• Journal of Microbiology and Biotechnology
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• 제27권10호
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• pp.1867-1876
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• 2017

Most of the biosynthetic pathways for secondary metabolites are influenced by carbon metabolism and supply of cytosolic NADPH. We engineered carbon distribution to the pentose phosphate pathway (PPP) and redesigned the host to produce high levels of NADPH and primary intermediates from the PPP. The main enzymes producing NADPH in the PPP, glucose 6-phosphate dehydrogenase (encoded by zwf1 and zwf2) and 6-phosphogluconate dehydrogenase (encoded by zwf3), were overexpressed with opc encoding a positive allosteric effector essential for Zwf activity in various combinations in Streptomyces lividans TK24. Most S. lividans transformants showed better cell growth and higher concentration of cytosolic NADPH than those of the control, and S. lividans TK24/pWHM3-Z23O2 containing zwf2+zwf3+opc2 showed the highest NADPH concentration but poor sporulation in R2YE medium. S. lividans TK24/pWHM3-Z23O2 in minimal medium showed the maximum growth (6.2 mg/ml) at day 4. Thereafter, a gradual decrease of biomass and a sharp increase of cytosolic NADPH and sedoheptulose 7-phosphate between days 2 and 4 and between days 1 and 3, respectively, were observed. Moreover, S. lividans TK24/pWHM3-Z23O2 produced 0.9 times less actinorhodin but 1.8 times more undecylprodigiosin than the control. These results suggested that the increased NADPH concentration and various intermediates from the PPP specifically triggered undecylprodigiosin biosynthesis that required many precursors and NADPH-dependent reduction reaction. This study is the first report on bespoke metabolic engineering of PPP routes especially suitable for producing secondary metabolites that need diverse primary precursors and NADPH, which is useful information for metabolic engineering in Streptomyces.

• Journal of Microbiology and Biotechnology
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• 제25권5호
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• pp.579-588
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• 2015

Fungi of the genus Pestalotiopsis have drawn attention for their capability to produce an array of bioactive secondary metabolites that have potential for drug development. Here, we report the determination of a polyketide derivative compound, pestalotiollide B, in the culture of the saprophytic fungus Pestalotiopsis microspora NK17. Structural information acquired by analyses with a set of spectroscopic and chromatographic techniques suggests that pestalotiollide B has the same skeleton as the penicillide derivatives, dibenzodioxocinones, which are inhibitors of cholesterol ester transfer protein (CETP), and as purpactins A and C', inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). Strain NK17 can make a fairly high yield of pestalotiollide B (i.e., up to 7.22 mg/l) in a constitutive manner in liquid culture. Moreover, we found that a putative histone deacetylase gene, designated as hid1, played a role in the biosynthesis of pestalotiollide B. In the hid1 null mutant, the yield of pestalotiollide B increased approximately 2-fold to 15.90 mg/l. In contrast, deletion of gene hid1 led to a dramatic decrease of conidia production of the fungus. These results suggest that hid1 is a modulator, concerting secondary metabolism and development such as conidiation in P. microspora. Our work may help with the investigation into the biosynthesis of pestalotiollide B and the development for new CETP and ACAT inhibitors.

• Journal of Microbiology and Biotechnology
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• 제15권5호
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• pp.1060-1066
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• 2005

Under environmental stress, such as strong irradiance or nitrogen deficiency, unicellular green algae of the genus Haematococcus accumulate secondary carotenoids, i.e. astaxanthin, in the cytosol. The induction and regulation of astaxanthin biosynthesis in microalgae has recently received considerable attention owing to the increasing use of secondary carotenoids as a source of pigmentation for fish aquacultures, and as a potential drug in cancer prevention as a free-radical quencher. Accordingly, this study generated expressed sequence tags (ESTs) from a library constructed from astaxanthin-induced Haematococcus pluvialis. Partial sequences were obtained from the 5' ends of 1,858 individual cDNAs, and then grouped into 1,025 non-overlapping sequences, among which 708 sequences were singletons, while the remainder fell into 317 clusters. Approximately $63\%$ of the EST sequences showed similarity to previously described sequences in public databases. H. pluvialis was found to consist of a relatively high percentage of genes involved in genetic information processing ($15\%$) and metabolism ($11\%$), whereas a relatively low percentage of sequences was involved in the signal transduction ($3\%$), structure ($2\%$), and environmental information process ($3\%$). In addition, a relatively large fraction of H. pluvialis sequences was classified as genes involved in photosynthesis ($9\%$) and cellular process ($9\%$). Based on this EST analysis, the full-length cDNA sequence for superoxide dismutase (SOD) of H. pluvialis was cloned, and the expression of this gene was investigated. The abundance of SOD changed substantially in response to different culture conditions, indicating the possible regulation of this gene in H. pluvialis.

