Biphenyl degradation by recombinant photosynthetic cyanobacterium Synechocystis sp. PCC6803 in an oligotrophic environment using unphysiological electron transfer.

Cyanobacteria are potentially useful photosynthetic microorganisms for bioremediation under oligotrophic environments. Here, the biphenyl degradation pathway genes of ß-proteobacterium Acidovorax sp. strain KKS102 were co-expressed in cyanobacterium Synechocystis sp. PCC6803 cells under control of the photo-inducible psbE promoter. In the KKS102 cells, biphenyl is dioxygenated by bphA1 and bphA2 gene products complex using electrons supplied from NADH via bphA4 and bphA3 gene products (BphA4 and BphA3, respectively), and converted to benzoic acid by bphB, bphC and bphD gene products. Unexpectedly, biphenyl was effectively hydroxylated in oligotrophic BG11 medium by co-expressing the bphA3, bphA1 and bphA2 genes without the bphA4 gene, suggesting that endogenous cyanobacteria-derived protein(s) can supply electrons to reduce BphA3 at the start of the biphenyl degradation pathway. Furthermore, biphenyl was converted to benzoic acid by cyanobacterial cells co-expressing bphA3, bphA1, bphA2, bphB, bphC and bphD. Structural gene-screening using recombinant Escherichia coli cells co-expressing bphA3, bphA1, bphA2, bphB and bphC suggested that petH, which encodes long- and short-type NADP-ferredoxin oxidoreductase isomers (FNRL and FNRS, respectively), and slr0600, which is annotated as an NADPH-thioredoxin reductase gene in CyanoBase, were BphA3-reducible proteins. Purified FNRL and FNRS, and the slr0600 gene product showed BphA3 reductase activity dependent on NADPH and the reduced form of glutathione, respectively, potentially shedding light on the physiological roles of the slr0600 gene product in cyanobacterial cells. Collectively, our results demonstrate the utility of Synechocystis sp. PCC6803 cells as a host for bioremediation of biphenyl compounds under oligotrophic environments without an organic carbon source.

Synthesis and characterization of bent fluorine-containing donor-π-acceptor molecules as intense luminophores with large Stokes shifts.

Herein, we prepared novel bent fluorine-containing donor-π-acceptor (D-π-A) molecules from commercially available octafluorocyclopentene using a facile two-step procedure, revealing that the above molecules absorb UV-light and exhibit yellow photoluminescence (PL) with high PL efficiencies (ΦPL) in solution. The corresponding Stokes shifts exceeded 10 000 cm-1, and the maximum PL wavelength (λPL) strongly depended on solvent polarity or intermolecular interactions in the solid state. On the basis of a Lippert-Mataga plot, PL was confidently assigned to radiative relaxation from an intramolecular charge-transfer excited state. Moreover, the synthesized luminophores showed intense PL even in the crystalline state and exhibited alkoxy chain length-dependent PL behavior (e.g., high ΦPL, λPL = 486-540 nm).

Complete Genome Sequence of a Microcystin-Degrading Bacterium, Sphingosinicella microcystinivorans Strain B-9.

Sphingosinicella microcystinivorans strain B-9 has the ability to degrade cyanobacterial hepatotoxic cyclic peptides, microcystins, and nodularins. This is the first report of the complete genome sequence of the microcystin-degrading bacterium.

Actinophage R4 integrase-based site-specific chromosomal integration of non-replicative closed circular DNA.

The actinophage R4 integrase (Sre)-based molecular genetic engineering system was developed for the chromosomal integration of multiple genes in Escherichia coli. A cloned DNA fragment containing two attP sites, green fluorescent protein (gfp) as a first transgene, and an antibiotic resistance gene as a selection marker was self-ligated to generate non-replicative closed circular DNA (nrccDNA) for integration. nrccDNA was introduced into attB-inserted E. coli cells harboring the plasmid expressing Sre by electroporation. The expressed Sre catalyzed site-specific integration between one of the two attP sites on nrccDNA and the attB site on the E. coli chromosome. The integration frequency was affected by the chromosomal location of the target site. A second nrccDNA containing two attB sites, lacZα encoding the alpha fragment of ß-galactosidase as a transgene, and another antibiotic resistance gene was integrated into the residual attP site on the gfp-integrated E. coli chromosome via one of the two attB sites according to reiterating site-specific recombination. The integrants clearly exhibited ß-galactosidase activity and green fluorescence, suggesting the simultaneous expression of multiple recombinant proteins in E. coli. The results of the present study showed that a step-by-step integration procedure using nrccDNA achieved the chromosomal integration of multiple genes.

Complete pyridine-nucleotide-specific conversion of an NADH-dependent ferredoxin reductase.

