broad range of bacteria, including Bradyrhizobium, A. brasilense, Rhizobium, E. cloacae, and cyanobacteria [4, 7, 18, 25, 40]. Tryptophan is first transaminated to IPA by an aminotransferase with broad substrate specificity for amino acids [13, 31, 46]. IPA proceeds via indole-3-acetaldehyde (IAAld) to IAA through the activities of indole-3-pyruvic acid decarboxylase (IpdC) and IAAld dehydrogenase, respectively [41]. It was suggested that the rate-limiting step of the IPA pathway in bacteria is the conversion of IPA to IAAld. Several decarboxylases encoded by ipdC have been isolated and characterized from E. cloacae, A. brasilense, Paenibacillus polymyxa, Methylobacterium extorquens, and E. herbicola [4, 7, 12, 19, 32]. Meanwhile, aldehyde dehydrogenase encoded by iad1, which is able to convert IAAld to IAA, was only reported in U. maydis [3, 35].

Fig. 1.Scheme of indole-3-acetic acid biosynthetic pathway from L-tryptophan in bacteria. Abbreviations: TRP, L-tryptophan; IPA, indole-3-pruvic acid; IAAld, indole-3-acetaldehyde; IAM, indole-3-acetamide.

Since IAA biosynthesis can be modulated by environmental stresses, including acidic pH, osmotic stress, and carbon limitation, plant extracts or specific compounds and/or the presence of plant surfaces, end-product IAA or intermediate, and genetic factors such as the location of IAA biosynthesis genes in the genome, the expression mode of genes, and transcriptional regulators, IAA-producing microbes isolated from rhizosphere produced very little IAA [41]. IAA biosynthesis via the IAM pathway has been attempted following expression of IaaM and IaaH; however, the production level was less than 1 g/l [6,23]. Meanwhile, the IPA pathway was induced by addition of tryptophan to the culture medium or carried out by amplification of ipdC in recombinant microbes [4,7,29,40]. When microbes were cultured in media with tryptophan, a mixture of indolic byproducts including tryptophol (TOL), IAAld, and indole- 3-lactic acid as well as IAA accumulated, and thus the production of IAA was very inefficient in these microbes [7,32]. To date, although it is necessary to functionally express three genes related to the IPA pathway, genes involved in the expression of the remaining part of this pathway have not yet been tried to produce IAA-producing microbes. In addition, the commercial production of IAA was performed by chemical synthesis until now.

Escherichia coli is widely used in the industrial production of many amino acids including phenylalanine and tryptophan and is a potential host for production of valuable metabolites [2,42,45]. In this study, we report, for the first time, efficient production of IAA from tryptophan via the IPA pathway by recombinant E. coli cells expressing AspC, IpdC, and Iad1. The aspC, ipdC, and iad1 genes obtained from E. coli, E. cloacae, and U . maydis, respectively, were functionally expressed under the control of promoter Psod, Ptac, and Ptac, respectively, in E. coli. Moreover, the complete conversion of tryptophan to IAA was achieved by deletion of gene tnaA in E. coli.

Table 1.aAmpR, ampicillin resistance; KanR, kanamycin resistance; Spc/StrR, spectinomycin/streptomycin resistance; CmR, chloramphenicol resistance. bDHT36 was kindly donated by Prof. Seon-won Kim working at Gyeongsang National University in South Korea. cPlasmid pTZiad1c was kindly donated by authors.

 

Materials and Methods

Bacterial Strains, Plasmids, and Recombinant DNA Techniques

Bacterial strains and plasmids used in this work are described in Table 1. E. coli Top 10 was employed as the host for general DNA manipulation and the source of aspC gene. E. coli XL-1 blue was used as the host for propagation of pCL1921 and its derivative [20]. E. coli DH5α and tnaA deletion strain DHT39 were used as hosts for IAA production. DNA templates of the sod promoter and ipdC gene were obtained from Corynebacterium glutamicum ATCC 13032 and E. cloacae ATCC 13047, respectively. Plasmids pKK223-3 and pTZiad1c were used as DNA templates for obtaining the tac promoter-multiple cloning sites-rrnB transcription terminator and iad1 gene from U. maydis, respectively [3]. Plasmids pKK223-3, pCES208, and pCL1921 were used for the cloning and expression of aspC, ipdC, and iad1 [27]. All general recombinant DNA techniques were carried out according to Sambrook and Russell [37]. PCR constructs were verified by DNA sequencing.

