Pivalic acid acts as a starter unit in a fatty acid and antibiotic biosynthetic pathway in Alicyclobacillus, Rhodococcus and Streptomyces

A biosynthetic pathway using pivalic acid as a starter unit was found in three bacterial species, Alicyclobacillus acidoterrestris, Rhodococcus erythropolis and Streptomyces avermitilis. When deuterium-labelled pivalic acid was added to A. acidoterrestris and R. erythropolis nutrient media it was incorporated into fatty acids to give rise to tert-butyl fatty acids (t-FAs). In addition, in R. erythropolis, pivalic acid was transformed into two starter units, i.e. isobutyric and 2-methylbutyric acid, which served as precursors of corresponding iso-even FAs and anteiso-FAs. In S. avermitilis the biosynthesis also yielded all three branched FAs; apart from this pathway, both pivalic and 2-methylbutyric acids were incorporated into the antibiotic avermectin.     

Identification and functional characterization of phenylglycine biosynthetic genes involved in pristinamycin biosynthesis in Streptomyces pristinaespiralis

Pristinamycin I (PI), a streptogramin type B antibiotic produced by Streptomyces pristinaespiralis, contains the aproteinogenic amino acid l-phenylglycine. Recent sequence analysis led to the identification of a set of putative phenylglycine biosynthetic genes. Successive inactivation of the individual genes resulted in a loss of PI production. Production was restored by supplementation with externally added l-phenylglycine, which demonstrates that these genes are involved in phenylglycine biosynthesis and thus probably disclosing the last essential pristinamycin biosynthetic genes. Finally, a putative pathway for phenylglycine synthesis is proposed.     

PCR screening of 3-amino-5-hydroxybenzoic acid synthase gene leads to identification of ansamycins and AHBA-related antibiotic producers in Actinomycetes

Aims:  The 3-amino-5-hydroxybenzoic acid (AHBA) synthase is one of the essential and unique enzymes for AHBA biosynthesis. The possibility of screening for ansamycin or AHBA-related antibiotic-producing strains from Actinomycetes by targeting an AHBA synthase gene was explored.
Methods and Results:  A pair of degenerated primers designed according to the conserved regions of five known AHBA synthases was used to detect AHBA synthase genes within the genomic DNA of Actinomycetes. PCR screening resulted in obtaining 33 AHBA synthase gene-positive strains from 2000 newly isolated Actinomycetes. Phylogenetic analysis of these gene fragments along with those involved in the biosynthesis of structurally determined ansamycins showed that the genes with close phylogenetic relationships might be involved in the biosynthesis of compounds with the same/similar structures. Four strains have been proved to be actual geldanamycin or rifamycin producers by chemical characterization of their fermentation products.
Conclusions:  The results confirmed the feasibility of using the AHBA synthase gene as a probe in polymerase chain reaction (PCR) screening of ansamycin or AHBA-related antibiotic-producing strains.
Significance and Impact of the Study:  The PCR screening of AHBA synthase gene represents a direct and sensitive molecular method for rapid detection of AHBA-related antibiotic-producing strains.     

Characterization of TioF, a tryptophan 2,3-dioxygenase involved in 3-hydroxyquinaldic acid formation during thiocoraline biosynthesis

Thiocoraline is a thiodepsipeptide antitumor agent that belongs to the family of bisintercalator natural products that bind duplex DNA through their two planar intercalating moieties. In thiocoraline, the 3-hydroxyquinaldic acid (3HQA) chromophores required for intercalation are derived from L-Trp. We have expressed the Micromonospora sp. ML1 tryptophan 2,3-dioxygenase(TDO) TioF, purified it from E. coli, and confirmed its role in the irreversible oxidation of L-Trp to N-formylkynurenine, the proposed first step during 3HQA biosynthesis. We have established that TioF is a catalyst with broader specificity than other TDOs, but that is less promiscuous than indoleamine 2,3-dioxygenases. TioF was found to display activity with various L-Trp analogs (serotonin, D-Trp, and indole). The TioF reaction products generated during this study will be used as substrates for subsequent analysis of the other enzymes involved in 3HQA biosynthesis.     

海洋破囊壶菌Thraustochytrium sp. FJN-10 DHA生物合成 途径相关延长酶的克隆与表达

二十二碳六烯酸(Docosahexaenoic acid,DHA)是具有各种重要生理功能的高度不饱和脂肪酸.以海洋真菌Thraustochytrium sp.FJN-10为研究对象,利用RT-PCR结合RACE,获得了两个碳链延长酶(TFD6和TFD5)的完整基因,其中TFD6 cDNA全长816 bp,编码271个氨基酸;TFD5 cDNA全长831 bp,编码276个氨基酸.将TFD6、TFD5酶切后分别连接到HindⅢ/Xba Ⅰ处理过的pYES2载体,醋酸锂法转化酿酒酵母感受态细胞,成功构建了延长酶酵母表达系统.气相色谱分析表明TFD6可延长C18:3n-6至C20:3n-6,TFD5可延长C20:5n-3至C22:5n-3.     

