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

( Elisa Friska Romasi ),( Jin Ho Lee ) 한국미생물 · 생명공학회 2013 Journal of microbiology and biotechnology Vol.23 No.12

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 Ptac, whereas AspC was efficiently expressed by Psod 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 DH5α 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 DH5α 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.

Liquid Chromatography-Mass/Mass Spectrometry (MS)와 Gas Chromatography-MS를 이용한 농축 액상 비료제품 중 Indole-3-acetic acid 및 Indole-3-butyric acid 정량분석능 비교

김진효 ( Jin Hyo Kim ),박종민 ( Jong Min Park ),최근형 ( Geun Hyoung Choi ),박연기 ( Yun Ki Park ),임건재 ( Geon Jae Im ),김두호 ( Doo Ho Kim ),권오경 ( Oh Kyung Kwon ) 한국응용생명화학회(구 한국농화학회) 2013 Journal of Applied Biological Chemistry (J. Appl. Vol.56 No.1

비료제품에는 의도적 혹은 비의도적으로 농약으로 등록된 indole-3-acetic acid (IAA) 및 indole-3-butyric acid (IBA)성분에 대한 잔류 가능성이 꾸준히 제기되고 있으나, 생장조정제가 아닌 비 료제품에서 mg/L 수준의 옥신류 잔류분석법이 마련되어 있지 않았다. 따라서, 본 연구에서는 생체시료에 미량 잔류하는 IAA 및 IBA 분석에 사용되어 온 liquid chromatography-mass/mass spectrometry (LC-MS/MS)와 gas chromatography-MS 기기분석 법을 활용하여 농축 액상비료제품에 적용할 수 있는지 조사하였으며, 액상 비료제품에 적용 가능한 정밀기기 분석법을 개발하고자 하였다. 수용액 상태의 시료에서 식물 생장호르몬인 IAA 와 IBA를 가장 손쉽게 정제할 수 있는 방법으로 hydrophile-lipophile balance (HLB) solid phase extraction를 활용하였으나, 제한된 조건에서 LC-MS/MS를 통한 정량분석은 적합한 회수율을 확보하지 못하였고, 정성분석만 가능함을 확인하였다. 반면, 비료제품 250배 희석액을 사용하여 HLB 정제, trimethylsilyl chloride을 이용한 methylation을 통한 GC-MS 분석에서는 검출 한계 1.4 mg/L과 93-107%의 회수율로 액상 비료제품에서 IAA 와 IBA 정량분석법을 확립할 수 있었다. In here, we investigated the quantitative analysis method of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) with liquid chromatography-mass/mass spectrometry (LCMS/ MS) or gas chromatography-MS. Two ways of clean-up pr℃ess were investigated for LC-MS/MS instrumental analysis of IAA, but both a simple dilution and hydrophile-lipophile balance (HLB) solid phase extraction (SPE) were not met the optimal recovery rates for quantitative analysis. On the other hand, the clean-up method for GC-MS was finally optimized through HLBSPE from 250-folds diluted sample and methylation with trimethylsilyl chloride in methanol for 4 h. The limit of detection for methyl ester of IAA and IBA were both 1.4 mg/L, and recovery rates showed 93-107% from the concentrated liquid fertilizer.

