• Journal of Microbiology and Biotechnology
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• 제23권12호
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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.

• 한국미생물·생명공학회지
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• 제48권3호
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• pp.373-382
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• 2020

Candidatus Carsonella ruddii is an endosymbiont that resides in specialized cells within the body cavity of plant sap-feeding insects called psyllids. The establishment of symbiotic associations is considered one of the key factors for the evolutionary success of psyllids, as it may have helped them adapt to imbalanced food resources like plant sap. Although C. ruddii is defined as a psyllid primary symbiont, the genes for some essential amino acid pathways are absent. Complete genome sequences of several C. ruddii strains have been published. However, in-depth intra-species comparison of C. ruddii strains has not yet been done. This study therefore aimed to perform a comparative genome analysis of six C. ruddii strains, allowing the interrogation of phylogenetic group, functional category of genes, and biosynthetic pathway analysis. Accordingly, overall genome size, number of genes, and GC content of C. ruddii strains were reduced. Phylogenetic analysis based on the whole genome proteomes of 30 related bacterial strains revealed that the six C. ruddii strains form a cluster in same clade. Biosynthetic pathway analysis showed that complete sets of genes for biosynthesis of essential amino acids, except tryptophan, are absent in six C. ruddii strains. All genes for tryptophan biosynthesis are present in three C. ruddii strains (BC, BT, and YCCR). It is likely that the host may depend on a secondary symbiont to complement its deficient diet. Overall, it is therefore possible that C. ruddii is being driven to extinction and replacement by new symbionts.

• Asian-Australasian Journal of Animal Sciences
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• 제33권11호
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• pp.1770-1778
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• 2020

Objective: This study was conducted to reveal potential metabolic differences of dairy cows fed corn stover (CS) and rice straw (RS) instead of alfalfa hay (AH) as main forage source. Methods: Thirty multiparous mid-late lactation Holstein dairy cows were selected and randomly assigned to three diets, AH, CS, or RS (n = 10). After 13 weeks of the feeding trial, coccygeal arterial and superficial epigastric venous plasma samples were collected before morning feeding for gas chromatography time-of-flight/mass spectrometry analyses. Results: In the artery, 8 and 13 metabolites were detected as differential metabolites between AH and CS, and between AH and RS, respectively. The relative abundance of phenylpropanoate (log₂fold change [FC]) = 1.30, 1.09), panthenol (log₂FC = 2.36, 2.20), threitol (log₂FC = 1.00, 1.07), and 3,7,12-trihydroxycoprostane (log₂FC = 0.79, 0.78) were greater in both CS and RS than in AH, and tyrosine (log₂FC = -0.32), phenylalanine (log₂FC = -0.30), and pyruvic acid (log₂FC = -0.30) were lower in RS than in AH. In the vein, 1 and 7 metabolites were detected as differential metabolites between AH and CS, and between AH and RS, respectively. By comparing AH and RS, we found that metabolic pathways of phenylalanine, tyrosine, and tryptophan biosynthesis and phenylalanine metabolism were enriched by integrative artery and vein analysis. Furthermore, AH and RS, arterial phenylpropanoate and 4-hydroxyproline were positively, and phenylalanine was negatively correlated with milk urea nitrogen. Finally, in AH and CS, arterial panthenol was negatively correlated with feed efficiency. Conclusion: Arterial metabolic profiles changed more than those in the veins from animals on three forage diets, differing in amino acids. We found that phenylalanine, tyrosine, and tryptophan biosynthesis and phenylalanine metabolism were restricted when cows were fed low-quality cereal straw diets.