Media and Conditions

E. coli was grown in LB medium (10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl) at 37℃. When needed, appropriate antibiotic(s) and/or IPTG were added to the medium. The amounts of added antibiotics were as follows; 100 mg/l ampicillin, 50 mg/l kanamycin, and 100 mg/l spectinomycin. C. glutamicum and E. cloacae were grown in LB agar medium and Nutrient Agar medium (5 g/l peptone, 3 g/l beef extract, 5 g/l NaCl, 15 g/l agar) at 30℃, respectively.

Enzymes and Chemicals

Restriction enzymes, pfu-x DNA polymerase, plasmid mini-prep kit, and gel extraction kit were purchased from New England Biolab (USA), Solgent Corp. (Korea), Intron (Korea), and Macrogen (Korea), respectively. L-Tryptophan, α-ketoglutarate, indole-3-pyruvate, indole-3-acetaldehyde, and NAD+ were all purchased from Sigma-Aldrich (St. Louis, MO, USA), and the other chemicals were from Junsei (Japan) or Samchun Chemicals (Korea).

Construction of IpdC, AspC, and Iad1 Expression Vectors

IpdC expression vector. For cloning of the ipdC gene by polymerase chain reaction (PCR), a 1.7 kb fragment from E. cloacae was amplified using primers P1 and P2 (Table 2), digested with EcoRI and HindIII, and ligated with pKK223-3/EcoRI/HindIII. The resulting plasmid, pKIP16, was introduced into E. coli Top 10, and transformants were selected on ampicillin-containing LB agar plates. To subclone the ipdC with Ptac and TrrnB in pKIP16, primers P3 and P4 were used. The 2.2 kb fragment digested with SalI and KpnI was subcloned into pCES208, and yielded pCTIP22.

AspC expression vector. The aspC gene from E. coli was cloned into pKK223-3 via PCR using primers P5 and P6. A PCR product with 1.2 kb was gel-purified, cut with EcoRI/HindIII, and inserted into pKK223-3, which was digested using the same enzymes. The Ptac-aspC-TrrnB in constructed plasmid pKAS12 was subcloned into pCES208 at BamHI and SalI sites. The PCR was performed using primer set P7 and P8, and the constructed plasmid was designated pCTAS18. In order to functionally express aspC in E. coli, the sod promoter (0.3 kb) derived from C. glutamicum and aspC-TrrnB (1.6 kb) was amplified using primer sets P9-P10 and P11-P12, respectively. The gel-purified PCR products Psod and aspC-TrrnB were combined and used for the 2nd round of PCR with P9 and P12. Plasmid pCSAS19 was constructed by cutting pCES208 with SalI and BamHI and followed by ligation with 1.9 kb of the Psod-aspC-TrrnB/SalI/ BamHI fragment.

Table 2.aRestriction enzyme sites are underlined.

Iad1 expression vector. The iad1 ORF of U. maydis was amplified using primer set P13-P14 and pTZiad1c as the template DNA. The PCR product was cut with EcoRI and PstI and cloned into the same restriction sites of pKK223-3 to yield pKID15. The iad1 harboring Ptac and TrrnB in pKID15 was amplified using PCR with primers P15 and P16, digested with XbaI and BamHI, and inserted into pCES208/XbaI/BamHI to generate pCTID21. In addition, to express the iad1 gene in the low copy plasmid, pCL1921, the 2.3 kb XbaI-KpnI digested fragment of pCTID21 was gel-purified and ligated with pCL1921 cut by XbaI and KpnI to yield pCLTID23.