Indole-3-acetic acid (IAA) 생성 Arthrobacter sp.의 분리 및 식물 생육촉진 효과

김다솜,신호용,한송이 한국응용과학기술학회 2022 한국응용과학기술학회지 Vol.39 No.6

An auxin-producing bacteria, KSD16, KSD33, and KSD36 were isolated from agricultural soil. The strain KSD16, KSD33, and KSD36 was classified as a strain of Arthrobacter sp. based on phylogenetic analysis of 16S rRNA gene. The isolated KDS16, KDS33, and KSD36 was confirmed to produce indole-3-acetic acid (IAA), which is one of the auxin hormones. When the concentration of IAA was assessed the maximum concentration of IAA, 206.62 mg L -1 , was detected from the culture broth incubated in R2A medium containing 0.1% L-tryptophan for 48 h at 28 ℃. To study the effect of IAA producing bacteria on germination rate, seeds of Mung bean were prepared for each treatment. KSD16, KSD33, and KSD36 showed significant increase in root length and number of adventitious roots than the controls. To investigate the growth-promoting effects on the crops, Arthrobacter species were placed in water cultures and seed pots of mung beans. In consequence, the seed germination of mung beans was 73.4% higher than the control. 농업 재배지 토양으로부터 auxin 생성세균 KSD16, KSD33 그리고 KSD36를 분리하였다. 분리균주 KSD16, KSD33 그리고 KSD36 는 16S rRNA 유전자 계통분석을 통해 Arthrobacter 속으로 분류되었다. 이들 균주는 R2A배지에 0.1% L-tryptophan를 첨가한 배지에서 28℃, 48 시간 배양한 결과, auxin 의 일종인 indole-3-acetic acid (IAA)를 204.4 mg L -1 생성하는 것으로 확인되었다. IAA 생성세균의 녹두종자 발아에 미치는 영향을 확인한 결과, Arthrobacter 속 균주 KSD16, KSD33 그리고 KSD36 는 대조군에 비해 뿌리길이와 발근수가 증가하였다. Arthrobacter 속 균주의 녹두 성장촉진 효과를 확인한 결과, 녹두의 발아율이 대조군보다 73.4 % 증가하는 특징을 나타내었다.

Suppression of Morningglory (Ipomoea Hederacea) Growth by Rhizobacteria and IAA-3-ACETIC Acid

Kim Su-Jung 한국유기농업학회 2006 韓國有機農業學會誌 Vol.14 No.4

Indole-3-acetic acid (IAA) biosynthesis by bacteria occurs widely in rhizospheres. Bacterial species able to synthesize IAAmay be exploited for beneficial interactions in crop management systems. The objective of this study was to determine the response of ivyleaf morningglory (Ipomoea hederacea) seedlings to IAA and to an IAA-producing rhizobacterum, Bradyrhizobium japonicum isolate GD3. IAA solution and isolate GD3 suppression of seedling growth measured as radicle length and biomass depended on IAA concentration. Seedling radicle length was significantly reduced by ca. 29% with more than 1.0 ㎛ of IAA solution, compared to the control, 48 h after application. The cell concentration at 50% growth reduction (GR₅₀) of the seedling radicle was IAA production by isolate GD3 at 104.82 cfu, the cell concentration for 50% growth reduction (GR₅₀) of seedling radicle was 0.24 ìM, which was much lower than the IAA solution concentration (117.48 ㎛) required for GR₅₀. Therefore, excess IAA production by isolate GD3 may be more detrimental to morningglory radicle growth than standard IAA solution. Results confirmed involvement of IAA in suppressive effects of isolate GD3 on morningglory seedlings grown in a hydroponic system.

Production of Indole-3-Acetic Acid by Enterobacter sp. DMKU-RP206 Using Sweet Whey as a Low-Cost Feed Stock

( Nantana Srisuk ),( Varunya Sakpuntoon ),( Pumin Nutaratat ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.9

In this study, we investigated Indole-3-acetic acid (IAA) production by a rice phylloplane bacteria, Enterobacter sp. DMKU-RP206, using sweet whey as a feed stock instead of lactose. We succeeded in using sweet whey for Enterobacter sp. DMKU-RP206 to produce 3,963.0 mg IAA/l with the optimal medium containing 1.48% sweet whey, 1.42% yeast extract and 0.88% L-tryptophan. The medium pH was adjusted to 6 and the culture conditions were shaking at 200 rpm on an orbital shaker at 30°C for 3 days. We also evaluated the effect of IAA in culture filtrates of Enterobacter sp. DMKU-RP206 on the promotion of jasmine rice growth in a pot experiment. Compared with the negative control (without IAA), the result showed that biosynthetic IAA produced by Enterobacter sp. DMKU-RP206 significantly increased the growth of jasmine rice (Oryza sativa L. cv. KDML105) in terms of length and dry weight of shoot. This work thus reveals the impact of IAA produced by Enterobacter sp. on the promotion of jasmine rice growth.

