Sequence-based screening for self-sufficient P450 monooxygenase from a metagenome library

Kim, B.S.,Kim, S.Y.,Park, J.,Park, W.,Hwang, K.Y.,Yoon, Y.J.,Oh, W.K.,Kim, B.Y.,Ahn, J.S. Published for the Society for Applied Bacteriology 2007 Journal of applied microbiology Vol.102 No.5

<P>Abstract</P><P>Aims: </P><P>Cytochrome P450 monooxygenases (CYPs) are useful catalysts for oxidation reactions. Self-sufficient CYPs harbour a reductive domain covalently connected to a P450 domain and are known for their robust catalytic activity with great potential as biocatalysts. In an effort to expand genetic sources of self-sufficient CYPs, we devised a sequence-based screening system to identify them in a soil metagenome.</P><P>Methods and Results: </P><P>We constructed a soil metagenome library and performed sequence-based screening for self-sufficient CYP genes. A new CYP gene, <I>syk181</I>, was identified from the metagenome library. Phylogenetic analysis revealed that SYK181 formed a distinct phylogenic line with 46% amino-acid-sequence identity to CYP102A1 which has been extensively studied as a fatty acid hydroxylase. The heterologously expressed SYK181 showed significant hydroxylase activity towards naphthalene and phenanthrene as well as towards fatty acids.</P><P>Conclusions: </P><P>Sequence-based screening of metagenome libraries is expected to be a useful approach for searching self-sufficient CYP genes. The translated product of <I>syk181</I> shows self-sufficient hydroxylase activity towards fatty acids and aromatic compounds.</P><P>Significance and Impact of the Study: </P><P>SYK181 is the first self-sufficient CYP obtained directly from a metagenome library. The genetic and biochemical information on SYK181 are expected to be helpful for engineering self-sufficient CYPs with broader catalytic activities towards various substrates, which would be useful for bioconversion of natural products and biodegradation of organic chemicals.</P>

Thiamine increases β-glucosidase production in the newly isolated strain of <i>Fomitopsis pinicola</i>

Joo, A.-R.,Lee, K.-M.,Sim, W.-I.,Jeya, M.,Hong, M.-R.,Kim, Y.-S.,Oh, D.-K.,Lee, J.-K. Published for the Society for Applied Bacteriology 2009 Letters in applied microbiology Vol.49 No.2

<P>Abstract</P><P>Aims: </P><P>To isolate a high &bgr;-glucosidase (BGL)-producing strain and to optimize BGL production in the isolated strain.</P><P>Methods and Results: </P><P>A high BGL-producing strain was isolated and identified as <I>Fomitopsis pinicola</I> KMJ812 based on its morphology and a comparison of sequence of its internal transcribed spacer rDNA gene. To increase BGL production, <I>F. pinicola</I> was supplemented with various vitamins. Supplementation with thiamine (20 mg l<SUP>−1</SUP>) improved BGL production in <I>F. pinicola</I> cultures by 3·7-fold to give a specific activity of 114·4 &mgr;mol min<SUP>−1</SUP> mg<SUP>−1</SUP> protein, one of the highest among BGL-producing micro-organisms. The increased production of BGL in the thiamine-supplemented culture was confirmed by 2D electrophoresis followed by MS/MS sequencing. The BGL purified from <I>F. pinicola</I> culture showed the highest catalytic efficiency ever reported.</P><P>Conclusion: </P><P>Supplemental thiamine remarkably increased BGL production by a novel BGL-producing strain, <I>F. pinicola</I> KMJ812.</P><P>Significance and Impact of the Study: </P><P>Our results provide a high BGL-producing strain and the production media for BGL production, and should contribute to better industrial production of glucose via biological processes.</P>

Biohydrogenation of linoleic acid by rumen fungi compared with rumen bacteria

Nam, I.S.,Garnsworthy, P.C. Published for the Society for Applied Bacteriology 2007 Journal of applied microbiology Vol.103 No.3