AspC-IpdC coexpression vector. In order to obtain the hybrid plasmid containing AspC and IpdC, the 2.2 kb ipdC fragment in pCTIP22 was digested using SalI and KpnI, gel-purified, and inserted into SalI/KpnI-cleaved pCSAS19, resulting in plasmid pCIA41.

Construction of tnaA Deletion Mutant

To construct a tnaA deletion mutant, the Kmr marker in E. coli DHT39 was removed using pCP20, which shows the temperature-sensitive replication nature and thermal induction of FLP recombinase [8,9].

Enzyme Assays

When the optical cell density of all of recombinant E. coli cells, except E. coli TOP 10/pCSAS19, reached 0.6 in LB medium at 37℃, 0.2 mM IPTG was added to the culture broth and cultivated for 5 h. Cells were harvested by centrifugation at 10,000 ×g for 5 min, washed with each reaction buffer, resuspended with the same buffer, and lysed by sonication. After centrifugation at 13,000 ×g for 30 min, the crude extracts were used for enzyme assays.

To determine the IpdC activity, coupled reaction with Iad1 was performed [3,38]. To a 1 ml reaction mixture, 50 mM phosphate buffer (pH6.5), 0.1 mM thiamine pyrophosphate, 0.1 mM sodium bisulfite, 0.1 mM magnesium sulfate, 0.5 mM IPA, 10 mM KCl, 1 mM NAD+, 0.05 ml crude extract containing IpdC, and 0.1 ml crude extract with Iad1 were added. The absorbance of NAD+ reduction was measured at 360 nm instead of 340 nm to remove the interference of IPA absorbance. One unit (U) of IpdC activity was defined as the amount of enzyme required to reduce 1 nmol NAD+ per minute per mg-protein at 360 nm by UV spectrophotometry (Shimadzu, UV-1800).

The AspC activity was assayed using cell-free extract of E. coli Top 10 containing plasmid pCES208, pCTAS18, or pCSAS19. The 1 ml reaction mixture consisted of 50 mM sodium phosphate (pH 7.5), 5 mM L-tryptophan, 5 mM α-ketoglutarate, 0.01 mM pyridoxal phosphate, and 0.1 ml of crude extract. After incubation at 30℃ for 15 min and 30 min, the reaction was stopped by the addition of 200 μl of 20% trichloroacetic acid, followed by centrifugation at 10,000 ×g for 10 min. The produced IPA in supernatant was determined at 327 nm using a UV spectrophotometer. One unit (U) of AspC activity was defined as the amount of mg-protein that catalyzed the formation of 1 nmol of IPA per minute.

Iad1 activity was determined as follows: 60 mM Tris-HCl (pH 8.0), 10 mM KCl, 1 mM indole-3-acetaldehyde, 1 mM NAD+, and 0.2 ml of crude extract were mixed in a 1 ml reaction tube [3]. The specific Iad1 activity (1 unit) was defined as the amount of enzyme required for reduction of 1 nmol NAD+ per minute per mg-protein at 340 nm by UV spectrophotometry. All of the enzyme assays described above were performed in triplicate .

Production and Analysis of IAA, TOL, and Tryptophan

For IAA production in a 250 ml flask, LB medium supplemented with 0~6 g/l tryptophan, 5 g/l KH2PO4 (pH 7.0), and appropriate antibiotics were used. One milliliter of overnight culture was added to an Δ-flask containing 30 ml of the medium and cultured for 48 h at 37℃ with vigorous shaking. If necessary, 0.2 mM IPTG was added after 1~2 h cultivation (OD600nm reaches to 0.6). To quantify IAA and TOL, 1.5 ml of the culture supernatant was adjusted to pH 2.5 with 10% H3PO4, extracted with 1 volume of ethyl acetate, and the solvent was then evaporated. The dried samples were dissolved in 1 ml of methanol, filtered, and analyzed on an Agilent 1200 HPLC (high-pressure liquid chromatography) system equipped with a C-18 column maintained at 50℃. HPLC was performed at a flow rate of 0.8 ml/min by using acetonitrile and 1% acetic acid (30:70 (v:v)) as the mobile phase. Tryptophan was analyzed by HPLC (Waters). All of the cell cultures were conducted in triplicate.