살코스키 시약과 Indole-3-acetic Acid의 반응 중 트립토판의 간섭

이슬 ( Seul Lee ),어나미까커날 ( Anamika Khanal ),여준구 ( Junkoo Yeo ),노그라도캐시린 ( Kathyleen Nogrado ),조아현 ( Ahyeon Cho ),송윤진 ( Yoonjin Song ),권미지 ( Miji Kwon ),이지훈 ( Ji-hoon Lee ) 한국환경농학회 2018 한국환경농학회 학술대회집 Vol.2018 No.-

Indole-3-acetic acid (IAA) is the most common auxin hormone produced in the plant and is very important because it regulates various aspects of plant growth and development. Tryptophan is the main precursor for IAA while IAA can be synthesized by two pathways i.e., tryptophan-dependent pathway and tryptophan-independent pathway. There have been many studies using Salkowski regent to measure the IAA production by bacteria. In this study, we have found that there was the interference of tryptophan on the colorimetric estimations of indole-3-acetic acid (IAA) by using Salkowski reagent (35% HClO4 and 10 mM FeCl3), which developed a pink color complex of tris-(indole-3-acetate) iron(III) with IAA. Moreover, we detected the effect of different concentration of tryptophan (100, 200, and 300 μM) on different concentration of IAA (100, 200, and 300 μM). The absorbance on IAA for the tested concentrations was found to be increased with the increasing concentration of tryptophan. It seems that both the IAA and tryptophan show the maximum absorbance at the similar wavelengths. It was also observed that the difference between the predicted and measured concentrations of IAA became larger as the predicted IAA concentration decreased. From this study, it is expected that tryptophan would interfere with accurate measurements of IAA, and therefore care should be taken for the measurement of IAA generated by the precursor, tryptophan.

Biosynthetic Pathway of Indole-3-Acetic Acid in Basidiomycetous Yeast Rhodosporidiobolus fluvialis

( Sakaoduoen Bunsangiam ),( Varunya Sakpuntoon ),( Nantana Srisuk ),( Takao Ohashi ),( Kazuhito Fujiyama ),( Savitree Limtong ) 한국균학회 2019 Mycobiology Vol.47 No.3

IAA biosynthetic pathways in a basidiomycetous yeast, Rhodosporidiobolus fluvialis DMKUCP293, 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.

반응표면분석법을 활용한 Pantoea agglomerans SRCM 119864의 Indole-3-acetic acid 생산 배지 최적화

정호진(Ho Jin Jeong),하광수(Gwangsu Ha),정수지(Su Ji Jeong),류명선(Myeong Seon Ryu),김진원(JinWon Kim),정도연(Do-Youn Jeong),양희종(Hee-Jong Yang) 한국생명과학회 2022 생명과학회지 Vol.32 No.11

본 연구에서는 토양으로부터 분리한 Indole-3-acetic acid (IAA) 고생산 균주인 Pantoea agglomerans SRCM 119864의 IAA 생산량을 증가시키기 위해 반응표면분석법을 활용하여 배지 조성 최적화를 수행하였다. Plackett-Burman design (PBD)을 이용하여 전구체인 L-tryptophan 이외의 IAA 생산에 영향을 주는 배지 성분 11개의 영향을 조사하였으며, 통계학적 분석을 통해 IAA 최적생산을 위한 배지 인자로 sucrose, tryptone, sodium chloride를 선정하였다. PBD에서 선정된 3가지 인자와 L-tryptophan의 농도 최적화를 수행하기 위해 적은 실험수로도 최적값을 분석할 수 있는 hybrid design을 설계하였다. 실험 모형에서 예측한 P. agglomerans SRCM 119864 균주의 IAA 최적 생산을 위한 배지 조성과 농도는 sucrose 13.38 g/l, tryptone 18.34 g/l, sodium chloride 9.71 g/l, L-tryptophan 6.25 g/l로 분석되었으며, 이때의 IAA 생산량은 64.34±1.07 mg/l로 예측되었다. ANOVA 분석을 통하여 실험 모형의 통계학적 유의성과 적합성을 검증하였으며, 설계한 최적 조성 배지에서 모델 검증실험을 수행하여 IAA 생산량을 측정한 결과, 예측된 IAA 생산량과 매우 유사함을 확인하였으며, 최종 최적화 수행을 통해 IAA 생산량을 기본 배지 대비 44.56% 증가시켰다. 본 연구를 통하여 토양에서 IAA를 고생산하는 균주를 선별하여 배지 조성 최적화를 수행하였으며, 이를 바탕으로 지속가능한 농업 및 작물 생산량 증대를 위한 산업화 기초자료로서 활용될 수 있을 것으로 기대된다. In this study, we optimized the composition of the indole-3-acetic acid (IAA) production medium using response surface methodology on Pantoea agglomerans SRCM 119864 isolated from soil. IAA-producing P. aglomerans SRCM 119864 was identified by 16S rRNA gene sequencing. There are 11 intermediate components known to affect IAA production, hence the effect of each component on IAA production was investigated using a Plackett-Burman design (PBD). Based on the PBD, sucrose, tryptone, and sodium chloride were selected as the main factors that enhanced the IAA production at optimal L-tryptophan concentration. The predicted maximum IAA production (64.34 mg/l) was obtained for a concentration of sucrose of 13.38 g/l, of tryptone of 18.34 g/l, of sodium chloride of 9.71 g/l, and of L-tryptophan of 6.25 g/l using a the hybrid design experimental model. In the experiment, the nutrient broth medium supplemented with 0.1% L-tryptophan as the basal medium produced 45.24 mg/l of IAA, whereas the optimized medium produced 65.40 mg/l of IAA, resulting in a 44.56% increase in efficiency. It was confirmed that the IAA production of the designed optimal composition medium was very similar to the predicted IAA production. The statistical significance and suitability of the experimental model were verified through analysis of variance (ANOVA). Therefore, in this study, we determined the optimal growth medium concentration for the maximum production of IAA, which can contribute to sustainable agriculture and increase crop yield.