Biosynthesis of antibiotic prodiginines in the marine bacterium <i>Hahella chejuensis</i> KCTC 2396

Kim, D.,Lee, J.S.,Park, Y.K.,Kim, J.F.,Jeong, H.,Oh, T.-K.,Kim, B.S.,Lee, C.H. Published for the Society for Applied Bacteriology 2007 Journal of applied microbiology Vol.102 No.4

<P>Abstract</P><P>Aims: </P><P><I>Hahella chejuensis</I> KCTC 2396 produces red pigments, showing antibacterial and algicidal activities. The main red-coloured metabolite of the pigments was identified as antibiotic prodigiosin. With the expectation that the red pigments are a mixture of a series of close relatives, the aim of the present study is to detect new antibiotic prodigiosin analogues and to analyse the biosynthetic pattern for prodiginines in KCTC 2396.</P><P>Methods and Results: </P><P>Except prodigiosin, the other constituents in the red pigments were confirmed as well-known dipyrrolyldipyrromethene prodigiosin, norprodigiosin, and undecylprodiginine. Additionally, four new prodigiosin analogues, each of which was distinguished from prodigiosin (C<SUB>5</SUB>), according to differences in alkyl chain length (C<SUB>3</SUB>–C<SUB>7</SUB>), were detected in small quantities by liquid chromatography mass spectrometry/mass spectrometry spectroscopy. Owing to the presence of a cytotoxic methoxy group, it is expected that all the new prodigiosin analogues are bioactive.</P><P>Conclusions: </P><P>Four characterized prodiginines, including prodigiosin and four new prodigiosin analogues are produced in different ratio in KCTC 2396. All of the prodiginines possess a common linear tripyrrolyl structure and a cytotoxic methoxy group.</P><P>Significance and Impact of the Study: </P><P>This study shows for the first time that KCTC 2396 is able to produce antibiotic prodigiosin, undecylprodiginine and new prodigiosin analogues in a mixture of pigments. It is also shown that KCTC 2396 possesses a novel system for the simultaneous production of multiple prodiginines in a single micro-organism.</P>

The effect of NADP-dependent malic enzyme expression and anaerobic C4 metabolism in <i>Escherichia coli</i> compared with other anaplerotic enzymes

Kwon, Y.-D.,Kwon, O.-H.,Lee, H.-S.,Kim, P. Published for the Society for Applied Bacteriology 2007 Journal of applied microbiology Vol.103 No.6

<P>Abstract</P><P>Aims: </P><P>To understand the modification of C4-metabolism under anaerobic glycolysis condition by overexpressing anaplerotic enzymes, which mediating carboxylation of C3 into C4 metabolites, in <I>Escherichia coli</I>.</P><P>Methods and Results: </P><P>Anaplerotic NADP-dependent malic enzyme (MaeB), as well as the other anaplerotic enzymes, including phosphoenolpyruvate carboxylase (Ppc), phosphoenolpyruvate carboxykinase (Pck) and NAD-dependent malic enzyme (MaeA), were artificially expressed and their C4 metabolism was compared in <I>E. coli</I>. Increasing MaeB expression enhanced the production of C4 metabolites by 2·4 times compared to the wild-type strain in anaerobic glucose medium with bicarbonate supplementation. In MaeB expression, C4 metabolism by supplementing 10 g l<SUP>−1</SUP> of NaHCO<SUB>3</SUB> was three times than that by no supplementation, which showed the greatest response to increased CO<SUB>2</SUB> availability among the tested anaplerotic enzyme expressions.</P><P>Conclusions: </P><P>The higher C4 metabolism was achieved in <I>E. coli</I> expressing increased levels of the NADPH-dependent MaeB. The greatest increase in the C4 metabolite ratio compared to the other tested enzymes were also found in <I>E. coli</I> with enhanced MaeB expression as CO<SUB>2</SUB> availability increased.</P><P>Significance and Impact of the Study: </P><P>The higher C4 metabolites and related biomolecule productions can be accomplished by MaeB overexpression in metabolically engineered <I>E. coli</I>.</P>