Protein Concentration Analysis and SDS-PAGE

The protein concentration of crude extracts was determined by using the Bio-Rad protein assay kit (Bio-Rad, USA) with bovine serum albumin as the standard. The protein expression was monitored using by 10% SDS-PAGE.

 

Results

Expression of IpdC, AspC, and Iad1 in E. coli

It is known that IAA biosynthesis through the IPA pathway requires three kinds of enzymes: tryptophan aminotransferase, IPA decarboxylase, and IAAld dehydrogenase [41]. A critical enzyme in this pathway is IpdC. At first, the IpdC from E. cloacae with a high specificity and affinity for IPA compared with other sources of IpdCs [7,12,38,40] was expressed under the control of Ptac in E. coli. An IPTGinduced crude extract of E. coli Top 10/pCTIP22 showed a noticeable band corresponding to a molecular mass of about 60 kDa using SDS-PAGE and 215.6 U/mg-protein of IpdC activity. These results demonstrate that IpdC in pCTIP22 is well expressed and catalyzes the decarboxylation of IPA (Fig. 2A, Table 3). Secondly, the aspC coding for aspartate aminotransferase from E. coli acting on a wide range of substrates, including aspartate, tryptophan, phenylalanine, and tyrosine, was cloned and expressed under the control of tac and sod promoters in E. coli [13,33]. As shown in Table 3, E. coli cells harboring plasmid pCSAS19, in which the aspC ORF linked with Psod, displayed a 2.1-fold increase of aspartate aminotransferase activity over the control strain, E. coli Top 10/pCES208. However, contrary to our expectations, cells bearing pCTAS18 with aspC attached to Ptac revealed a small increase of AspC activity compared with the control strain. SDS-PAGE analysis supported that the crude extract of E. coli cells containing plasmid in which the aspC was fused to Psod instead of Ptac exhibited a thick band with molecular mass of about 40 kDa (Fig. 2B). Based on SDS-PAGE analysis and the activity assay, AspC was efficiently produced by the Psod-controlled expression system in E. coli. Finally, the produced indole-3-acetaldehyde can be converted to indole-3-acetic acid by IAAld dehydrogenase, which was characterized in U. maydis. Thus, the iad1 fused to Ptac was introduced into pCES208 and pCL1921, a low-copy plasmid, resulting in pCTID21 and pCLTID23, respectively. As shown in Fig. 2C, a distinct band corresponding to a molecular mass of about 55 kDa appeared in the crude extract of E. coli Top 10/pCTID21. In addition, the IAAld activity of E. coli Top 10/pCTID21 was shown to be 272 U/mg-protein (Table 3). On the other hand, E. coli Top 10/pCES208 exhibited no detectable activity. Thus, according to the enzyme assays and SDSPAGE analyses, IpdC and Iad1 were well expressed by Ptac, whereas AspC was efficiently expressed by the Psod promoter in E. coli.

Fig. 2.SDS-PAGE of IpdC, AspC, and Iad1 expressions in E. coli Top 10. Proteins were separated by 10% SDS-PAGE. (A) IpdC expression. Lanes: 1, E. coli Top 10/pCES208; 2, Top 10/pCTIP22. (B) AspC expression. Lanes: 1, Top 10/pCES208; 2, Top 10/pCTAS18; 3, Top 10/pCSAS19. (C) Iad1 expression. Lanes: 1, Top 10/pCES208; 2, Top 10/pCTID21. The arrows indicate thick bands corresponding to the expressed IpdC, AspC, and Iad1 on SDS-PAGE.