Organization and expression of genes involved in the biosynthesis of 987P fimbriae

Summary A chromosomal DNA segment encoding the biosynthesis of 987P fimbriae was isolated by cosmid-cloning and subsequent subcloning into pBR322. The 12 kb DNA segment expressed five polypeptides with apparent molecular weights of 81,000, 39,000, 28,500, 20,500, and 16,500, respectively. The location of the corresponding genes was determined by insertional mutagenesis using Tn5. The 20.5 K polypeptide was identified as the 987P fimbrial subunit by its reaction with specific anti-987P antibodies. The 81, 39, and 28.5 K polypeptides appeared to be accessory proteins involved in 987P production.     

Two dioxygenase genes,Ids3 and Ids2, from Hordeum vulgare are involved in the biosynthesis of mugineic acid family phytosiderophores

A cDNA clone, Ids3 (iron deficiency-specific clone 3), was isolated from an Fe-deficient-root cDNA library of Hordeum vulgare. Ids3 encodes a protein of 339 amino acids with a calculated molecular mass of 37.7 kDa, and its amino acid sequence shows a high degree of similarity with those of plant and fungal 2-oxoglutarate-dependent dioxygenases. One aspartate and two histidine residues for ferrous Fe binding (Asp-211, His-209, His-265) and arginine and serine residues for 2-oxoglutarate binding (Arg-275, Ser-277) are conserved in the predicted amino acid sequence of Ids3. Ids3 expression was rapidly induced by Fe deficiency, and was suppressed by re-supply of Fe. Among eight graminaceous species tested, Ids3 expression was observed only in Fe-deficient roots of H. vulgare and Secale cereale, which not only secrete 2-deoxymugineic acid (DMA), but also mugineic acid (MA) and 3-epihydroxymugineic acid (epiHMA, H. vulgare), and 3-hydroxymugineic acid (HMA, S. cereale). The Ids3 gene is encoded on the long arm of chromosome 4H of H. vulgare, which also carries the hydroxylase gene that converts DMA to MA. Moreover, the Ids2 gene, which is the plant dioxygenase with the highest homology to Ids3, is encoded on the long arm of chromosome 7H of H. vulgare, which carries the hydroxylase gene that converts MA to epiHMA. The observed expression patterns of the Ids3 and Ids2 genes strongly suggest that IDS3 is an enzyme that hydroxylates the C-2 positions of DMA and epiHDMA, while IDS2 hydroxylates the C-3 positions of MA and DMA.     

Carotenoid biosynthetic genes in Brassica rapa: comparative genomic analysis,phylogenetic analysis,and expression profiling

Background

Carotenoids are isoprenoid compounds synthesized by all photosynthetic organisms. Despite much research on carotenoid biosynthesis in the model plant Arabidopsis thaliana, there is a lack of information on the carotenoid pathway in Brassica rapa. To better understand its carotenoid biosynthetic pathway, we performed a systematic analysis of carotenoid biosynthetic genes at the genome level in B. rapa.

Results

We identified 67 carotenoid biosynthetic genes in B. rapa, which were orthologs of the 47 carotenoid genes in A. thaliana. A high level of synteny was observed for carotenoid biosynthetic genes between A. thaliana and B. rapa. Out of 47 carotenoid biosynthetic genes in A. thaliana, 46 were successfully mapped to the 10 B. rapa chromosomes, and most of the genes retained more than one copy in B. rapa. The gene expansion was caused by the whole-genome triplication (WGT) event experienced by Brassica species. An expression analysis of the carotenoid biosynthetic genes suggested that their expression levels differed in root, stem, leaf, flower, callus, and silique tissues. Additionally, the paralogs of each carotenoid biosynthetic gene, which were generated from the WGT in B. rapa, showed significantly different expression levels among tissues, suggesting differentiated functions for these multi-copy genes in the carotenoid pathway.

Conclusions

This first systematic study of carotenoid biosynthetic genes in B. rapa provides insights into the carotenoid metabolic mechanisms of Brassica crops. In addition, a better understanding of carotenoid biosynthetic genes in B. rapa will contribute to the development of conventional and transgenic B. rapa cultivars with enriched carotenoid levels in the future.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1655-5) contains supplementary material, which is available to authorized users.     