• KSBB Journal
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• 제15권5호
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• pp.507-513
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• 2000

고지혈증 치료제인 lovastatin을 Aspergillus terreus로부터 생산하기 위해 발효조 배양설험에서 교반속도와 pH에 대한 영향올 조사하였다. 최적 교반속도는 400 rpm이었고 pH는 5.8로 유지하였을 때 lovastatin 생산이 최대였으며, 교반속도 보다 pH가 lovastatin 생산에 더 많은 영향을 미치는 것으로 나타났다 L-tryptop뼈n과 L-histi빼le의 첨가시기에 따른 lovastatin 생산량을 실험한 결과, 둘다 발효초기부터 첨가하여 배양하 는 것이 효과적이었다 L-tryptophan을 발효초기에 첨가한 최 적배지와 최적 환경조건인 교반속도 400 rpm, pH 5.8에서 회 분식 배양을 수행한 결과 기본배지를 이용하여 실험한 배양 결과보다 약 10배 정도 많은 836 mg/L이었고, 생산성은 3.5 mg/L.hr였다

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1142-1149
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• 2009

Several bioactive metabolites, including pyrrolnitrin, N-acylhomoserine lactones, and polyhydroxyalkanoates were isolated from Burkholderia sp. O33. Effects of various nutrients, including sugars, gluconolactone, glycerol, tryptophan, chloride, and zinc were investigated in relation to the production of these metabolites. Logarithmic increase of pyrrolnitrin was observed between 2-5 days and reached a maximum at 7-10 days. Tryptophan concentration reached the maximum at 3 days, whereas 7-chlorotryptophan was gradually increased throughout the studies. Among various carbon sources, gluconolactone, trehalose, and glycerol enhanced pyrrolnitrin production, whereas strong inhibitory effects were found with glucose. Relative concentrations of pyrrolnitrin and its precursors were in the order of pyrrolnitrin$\gg$dechloroaminopyrrolnitrin or aminopyrrolnitrin throughout the experiments. Among three N-acylhomoserine lactones, the N-octanoyl analog was the most abundant quorum sensing signal, of which the concentrations reached the maximum in 2-3 days, followed by a rapid dissipation to trace level. No significant changes in pyrrolnitrin biosynthesis were observed by external addition of N-acylhomoserine lactones. Polyhydroxyalkanoates accumulated up to 3-4 days and decreased slowly thereafter. According to the kinetic analyses, no strong correlations were found between the levels of pyrrolnitrin, N-acylhomoserine lactones, and polyhydroxyalkanoates.

• Journal of Microbiology and Biotechnology
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• 제20권9호
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• pp.1259-1265
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• 2010

The filamentous cyanobacterium Arthrospira platensis strain MMG-9 was isolated from a rice field. The ability of this strain to synthesize the bioactive compound indole-3-acetic acid (IAA) was demonstrated. IAA was extracted from the culture of A. platensis strain MMG-9 and its identity was confirmed by thin-layer chromatography (TLC) as well as by high-performance liquid chromatography (HPLC). The IAA precursor L-tryptophan was required for IAA biosynthesis. Released IAA increased with the increase of the initial concentration of L-tryptophan in the medium and with the incubation time. A. platensis strain MMG-9 accumulated more IAA than it released into the medium. The bioactivity of the secreted IAA was shown by its effect on the formation of roots by Pisum sativum. There was a significant positive effect of the supernatant of cultures of A. platensis strain MMG-9 on the number of lateral roots of P. sativum, whereas a negative effect on root length was observed.

• 한국미생물·생명공학회지
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• 제47권2호
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• pp.242-249
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• 2019

Biosynthesis of indole-3-acetate (IAA) from L-tryptophan via indole-3-pyruvate pathway requires three enzymes including aminotransferase, indole-3-pyruvate decarboxylase, and indole-3-acetate dehydrogenase. To establish a bio-based production of IAA, the aspC, ipdC, and iad1 from Escherichia coli, Enterobacter cloacae, and Ustilago maydis, respectively, were expressed under control of the tac, ilvC, and sod promoters in C. glutamicum. Cells harboring ipdC produced tryptophol, indicating that the ipdC product is functional in this host. Analyses of SDS-PAGE and enzyme activity revealed that genes encoding AspC and Iad1 were efficiently expressed from the sod promoter, and their enzyme activities were 5.8 and 168.5 nmol/min/mg-protein, respectively. The final resulting strain expressing aspC, ipdC, and iad1 produced 2.3 g/l and 7.3 g/l of IAA from 10 g/l L-tryptophan, respectively, in flask cultures and a 5-L bioreactor.