Interactive effect of indole-3-acetic acid and diethyl aminoethyl hexanoate on the growth and fatty acid content of some microalgae for biodiesel production

Salama, El-Sayed,Jeon, Byong-Hun,Chang, Soon Woong,Lee, Sang-hun,Roh, Hyun-Seog,Yang, Il-Seung,Kurade, Mayur B.,El-Dalatony, Marwa M.,Kim, Do-Hyeon,Kim, Ki-Hyun,Kim, Sunjoon Elsevier 2017 Journal of cleaner production Vol.168 No.-

<P><B>Abstract</B></P> <P>Enhancement of microalgal growth and fatty acid production is essential for development of a microalgae-based biodiesel production platform. Three different microalgal species (<I>Scenedesmus obliquus</I> GU732418, <I>Ourococcus multisporus</I> GU732424 and <I>Chlorella vulgaris</I> FR751187) were individually cultivated in media containing both indole-3-acetic acid (IAA) and diethyl aminoethyl hexanoate (DAH) at concentrations of 10<SUP>−8</SUP>−10<SUP>−4</SUP> M. Combined phytohormones (10<SUP>−8</SUP> to 10<SUP>−5</SUP> M) increased the growth of all three species compared to growth in media without phytohormones. IAA and DAH supported the maximum growth of <I>S</I>. <I>obliquusi</I> (38.12 × 10<SUP>6</SUP> cells mL<SUP>−1</SUP>) at 10<SUP>−8</SUP> M, <I>O</I>. <I>multisporus</I> (85.89 × 10<SUP>6</SUP> cells mL<SUP>−1</SUP>) at 10<SUP>−6</SUP> M, and <I>C. vulgaris</I> (4.09 × 10<SUP>6</SUP> cells mL<SUP>−1</SUP>) at 10<SUP>−5</SUP> M. Addition of 10<SUP>−7</SUP> M IAA and DAH also assisted the removal of Zn<SUP>2+</SUP> (97%), K<SUP>+</SUP> (88%) and Mg<SUP>2+</SUP> (99%) from the media by <I>S</I>. <I>obliquus</I>. The highest removal of Zn<SUP>2+</SUP>, K<SUP>+</SUP>, and Mg<SUP>2+</SUP> by <I>C</I>. <I>vulgaris</I> was achieved at 10<SUP>−5</SUP> M IAA and DAH. Under all experimental conditions (10<SUP>−8</SUP>−10<SUP>−4</SUP> M IAA and DAH) the amounts of poly-unsaturated fatty acids were significantly increased. Palmitic acid, linoleic acid and γ-linolenic acid were the major fatty acids, accounting for 11.75–21.55%, 2.55–6.73%, and 52.93–75.89% of the total fatty acid content, respectively. The fatty acids that accumulated in <I>O</I>. <I>multisporus</I> and <I>C</I>. <I>vulgaris</I> were found to be suitable for production of high quality biodiesel with characteristics equivalent to crop seed oil-derived biodiesel.</P> <P><B>Highlights</B></P> <P> <UL> <LI> IAA and DAH in the range of 10<SUP>−8</SUP>−10<SUP>−5</SUP> M enhanced the growth of algae. </LI> <LI> PUFAs was increased by growth in medium containing both IAA and DAH. </LI> <LI> Accumulated fatty acids in algae are suitable for production of high quality biodiesel. </LI> </UL> </P>