• Journal of Nutrition and Health
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• 제33권8호
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• pp.802-812
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• 2000

The purpose of this study was to examine the effect of resistant starch(RS) in hyperchlesterolemia and colon cancer. The subjects of this study was eight college women participating in the general starch diet(GSD) period for 5 days and resistant starch diet(RSD) period for 7 days. RSD contains 30g or the RS. On the last day of each program blood were collected. And for the last 3 days of each diet period, the amount of all the food consumed by the subjects and feces were collected. Food was measured to determine and compared the energy, protein and fat intakes. The amount of total cholesterol, HDL-cholesterol, LDL-cholesterol and volatile fatty acids in plasma and the amounts of bile acids in feces were measured by gas chromatography. The results obtained were as follows, Daily energy intake was higher in the RSD compared with the GSD, Protein and fat intakes were lower in the RSD compared with the compared with the GSD. Volatile fatty acid contents in plasma, the amounts of acetic acid, propionic acid and valeric acid were higher in the RSD compared with the GSD. The amounts of bile acids in feces, cholic acid, chenodeoxycholic acid and lithocholic acid were higher in the RSD compared with the GSD, But the amount of deoxycholic acid n the RSD period was significantly low. Secondary/primary ratios of bile acids was lower in the RSD compared with GSD, respectively. We speculate that , RS consumption decreases colonic mucosal proliferation as a result of the decreased formation of cytotoxic secondary bile acids. Thus, RS intakes may contribute the prevention of heart disease and colon cancer in humans. (Korean J Nutrition 33(8) : 802-812, 2000)

• Molecules and Cells
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• 제23권2호
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• pp.228-238
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• 2007

Since molecular structure of hnRNP is not available in foreseeable future, it is best to construct a working model for hnRNP structure. A geometric problem, assembly of $700{\pm}20$ nucleotides with 48 proteins, is visualized by a frame work in which all the proteins participate in primary binding, followed by secondary, tertiary and quaternary binding with neighboring proteins without additional import. Thus, 40S hnRNP contains crown-like secondary structure (48 stemloops) and appearance of 6 petal (octamers) rose-like architectures. The proteins are wrapped by RNA. Co-transcriptional folding for RNP fibril of FMR1 gene can produce 2,571 stem-loops with frequency of 1 stem-loop/15.3 nucleotides and 53 40S hnRNP beaded structure. By spliceosome driven reactions, there occurs removal of 16 separate lariated RNPs, joining 17 separate beaded exonic structures and anchoring EJC on each exon junction. Skipping exon 12 has 5'GU, 3'AG and very compact folding pattern with frequency of 1 stem-loop per 12 nucleotides in short exon length (63 nucleotides). 5' end of exon 12 contains SS (Splicing Silencer) element of UAGGU. In exons 10, 15 and 17 where both regular and alternative splice sites exist, SS (hnRNP A1 binding site) is observed at the regular splicing site. End products are mature FMR-1 mRNP, 4 species of Pri-microRNAs derived from introns 7,9,15 and 3'UTR of exon17, respectively. There may also be some other regulatory RNAs containing ALU/Line elements as well.