Docosapentaenoic acid (22:5n-3) down-regulates the expression of genes involved in fat synthesis in liver cells

Previous studies have shown that Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) exhibit triacylglycerol (TAG) lowering effect in vitro and in vivo by down-regulating the Sterol Regulating Element Binding Protein (SREBP-1c) and reducing the expression levels of lipogenic genes. However, there is no evidence on the effect of Docosapentaenoic Acid (DPA) on SREBP-1c expression levels. DPA is a long chain n-3 fatty acid present in our diet through fish, red meat and milk of ruminant animals. Therefore, this study aimed to elucidate the effect of DPA on liver fatty acid synthesis in an in vitro model using rat liver cells. Our results suggested that DPA incubation (50μM) for 48h (like EPA and DHA) caused a significant decrease in the mRNA expression levels of SREBP-1c, 3-Hydroxy-3-Methyl-Glutaryl-Coenzyme A reductase (HMG-CoA reductase), Acetyl Coenzyme A Carboxylase (ACC-1) and Fatty Acid Synthase (FASn) compared with Oleic Acid (OA) and also a decrease in the protein levels of SREBP-1 and ACC-1. A time-course fatty acid analysis showed that DPA and EPA are interconvertable in the cells; however, after 8h of incubation with DPA, the cell phospholipids contained mainly DPA. The gene expression profiling of the lipogenic genes repeated at 8h confirmed that the inhibitory effect of DPA on mRNA expression levels of the lipogenic genes was most likely due to DPA itself and not due to its conversion into EPA.     

Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii.

DNA sequencing of the region upstream from the Azotobacter vinelandii operon (modEABC) that contains genes for the molybdenum transport system revealed an open reading frame (modG) encoding a hypothetical 14-kDa protein. It consists of a tandem repeat of an approximately 65-amino-acid sequence that is homologous to Mop, a 7-kDa molybdopterin-binding protein of Clostridium pasteurianum. The tandem repeat is similar to the C-terminal half of the product of modE. The effects of mutations in the mod genes provide evidence for distinct high- and low-affinity Mo transport systems and for the involvement of the products of modE and modG in the processing of molybdate. modA, modB, and modC, which encode the component proteins of the high-affinity Mo transporter, are required for 99Mo accumulation and for the nitrate reductase activity of cells growing in medium with less than 10 microM Mo. The exchange of accumulated 99Mo with nonradioactive Mo depends on the presence of modA, which encodes the periplasmic molybdate-binding protein. 99Mo also exchanges with tungstate but not with vanadate or sulfate. modA, modB, and modC mutants exhibit nitrate reductase activity and 99Mo accumulation only when grown in more than 10 microM Mo, indicating that A. vinelandii also has a low-affinity Mo uptake system. The low-affinity system is not expressed in a modE mutant that synthesizes the high-affinity Mo transporter constitutively or in a spontaneous tungstate-tolerant mutant. Like the wild type, modG mutants only show nitrate reductase activity when grown in > 10 nM Mo. However, a modE modG double mutant exhibits maximal nitrate reductase activity at a 100-fold lower Mo concentration. This indicates that the products of both genes affect the supply of Mo but are not essential for nitrate reductase cofactor synthesis. However, nitrogenase-dependent growth in the presence or absence of Mo is severely impaired in the double mutant, indicating that the products of modE and modG may be involved in the early steps of nitrogenase cofactor biosynthesis in A. vinelandii.     

Deletion of the pyc gene blocks clavulanic acid biosynthesis except in glycerol-containing medium: evidence for two different genes in formation of the C3 unit

The beta-lactamase inhibitor clavulanic acid is formed by condensation of a pyruvate-derived C3 unit with a molecule of arginine. A gene (pyc, for pyruvate converting) located upstream of the bls gene in the clavulanic acid gene cluster of Streptomyces clavuligerus encodes a 582-amino-acid protein with domains recognizing pyruvate and thiamine pyrophosphate that shows 29.9% identity to acetohydroxyacid synthases. Amplification of the pyc gene resulted in an earlier onset and higher production of clavulanic acid. Replacement of the pyc gene with the aph gene did not cause isoleucine-valine auxotrophy in the mutant. The pyc replacement mutant did not produce clavulanic acid in starch-asparagine (SA) or in Trypticase soy broth (TSB) complex medium, suggesting that the pyc gene product is involved in the conversion of pyruvate into the C3 unit of clavulanic acid. However, the beta-lactamase inhibitor was still formed at the same level as in the wild-type strain in defined medium containing D-glycerol, glutamic acid, and proline (GSPG medium) as confirmed by high-pressure liquid chromatography and paper chromatography. The production of clavulanic acid by the replacement mutant was dependent on addition of glycerol to the medium, and glycerol-free GSPG medium did not support clavulanic acid biosynthesis, suggesting that an alternative gene product catalyzes the incorporation of glycerol into clavulanic acid in the absence of the Pyc protein. The pyc replacement mutant overproduces cephamycin.