Comparison of Plant Growth Promoting Methylobacterium spp. and Exogenous Indole-3-Acetic Acid Application on Red Pepper and Tomato Seedling Development

Hari P. Deka Boruah,Puneet S. Chauhan,Woo-Jong Yim(임우종),Gwang-Hyun Han(한광현),Tong-Min Sa(사동민) 한국토양비료학회 2010 한국토양비료학회지 Vol.43 No.1

무균 및 온실조건에서 indole-3-acetic acid (IAA)의 처리와 1-aminocyclopropane-1-carboxylate deaminase (ACCD) 및 IAA 활성을 갖는 Methylobacterium 균주 접종 시 토마토와 고추의 생장을 비교 평가하였다. 무균조건에서 1.0 ㎍ mL?¹의 IAA는 고추와 토마토의 뿌리생장을 촉진시키는데 비해 10.0 ㎍ mL?¹ 이상의 높은 농도에서는 뿌리생장이 억제되었다. 그러나 높은 ACCD 활성을 갖고, IAA 활성은 낮거나 가지고 있지 않은 Methylobacterium 균주들을 접종하였을 때에는 고추와 토마토 모두 IAA 처리구 보다 뿌리생장이 증진되는 것을 확인하였다. 마찬가지로 온실조건에서 Methylobacterium 균주들을 접종했을 때, 마디길이와 잎의 크기 그리고 단위 면적당 잎의 무게(SLW)에서 유의성 있는 증진효과를 보였다. 전반적인 식물 생장에서 저농도의 IAA 처리 효과는 Methylobacterium의 효과와 비슷한 경향을 나타냈다. 유묘의 지상부 길이는 ACCD 활성과 IAA 생산능을 갖는 Methylobacterium 균주 처리구에서 유의성 있는 증가를 확인할 수 있었으며, 전체 건물중 또한 Methylobacterium 처리 시 유의성 있는 증진 효과를 확인 할 수 있었다. 하지만 고농도의 IAA는 고추와 토마토의 생물량을 억제시켰다. 이러한 결과는 접종 균주의 IAA와 ACCD가 고추와 토마토 유묘의 생장 증진에 영향을 끼친다는 것을 증명한다. A comparative study was performed in gnotobiotic and greenhouse conditions to evaluate the effect of exogenous application of indole-3-acetic acid (IAA) and inoculation of Methylobacterium spp. possessing 1-aminocyclopropane-1-carboxylate deaminase (ACCD) and IAA activity on red pepper and tomato seedling growth and development. Application of 1.0 ㎍ mL?¹ IAA positively influenced root growth while high concentrations (>10.0 ㎍ mL?¹) suppressed root growth of red pepper and tomato under gnotobiotic condition. On the other hand, inoculation of Methylobacterium strains with ACCD activity and IAA or without IAA enhanced root growth in both plants. Similarly, under greenhouse condition the inoculation of Methylobacterium sp. with ACCD activity and IAA enhanced plant fitness recorded as average nodal length and specific leaf weight (SLW) but the effect is comparable with the application of low concentrations of IAA. Seedling length was significantly increased by Methylobacterium strains while total biomass was enhanced by Methylobacterium spp. and exogenous applications of < 10.0 ㎍ mL?¹ IAA. High concentrations of IAA retard biomass accumulation in red pepper and tomato. These results confirm that bacterial strains with plant growth promoting characters such as IAA and ACCD have characteristic effects on different aspects of growth of red pepper and tomato seedlings which is comparable or better than exogenous applications of synthetic IAA.