Table 3.aWhen the optical cell density of all of recombinant E. coli cells except TOP 10/pCSAS19 reached 0.6 in LB medium at 37℃, 0.2 mM IPTG was added to the culture broth and cultivated for 5 h. bEach value represents the mean and standard error. cNot detectable. dThe IpdC activity was measured by absorbance of NAD+ reduction at 360 nm instead of 340 nm to remove the interference of IPA absorbance. eThe produced IPA determined at 327 nm using UV spectrophotometer. fThe Iad1 activity was determined by reduction of NAD+ at 340 nm.

Production of IAA by Recombinant E. coli DH5α Strains

As a result of IpdC overexpression, with the help of endogenous aminotransferases inE. coli, DH5α/pCTIP22 produced 0.19 g/l of TOL as a main product and 0.008 g/l of IAA from the complete consumption of 2 g/l tryptophan (Fig. 3A) [13]. To improve IAA production, the aspC was linked with a strong sod promoter in pCSAS19 and combined with ipdC attached to Ptac, yielding plasmid pCIA41. The amplified AspC with IpdC in DH5α/pCIA41 resulted in a 2.5-fold and a 1.9-fold increase of TOL and IAA production (0.46 g/l and 0.015 g/l), respectively, over the control strain without AspC overexpression. To further increase IAA production, Iad1-expressing plasmid pCLTID23, which has a different replication of origin compared with that of pCES208, was introduced in DH5α/pCIA41. Recombinant E. coli IAA51 expressing IpdC, AspC, and Iad1 produced 1.1 g/l of IAA with the stable maintenance of both plasmids, resulting in a 77-fold increase over the strain DH5α/pCIA41, and 0.13 g/l of TOL. Indeed, E. coli has several types of alcohol dehydrogenases (ADHs), which may easily catalyze the reduction of IAAld to TOL [2]. In this sense, the overexpression of Iad1 in IAA production contributes to the oxidation of IAAld into IAA, instead of reduction to TOL by ADH(s), and consequently results in a higher accumulation of IAA.

Fig. 3.Production of IAA and TOL by recombinant E. coli DH5α and DHT39 strains. (A) Recombinant E. coli DH5α containing pCES208 (control strain), pCTIP22 (with IpdC expression), pCIA41 (with IpdC and AspC expression), or pCIA41 + pCLTID23 (with IpdC, AspC, and Iad1 expression). (B) Recombinant E. coli DHT39 containing pCES208, pCTIP22, pCIA41, or pCIA41 + pCLTID23. All of the cells were cultured in 30 ml of LB medium supplemented with 2 g/l tryptophan and 5 g/l KH2PO4 for 48 h in a shaking incubator at 37℃. IAA and TOL concentrations were determined by HPLC analysis.

Production of IAA by Recombinant E. coli DHT39 Strains

Since E. coli can produce tryptophanase, which degrades tryptophan to indole, the conversion yield (mol/mol%) from tryptophan to IAA in E. coli IAA51 was only about 66% [21]. In order to interrupt the formation of the byproduct, indole, the tnaA gene of DH5α was deleted, and the gene dosage effects of IpdC, AspC, and Iad1 (Fig. 3B) were evaluated. The expression of IpdC and coexpression of IpdC and AspC led to the production of 0.32 g/l TOL and 0.83 g/l TOL as a main product, respectively, while 0.062 g/l and 0.031 g/l of IAA were accumulated in IpdC, and IpdC/AspC expressed strains, respectively. In addition, the combined expression effect of IpdC, AspC, and Iad1 resulted in the production of 1.8 g/l IAA and 0.032 g/l TOL. The conversion yield for IAA by E. coli IAA68 was about 105% from tryptophan, which is a 1.6-fold increase compared with that in wild-type DH5α harboring the same plasmids.

Fig. 4.Production of IAA by recombinant E. coi IAA68 during cultivation in LB medium. E. coli IAA68 was cultured in LB medium with 2 g/l tryptophan and 5 g/l KH2PO4 for 48 h at 37℃.