• KSBB Journal
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• 제10권1호
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• pp.89-96
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• 1995

A rabidopsis thaliana의 trpl 변이 식물체는 uv 하에서 푸른 형광 빛을 발하며, phosphoribosyl anthranilate transferase를 encoding하는 유전자가 결여되어 있다. 이 유전자를 PATl이라고 하며, 많 은 미 생물에서 phospho ribosyl anthranilate trans ferase와 homologous하다. 트럽토판 생합성에서 이 효소가 결여되면 anthranilate가 축적되며 형광 빛 을 발하게 된다. PATl 의 유전자 조절을 알아보기 위하여, PATl 유전자의 promoter를 단계 별로 삭제하여 트립토판 변이 식물체의 형질전환과 재분화를 시도하였다. 이 러한 유전자 조작을 통하여 이 유전자의 발현 양상을 조절하는 promoter 요소와 작용을 확인할 수 있으리라고 생각된다. 또한 PAT의 항체를 사용한 Immunoassay를 통하여 형질전환체 의 단백질 양의 변화를 분석한 결과 PATl의 완전 한 promoter를 가진 pHSI07의 형질전환체는 대조구보다 2배의 단백질 양을 나타냄으로서 2쌍의 PAT 유전자가 발현되었음을 알 수 있었다. PATl 은 selection marker와 reporter 유전자로서 분자유 전학연구에 공헌할 수 였으리라고 생각된다.

• Applied Biological Chemistry
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• 제33권1호
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• pp.68-72
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• 1990

저온에서 일어나는 식물생육의 억제와 식물생장조절물질의 하나인 IAA 수준의 조절을 알아보기 위하여 암실재배한 백색 완두유묘를 재료로 저온처리에 따른 생육과 IAA 수준의 변화를 조사하였다. $25^{\circ}C$에서 성장한 백색 완두유묘는 $5^{\circ}C$의 저온에 접하면 생장이 거의 정지하였다. 유리 IAA 수준은 생체중 기준 상온묘의 17.6 ng/g에서 3일간 저온처리시에 4.9 ng/g으로, 총 IAA 수준은 113.2 ng/g에서 60.8 ng/g으로 현저하게 감소하였다. IAA의 전구체인 트립토판 수준은 생체중 기준 상온묘의 583 nmol/g에서 저온처리시에 414 nmol/g으로 약 30 % 감소하였으며, 시킴산경로에 의하여 트립토판과 함께 생합성되는 페닐알라닌과 티로신 수준도 감소하였다. 이 결과는 저온에서 IAA의 전구체인 트립토판 생합성의 억제를 통하여 IAA 수준을 일부 하향조절할 수 있을 것임을 시사한다.

• The Korean Journal of Physiology and Pharmacology
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• 제3권5호
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• pp.501-505
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• 1999

Tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin biosynthesis, is primarily expressed in serotonergic neurons of the raphe nuclei. Simple tandem repeat polymorphisms, typically one to four nucleotides long, are tandemly repeated several times and often characterized by many alleles. To identify the presence of polymorphic repeats, we sequenced the 5'-upstream region of the mouse TPH gene. For the detection of any allelic variants, polymerase chain reaction, nonisotopic single-strand conformation polymophism, and DNA sequencing analyses of the tandem repeat sequences were performed using genomic DNA extracted from 60 ICR mice. Two dinucleotide repeats, $5'-(AC/TG)_{22}-3'$ and $5'-(GT/CA)_{17}3',$ were identified at approximately - 5.7 kb and - 3.4 kb upstream from the transcriptional initiation site of the mouse TPH gene, respectively. Minor allelic variants, $5'-(AC/TG)_{21}-3'$ and $5'-(GT/CA)_{18}-3',$ were observed in heterozygous pairs from 3 of 60 and 1 of 60 ICR mice, respectively. The identification of these microsatellites in the mouse TPH promoter raises the possibility that identical and/or other polymorphic sequences might exist in the upstream region of the human TPH gene.