• Journal of Applied Biological Chemistry
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• v.43 no.4
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• pp.269-272
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• 2000

The ginseng growth-promoting bacterium Pseudomonas fluorescens KGPP 207 synthesized indole-3- acetic acid (IAA) from L-tryptophan, indole-3-pyruvic acid (IPyA), and indole-3-acetaldehyde (IAAld), but not from indole-3-acetamide (lAM) and other intermediates of various IAA biosynthetic pathways in the experiment with indole compound supplemented cell suspensions. TLC, HPLC, and GC-MS analyses revealed the presence of IPyA, indole-3-ethanol, indole-3-lactic acid and its methyl ester, IAA and its methyl, and ethyl esters in the culture supernatant of the bacterium. IAAld was detected in the supernatant using sodium bisulfite and TLC. The results indicate that unlike gall-forming bacteria which can synthesize IAA by lAM, the indole-3-pyruvic acid pathway is the route for IAA biosynthesis in this beneficial strain of P. fluorescens.

• Journal of Microbiology and Biotechnology
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• v.23 no.12
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• pp.1726-1736
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• 2013

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.

• Mycobiology
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• v.47 no.3
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• pp.292-300
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• 2019

IAA biosynthetic pathways in a basidiomycetous yeast, Rhodosporidiobolus fluvialis DMKU-CP293, were investigated. The yeast strain showed tryptophan (Trp)-dependent IAA biosynthesis when grown in tryptophan supplemented mineral salt medium. Gas chromatography-mass spectrometry was used to further identify the pathway intermediates of Trpdependent IAA biosynthesis. The results indicated that the main intermediates produced by R. fluvialis DMKU-CP293 were tryptamine (TAM), indole-3-acetic acid (IAA), and tryptophol (TOL), whereas indole-3-pyruvic acid (IPA) was not found. However, supplementation of IPA to the culture medium resulted in IAA peak detection by high-performance liquid chromatography analysis of the culture supernatant. Key enzymes of three IAA biosynthetic routes, i.e., IPA, IAM and TAM were investigated to clarify the IAA biosynthetic pathways of R. fluvialis DMKU-CP293. Results indicated that the activities of tryptophan aminotransferase, tryptophan 2-monooxygenase, and tryptophan decarboxylase were observed in cell crude extract. Overall results suggested that IAA biosynthetic in this yeast strain mainly occurred via the IPA route. Nevertheless, IAM and TAM pathway might be involved in R. fluvialis DMKU-CP293.

• Journal of Microbiology and Biotechnology
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• v.18 no.7
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• pp.1235-1244
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• 2008

Indole-3-acetic acid (IAA) is produced commonly by plants and many bacteria, however, little is known about the genetic basis involving the key enzymes of IAA biosynthetic pathways from Bacillus spp. IAA intermediates from the Gram-positive spore-forming bacterium Paenibacillus polymyxa E681 were investigated, which showed the existence of only an indole-3-pyruvic acid (IPA) pathway for IAA biosynthesis from the bacterium. Four open reading frames (ORFs) encoding indole-3-pyruvate decarboxylase-like proteins and putative indole-3-pyruvate decarboxylase (IPDC), a key enzyme in the IPA synthetic pathway, were found on the genome sequence database of P. polymyxa and cloned in Escherichia coli DH5$\alpha$. One of the ORFs, PP2_01257, was assigned as probable indole-3-pyruvate decarboxylase. The ORF consisted of 1,743 nucleotides encoding 581 amino acids with a deduced molecular mass of 63,380 Da. Alignment studies of the deduced amino acid sequence of the ORF with known IPDC sequences revealed conservation of several amino acids in PP2_01257, essential for substrate and cofactor binding. Recombinant protein, gene product of the ORF PP2_01257 from P. polymyxa E681, was expressed in E. coli BL21 (DE3) as a glutathione S-transferase (GST)-fusion protein and purified to homogeneity using affinity chromatography. The molecular mass of the purified enzyme showed about 63 kDa, corresponding closely to the expected molecular mass of IPDC. The indole-3-pyruvate decarboxylase activity of the recombinant protein, detected by HPLC, using IPA substrate in the enzyme reaction confirmed the identity and functionality of the enzyme IPDC from the E681 strain.

• Molecules and Cells
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• v.41 no.12
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• pp.1033-1044
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• 2018

As sessile organisms, plants have evolved to adjust their growth and development to environmental changes. It has been well documented that the crosstalk between different plant hormones plays important roles in the coordination of growth and development of the plant. Here, we describe a novel recessive mutant, mildly insensitive to ethylene (mine), which displayed insensitivity to the ethylene precursor, ACC (1-aminocyclopropane-1-carboxylic acid), in the root under the dark-grown conditions. By contrast, mine roots exhibited a normal growth response to exogenous IAA (indole-3-acetic acid). Thus, it appears that the growth responses of mine to ACC and IAA resemble those of weak ethylene insensitive (wei) mutants. To understand the molecular events underlying the crosstalk between ethylene and auxin in the root, we identified the MINE locus and found that the MINE gene encodes the pyridoxine 5′-phosphate (PNP)/pyridoxamine 5′-phosphate (PMP) oxidase, PDX3. Our results revealed that MINE/PDX3 likely plays a role in the conversion of the auxin precursor tryptophan to indole-3-pyruvic acid in the auxin biosynthesis pathway, in which TAA1 (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1) and its related genes (TRYPTOPHAN AMINOTRANSFERASE RELATED 1 and 2; TAR1 and TAR2) are involved. Considering that TAA1 and TARs belong to a subgroup of PLP (pyridoxal-5′-phosphate)-dependent enzymes, we propose that PLP produced by MINE/PDX3 acts as a cofactor in TAA1/TAR-dependent auxin biosynthesis induced by ethylene, which in turn influences the crosstalk between ethylene and auxin in the Arabidopsis root.