Effect of Tryptophan on IAA Production

We investigated cell growth and IAA production profiles during culture in LB medium with 2 g/l of tryptophan, a starting substrate of IAA biosynthesis (Fig. 4). Cell growth of E. coli IAA68 rapidly increased and reached OD 5~6 after 7 h and was maintained for 48 h with the stable maintenance of two plasmids. IAA was produced following IPTG induction, and increased by degrees with cultivation, and produced 1.65~1.8 g/l for 12~48 h. Tryptophan gradually decreased with the increase of cell OD and IAA concentration and was completely consumed by 12 h cultivation. TOL, a byproduct, showed less than 0.7 g/l accumulation in the final culture. The conversion yield for IAA from tryptophan was maintained between 94~102% from 12~48 h. When 0 to 6 g/l of tryptophan was initially added to the LB medium, the IAA concentration was enhanced from the increased tryptophan concentration, and reached 3.0 g/l with the consumption of 3.6 g/l tryptophan when 4 g/l of tryptophan was added (Fig. 5). At 6 g/l of tryptophan, IAA68 produced 2.9 g/l of IAA while consuming 2.7 g/l of tryptophan for 24 h of cultivation, and there was no further accumulation of IAA and consumption of tryptophan with prolonged culture for 24 h.

Fig. 5.Production of IAA by recombinant E. coli IAA68 in LB medium with different concentrations of tryptophan. E. coli IAA68 was cultured in LB medium with 0~6 g/l tryptophan and 5 g/l KH2PO4 for 48 h at 37℃.

 

Discussion

To develop IAA-producing recombinant cells, IpdC, AspC, and Iad1 were functionally expressed in E. coli. Since IAAld produced by IpdC could not be analyzed by HPLC, as it was probably unstable and tended to spontaneously convert to IAA and TOL, the effect of IpdC expression in E. coli was evaluated by analyzing the presence of IAA and TOL [32]. As a result of amplification of IpdC, IAA as a minor product was accumulated in the culture broth, indicating that IpdC from E. cloacae was efficiently expressed and functioning in E. coli. Furthermore, the produced IAAld was primarily reduced into TOL and not oxidized into IAA. E. coli has several types of ADHs such as adhE, adhP, eutG, yiaY, yqhD, and yjgB, which show distinct structural differences, different substrate specificities, and different use of cofactors and metals [2,15]. Thus, IAAld made by IpdC-expressing cells is mainly reduced into TOL, whereas some IAA may be produced from the instability of IAAld or minor functions of unknown aldehyde dehydrogenases.

The effect of AspC expression was evaluated from the total sum of TOL and IAA following the reasoning described above. Although E. coli has several aminotransferases [13], the introduction of AspC along with IpdC exhibits the beneficial effect in the production of TOL and IAA, which demonstrates that any additional expression of AspC is necessary to generate IPA from tryptophan. According to the SDS-PAGE and activity assay analyses, AspC expressed under the control of Ptac could not be produced and worked in E. coli in which the tac promoter functions, whereas the Psod expression system attached to aspC ORF resulted in efficiently expressed AspC. Recently, many studies have shown that protein expression levels are strongly influenced by the secondary structure around the translational initiation region (TIG) as well as by the promoter strength [28,36,39]. The minimal folding free energy, ΔG, for an mRNA secondary structure of 80 nucleotides (nt) within the 40 nt of 3’ region in the tac promoter and 40 nt of the initiation region in the aspC ORF was calculated based on the Mfold web server [49]. As a result, ΔG for 80 nt in Ptac-aspC was - 17.0 kcal/mol, suggesting that the mRNA secondary structure of TIG in Ptac-aspC is very stable and consequently AspC expression by Ptac is very weak. Meanwhile, the Psod originating from C. glutamicum is known as a strong promoter in C. glutamicum and functions in both C. glutamicum and E. coli (unpublished data) [5]. Thus, Psod was employed to express the aspC gene in this study. The calculated ΔG for 80 nt in Psod-aspC was between -11.03 and -9.22 kcal/mol. According to previous reports, changing the ΔG near a hairpin structure with the Shine-Dalgarno sequence by +1.4 kcal/mol resulted in a 10-fold increase in the translation-initiation rate [10]. Thus, we suggest that the weak expression of AspC by Ptac is caused by a more stable mRNA secondary structure of TIP in Ptac-aspC than that in Psod-aspC.

