RESUMO
This study reports the production of astaxanthin in the photosynthetic bacterium Rhodovulum sulfidophilum, which has adequate precursor pools and storage capabilities for heterologous carotenoid production. Chemical mutagenesis was carried out using ethylmethane sulfonate to produce mutants with a modified carotenoid biosynthesis pathway downstream of phytoene. Stable green- and gray-colored mutants were selected. Green mutants contained neurosporene or chloroxanthin as their major carotenoid (>90%), while the gray mutants accumulated phytoene. We previously demonstrated the production of beta-carotene in Rhodovulum sulfidophilum by cloning the Erythrobacter longus crtI (phytoene dehydrogenase) and crtY (lycopene cyclase) genes. In the present study, an expression vector for astaxanthin production was constructed that contained the Paracoccus crtW (beta-carotene oxygenase) and crtZ (beta-carotene hydroxylase) genes in addition to the E. longus crtI and crtY genes. A transconjugant, which can synthesize astaxanthin, was successfully generated (2.0 microg g(-1) DCW).
Assuntos
Rhodovulum/genética , Rhodovulum/metabolismo , Carotenoides/biossíntese , Expressão Gênica , Genes Bacterianos , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Mutagênese , Oxigenases/biossíntese , Oxigenases/genética , Oxigenases/metabolismo , Paracoccus/genética , Fotossíntese , Rhodovulum/enzimologia , Sphingomonadaceae/genética , Xantofilas/biossínteseRESUMO
Limitations in obtaining sufficient specimens and difficulties in extracting high quality DNA from environmental samples have impeded understanding of the structure of microbial communities. In this study, multiple displacement amplification (MDA) using phi29 polymerase was applied to overcome these hindrances. Optimization of the reaction conditions for amplification of the bacterial genome and evaluation of the MDA product were performed using cyanobacterium Synechocystis sp. strain PCC6803. An 8-h MDA reaction yielded a sufficient quantity of DNA from an initial amount of 0.4 ng, which is equivalent to approximately 10(5) cells. Uniform amplification of genes randomly selected from the cyanobacterial genome was confirmed by real-time polymerase chain reaction. The metagenome from bacteria associated with scleractinian corals was used for whole-genome amplification using phi29 polymerase to analyse the microbial diversity. Unidentified bacteria with less than 93% identity to the closest 16S rDNA sequences deposited in DNA Data Bank of Japan were predominantly detected from the coral-associated bacterial community before and after the MDA procedures. Sequencing analysis indicated that alpha-Proteobacteria was the dominant group in Pocillopora damicornis. This study demonstrates that MDA techniques are efficient for genome wide investigation to understand the actual microbial diversity in limited bacterial samples.
Assuntos
Antozoários/microbiologia , Técnicas Bacteriológicas/métodos , DNA Polimerase Dirigida por DNA/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Ribossômico 16S/genética , Animais , Fagos Bacilares/enzimologia , Biodiversidade , Primers do DNA , Ecossistema , Biblioteca Gênica , Dados de Sequência Molecular , FilogeniaRESUMO
Siderophore activity was detected in the culture supernatant of the magnetotactic bacterium Magnetospirillum magneticum AMB-1. Here we report the first structural elucidation of a siderophore produced by a magnetotactic bacterium. The structure of the purified compound was 3,4-dihydroxybenzoic acid as determined by nuclear magnetic resonance (NMR) and electro-spray ionization mass spectroscopy (ESI-MS).