Growth inhibition of bloom-forming cyanobacterium <i>Microcystis aeruginosa</i> by rice straw extract

Park, M.-H.,Han, M.-S.,Ahn, C.-Y.,Kim, H.-S.,Yoon, B.-D.,Oh, H.-M. Published for the Society for Applied Bacteriology 2006 Letters in applied microbiology Vol.43 No.3

<P>Abstract</P><P>Aims: </P><P>To inhibit the growth of the bloom-forming cyanobacterium <I>Microcystis aeruginosa</I> using a rice straw extract.</P><P>Methods and Results: </P><P>The cell numbers of the algal strain <I>M. aeruginosa</I> UTEX 2388 significantly decreased after treatment with different concentrations (0·01, 0·1, 1 and 10 mg l<SUP>−1</SUP>) of a rice straw extract for an 8-day cultivation period. Among seven tested allelochemicals from rice straw, salicylic acid at 0·1 mg l<SUP>−1</SUP> exhibited the highest allelopathic activity (26%) on day 8. A synergistic effect on algal growth inhibition was found when adding two or three phenolic compounds from the rice straw.</P><P>Conclusions: </P><P>The growth of <I>M. aeruginosa</I> was inhibited by rice straw extract concentrations ranging from 0·01 to 10 mg l<SUP>−1</SUP>. This activity was due to the synergistic effects of various phenolic compounds in the rice straw.</P><P>Significance and Impact of the Study: </P><P>The identification of rice straw as an effective material for the growth inhibition of <I>M. aeruginosa</I> implies it may have the potential to be used as an environment-friendly biomaterial for controlling the algal bloom of <I>M. aeruginosa</I> in eutrophic water.</P>

Activity of some aminoglycoside antibiotics against true fungi, <i>Phytophthora</i> and <i>Pythium</i> species

Lee, H.B.,Kim, Y.,Kim, J.C.,Choi, G.J.,Park, S.-H.,Kim, C.-J.,Jung, H.S. Published for the Society for Applied Bacteriology 2005 Journal of applied microbiology Vol.99 No.4

<P>Abstract</P><P>Aims: </P><P>To investigate the <I>in vitro</I> antifungal and antioomycete activities of some aminoglycosides against true fungi and <I>Phytophthora</I> and <I>Pythium</I> species and to evaluate the potential of the antibiotics against <I>Phytophthora</I> late blight on plants.</P><P>Methods and Results: </P><P>Antifungal and antioomycete activities of aminoglycoside antibiotics (neomycin, paromomycin, ribostamycin and streptomycin) and a paromomycin-producing strain (<I>Streptomyces</I> sp. AMG-P1) against <I>Phytophthora</I> and <I>Pythium</I> species and 10 common fungi were measured in potato dextrose broth (PDB) and on seedlings in pots. Paromomycin was the most active against <I>Phytophthora</I> and <I>Pythium</I> species with a minimal inhibitory concentration of 1–10 <I>μ</I>g ml<SUP>−1</SUP> in PDB, but displayed low to moderate activities towards other common fungi at the same concentration. Paromomycin also showed potent <I>in vivo</I> activity against red pepper and tomato late blight diseases with 80 and 99% control value, respectively, at 100 <I>μ</I>g ml<SUP>−1</SUP>. In addition, culture broth of <I>Streptomyces</I> sp. AMG-P1 as a paromomycin producer exhibited high <I>in vivo</I> activity against late blight at 500 <I>μ</I>g freeze-dried weight per millilitre.</P><P>Conclusions: </P><P>Among tested aminoglycoside antibiotics, paromomycin was the most active against oomycetes both <I>in vitro</I> and <I>in vivo</I>.</P><P>Significance and Impact of the Study: </P><P>Data from this study show that aminoglycoside antibiotics have <I>in vitro</I> and <I>in vivo</I> activities against oomycetes, suggesting that <I>Streptomyces</I> sp. AMG-P1 may be used as a biocontrol agent against oomycete diseases.</P>