• 생명과학회지
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• 제25권6호
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• pp.615-623
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• 2015

본 연구는 시멘트 노출에 따른 참갈겨니 조직의 형태 생리적 변화를 분석하여 용해된 시멘트 분말이 어류에 미치는 영향을 조사하고자 하였다. 용해된 시멘트 분말에 노출된 아가미는 노출기간이 길어질수록 이차새변의 간격이 불규칙해지고, 점액세포의 활성도 증가하고 있으며, 곤봉화, 부종, 상피세포의 박리가 이차새변에서 관찰되었다. 신장 조직에서는 보우만주머니 공간이 넓게 관찰되었고, 표피 조직은 표피층의 두께가 감소하고 진피층의 배열이 불규칙해지는 것으로 관찰되었다. 항산화효소와 LDH의 활성은 조직 및 노출기간에 따라 활성에 차이가 있는 것으로 나타났다. 표피조직에서 용해된 시멘트 분말에 의해 발현이 증가되는 단백질은 근단백질 생성과 관련된 스트레스 단백질로 확인되었고 발현이 저하된 단백질들은 해당과정과 에너지 대사에 관여하는 단백질로 조사되었다. 이러한 결과로 보아 시멘트 노출에 따른 스트레스는 참갈겨니 조직의 형태적 변형과 생리적 기능의 약화를 초래하여 어류의 생존에 커다란 위협이 될 요인으로 작용할 수 있을 것으로 사료된다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권2호
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• pp.162-165
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• 2005

A series of fluorine and hydroxyl containing jasmonate derivatives, which were chemically synthesized in our institute, were investigated for their effects on the biosynthesis and heterogeneity of ginsenosides in suspension cultures of Panax notoginseng cells. Com-pared to the control (without addition of elicitors), $100{\mu}M$ of each of the jasmonate was added on day 4 to the suspension cultures of P. notoginseng cells. It was observed that, jasmonates greatly enhanced the ginsenoside content and the ratio of Rb group to Rg group (i.e. $(Rb_1\;+\;Rd)/(Rg_1\;+\;Re)$ in the P. notoginseng cells. Some of the synthetic jasmonates, such as pentafluoropropyl jasmonate (PFPJA), 2-hydroxyethyl jasmonate (HEJA) and 2-hydroxye-thoxyethyl jasmonate (HEEJA), could promote the ginsenoside content to $2.55\;\pm\;0.11,\;3.65\;\pm\;0.13\;and\;2.94\;\pm\;0.06$mg/100 mg DW, respectively, compared to that of $0.64\;\pm\;0.06$mg/100 mg DW for the control and $2.17\;\pm\;0.04$ mg/100 mg DW by the commercially available methyl jasmonate (MJA); and they could change the respective Rb:Rg ratio to $1.60\;\pm\;0.04,\;1.87\;\pm\;0.01\;and\;1.56\;\pm\;0.05$, compared to that of $0.47\;\pm\;0.01$ for the control and $1.42\;\pm\;0.06$ by MJA. The results suggest that suitable esterification of MJA with fluorine or hydroxyl group could in-crease the elicitation activity to induce plant secondary metabolism. The information obtained from this study is useful for hyper-production of heterogeneous products by plant cell cultures.

• 원예과학기술지
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• 제34권1호
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• pp.46-54
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• 2016

2-Oxoglutarate (2OG) acts as a signaling molecule and plays a critical role in secondary metabolism in a variety of organisms, including plants. Six 2-oxoglutarate (2OG) and Fe(II) oxygenase (2OGO) genes, VlCE2OGO1 [Vitis labruscana 2-oxoglutarate (2OG) and Fe(II) oxygenase 1], VlCE2OGO2, VlCE2OGO3, VlCE2OGO4, VlCE2OGO5, and VlCE2OGO6, which show different expression patterns upon transcriptome analysis of 'Campbell Early' grapevine exposed to low temperature for 4 weeks, were analyzed for their structure and expression. Comparison of the deduced amino acid sequences of the 2OGO genes from the V. labruscana transcripts revealed sequence similarities of 38.6% (VlCE2OGO1 and VlCE2OGO2) to 19.2% (VlCE2OGO2 and VlCE2OGO3). The lengths of these genes ranged from 1053 to 2298 bp, and they encoded 316 to 380 amino acids. The prediction of the secondary structure of the encoded proteins by Self-Optimized Prediction Method with Alignment (SOPMA) indicated that all the genes contained alpha helix (23.95 to 41.71%), extended strand (16 to 22.34%), beta turn (6.65 to 9.22%), and random coil (32.97 to 51.58%) in the analysis. Specific primers from unique regions in each gene obtained by alignment of nucleotide sequences were used in real time PCR for analysis of gene expression. All tested genes showed differential expression in grapevines exposed to low temperature. Of the six transcripts, VlCE2OGO1, VlCE2OGO2, and VlCE2OGO3 were up-regulated and VlCE2OGO4, VlCE2OGO5, and VlCE2OGO6 were down-regulated in response to cold treatments at all tested time points. The 2OG genes can be used for elucidation of mechanisms of tolerance to cold and as valuable molecular genetic resources for selection in breeding programs for cold-hardy grapevines.