Since iad1 was only characterized from U. maydis, this gene was expressed in pCL1921 and its gene dosage effect evaluated. In fact, the expressed Iad1 competes with endogenous ADHs in E.coli for the substrate IAAld [2,15]. By introducing Iad1 in IpdC and AspC-expressing E. coli, the production of IAA was greatly increased, whereas little amount of TOL was detected. Hence, the expressed Iad1 could successfully catalyze the IAAld oxidation, thereby exceeding the reduction by endogenous ADHs. Furthermore, the deletion strain of gene tnaA that catalyzes indole formation led to a higher increase of IAA concentration and conversion yield than the wild-type strain. The production of 3 g/l IAA from 4 g/l tryptophan is the highest level observed when compared with previous reports in which P. agglomerans produced 2.2 g/l of IAA [1]. The measured conversion yield of tryptophan to IAA is about 105%, which is beyond theoretical yield. The added tryptone and yeast extract in LB medium may contribute to the supply of tryptophan and result in the increase of IAA production yield.

E. coli is used in the industrial production of tryptophan, thereby offering distinct advantages for developing IAAoverproducing E. coli from glucose by direct fermentation. It has been reported that 49 g/l of L-tryptophan was accumulated in culture broth by metabolically engineered E.coli [45]. In the next study, we are expecting to develop IAA-producing E. coli with enhanced potential by introducing three genes, aspC, ipdC, and iad1, into a tryptophanproducing E. coli. Additionally, during creating IAAproducing E. coli via the IPA pathway, E. coli DHT39 with pCIA41 produced 0.83 g/l of TOL from 2 g/l of tryptophan. TOL is one of the aromatic alcohols, identified as an antioxidant, and used in the synthesis of the drug indoramin [2,22]. Accordingly, we are looking forward to developing a new recombinant E. coli producing TOL through the overexpression of ADH as well as by inserting AspC and IpdC into tryptophan-producing E. coli.

Until now, many IAA-producing microbes have been developed for growth-stimulating agents in the rhizosphere. E. coli IAA68 may be not easy for the plant to exploit directly, due to a low survival rate under natural conditions. However, the constructed expression plasmids, except for pCLTID23 and the Ptac and Psod promoters, used in this work are functioning in C. glutamicum, a representative grampositive soil bacterium [5]. Thus, we can develop IAAproducing C. glutamicum using constructed vectors, thereby providing the possibility of growth-stimulating bacterium for plants in the rhizosphere.

The development of IAA-producing microbes via the IPA pathway usually has been confined to screening IAAproducing microbes in the rhizosphere and expressing a variety of IpdCs from E. cloacae, A. brasilense, P. polymyxa, E. herbicola, and M. extorquens for each host strain [4,7,12,19,32]. When cells were cultured in tryptophan-containing media, a mixture of indolic compounds including IAA, TOL, IAAld, and indole-3-lactic acid was accumulated, and thus the production of IAA was very inefficent [7,32]. In this study, we sucessufully developed IAA-producing strains by the functional coexpressions of aspC, ipdC, and iad1 genes from E. coli, E. cloacae, and U. maydis, respectively, in a tnaA-deleted E. coli. Moreover, E. coli IAA68 achieved the complete conversion of tryptophan to IAA. Finally, IAA68 produced 3 g/l of IAA in LB medium with 4 g/l of added tryptophan for 24 h. These results clearly demonstrate that the production of IAA from tryptophan was successfully achieved by recombinant E. coli IAA68 expressing AspC, IpdC, and Iad1.