• Genes and Genomics
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• 제40권12호
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• pp.1287-1300
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• 2018

Hydrogen sulfide ($H_2S$), a small bioactive gas, has been proved functioning in plant growth and development as well as alleviation of abiotic stresses, which including promoting seed germination, accelerating embryonic root growth, regulating flower senescence, inducing stomatal closure, and defending drought, heat, heavy metals and osmotic stresses etc. However, the molecular functioning mechanism of $H_2S$ was still unclear. The primary objective of this research was to analyze the transcriptional differences and functional genes involved in the $H_2S$ responses. In details, 4-week-old plantlets in tissue culture of grapevine (Vitis vinifera L.) cultivar 'Zuoyouhong' were sprayed with 0.1 mM NaHS for 12 h, and then transcriptome sequencing and qRT-PCR analysis were used to study the transcriptional differences and functional genes involved in the $H_2S$ responses. Our results indicated that 650 genes were differentially expressed after $H_2S$ treatment, in which 224 genes were up-regulated and 426 genes were down-regulated. The GO enrichment analysis and KEGG enrichment analysis results indicated that the up-regulated genes after $H_2S$ treatment focused on carbon metabolism, biosynthesis of amino acids, and glycolysis/gluconeogenesis, and the down-regulated genes were mainly in metabolic pathways, biosynthesis of secondary metabolites, and plant hormone signal transduction. Analyzing the transcription factor coding genes in details, it was indicated that 10 AP2/EREBPs, 5 NACs, 3 WRKYs, 3 MYBs, and 2 bHLHs etc. transcription factor coding genes were up-regulated, while 4 MYBs, 3 OFPs, 3 bHLHs, 2 AP2/EREBPs, 2 HBs etc. transcription factor coding genes were down-regulated. Taken together, $H_2S$ increased the productions in secondary metabolites and a variety of defensive compounds to improve plant development and abiotic resistance, and extend fruits postharvest shelf life by regulating the expression of AP2/EREBPs, WRKYs, MYBs, CABs, GRIP22, FERRITINs, TPSs, UGTs, and GHs etc.

• Journal of Microbiology and Biotechnology
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• 제31권4호
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• pp.510-519
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• 2021

The pathological impact of haze upon the phyllosphere microbiota awaits investigation. A moderate degree of haze environment and a clean control were selected in Chengdu, China. Artemisia argyi, a ubiquitously distributed and extensively applied Chinese herb, was also chosen for experiment. Total genome DNA was extracted from leaf samples, and for metagenome sequencing, an Illumina HiSeq 2500 platform was applied. The results showed that the gene numbers of phyllosphere microbiota derived from haze leaves were lower than those of the clean control. The phyllosphere microbiota derived from both haze and clean groups shared the same top ten phyla; the abundances of Proteobacteria, Actinomycetes and Anorthococcuso of the haze group were substantially increased, while Ascomycetes and Basidiomycetes decreased. At the genus level, the abundances of Nocardia, Paracoccus, Marmoricola and Knoelia from haze leaves were markedly increased, while the yeasts were statistically decreased. KEGG retrieval demonstrated that the functional genes were most annotated to metabolism. An interesting find of this work is that the phyllosphere microbiota responsible for the synthesis of primary and secondary metabolites in A. argyi were significantly increased under a haze environment. Relatively enriched genes annotated by eggNOG belong to replication, recombination and repair, and genes classified into the glycoside hydrolase and glycosyltransferase enzymes were significantly increased. In summary, we found that both structure and function of phyllosphere microbiota are globally impacted by haze, while primary and secondary metabolites responsible for haze tolerance were considerably increased. These results suggest an adaptive strategy of plants for tolerating and confronting haze damage.