The coenzyme specificity of enzymes is one of the critical parameters for the engineered production of biological compounds using bacteria. Since NADPH is produced abundantly in photosynthetic organisms, conversion of an NADH-specific enzyme into an NADPH-specific one is a useful approach for the efficient carbon-neutral production of biological compounds in photosynthetic organisms. In the present study, an NADH-specific ferredoxin reductase component, BphA4 of biphenyl dioxygenase BphA from Acidovorax sp. strain KKS102, was changed to an NADPH-dependent form using a method combining structure-based systematic mutations and site-directed random mutagenesis. The resultant CRG mutant, in which Glu175-Thr176-Gln177 of an NADH-recognition loop in the wild-type BphA4 was replaced with Cys175-Arg176-Gly177, was highly specific and active for NADPH, and its biochemical and structural properties for NADPH were nearly the same as those of the wild-type BphA4 for NADH. In addition, this mutation project was assessed by a semi-empirical prediction method of mutation effects, and the results suggested that the CRG mutant was one of the best NADPH-specific mutants.

Construction of a stepwise gene integration system by transient expression of actinophage R4 integrase in cyanobacterium Synechocystis sp. PCC 6803.

The integrase of actinophage R4, which belongs to the large serine-recombinase family, catalyzes site-specific recombination between two distinct attachment site sequences of the phage (attP) and actinomycete Streptomyces parvulus 2297 chromosome (attB). We previously reported that R4 integrase (Sre) catalyzed site-specific recombination both in vivo and in vitro. In the present study, a Sre-based system was developed for the stepwise site-specific integration of multiple genes into the chromosome of cyanobacterium Synechocystis sp. PCC 6803 (hereafter PCC 6803). A transgene-integrated plasmid with two attP sites and a non-replicative sre-containing plasmid were co-introduced into attB-inserted PCC 6803 cells. The transiently expressed Sre catalyzed highly efficient site-specific integration between one of the two attP sites on the integration plasmid and the attB site on the chromosome of PCC 6803. A second transgene-integrated plasmid with an attB site was integrated into the residual attP site on the chromosome by repeating site-specific recombination. The transformation frequencies (%) of the first and second integrations were approximately 5.1 × 10(-5) and 8.2 × 10(-5), respectively. Furthermore, the expression of two transgenes was detected. This study is the first to apply the multiple gene site-specific integration system based on R4 integrase to cyanobacteria.

Volatile organic compounds derived from 2-keto-acid decarboxylase in Microcystis aeruginosa.

Volatile organic compounds (VOCs), 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol, were detected together with ß-cyclocitral from the cyanobacterium Microcystis aeruginosa NIES-843. These alcohols were optimally produced after 35 d of culture, during which nitrate nitrogen in the cultured broth became exhausted. Additionally, these alcohols were definitely produced using the 2-keto-acid decarboxylase (MaKDC) in Microcystis strains. These results suggested that these VOCs from Microcystis are significant for their lifecycle, because these compounds are not produced by any other genus of cyanobacteria. This is the first report of 2-keto-acid decarboxylase producing 3-methyl-1-butanol and 2-phenylethanol by an oxygenic photosynthetic microorganism.

Characterization of the locus of genes encoding enzymes producing heptadepsipeptide micropeptin in the unicellular cyanobacterium Microcystis.

The gene cluster involved in producing the cyclic heptadepsipeptide micropeptin was cloned from the genome of the unicellular cyanobacterium Microcystis aeruginosa K-139. Sequencing revealed four genes encoding non-ribosomal peptide synthetases (NRPSs) that are highly similar to the gene cluster involved in cyanopeptolins biosynthesis. According to predictions based on the non-ribosomal consensus code, the order of the mcnABCE NPRS modules was well consistent with that of the biosynthetic assembly of cyclic peptides. The biochemical analysis of a McnB(K-139) adenylation domain and the knock-out of mcnC in a micropeptin-producing strain, M. viridis S-70, revealed that the mcn gene clusters were responsible for the production of heptadepsipeptide micropeptins. A detailed comparison of nucleotide sequences also showed that the regions between the mcnC and mcnE genes of M. aeruginosa K-139 retained short stretches of DNA homologous to halogenase genes involved in the synthesis of halogenated cyclic peptides of the cyanopeptolin class including anabaenopeptilides. This suggests that the mcn clusters of M. aeruginosa K-139 have lost the halogenase genes during evolution. Finally, a comparative bioinformatics analysis of the congenial gene cluster for depsipetide biosynthesis suggested the diversification and propagation of the NRPS genes in cyanobacteria.

Ribosome-binding site interference caused by Shine-Dalgarno-like nucleotide sequences in Escherichia coli cells.

Two-cistronic expression plasmids are useful for high-level expression of heterologous genes in Escherichia coli cells by preventing the inhibition of translational initiation. In the process of constructing a two-cistronic expression plasmid pCbSTCR-4 containing the fragments of the porcine cytochrome b(5) (Psb5) and NADPH-cytochrome P450 reductase (PsCPR) genes as the first and second cistrons, respectively, the presence of a specific region in the first cistron that lowered the accumulation level of the PsCPR was suggested [Kimura, S., et al. (2005) J. Biochem. 137, 523-533]. In this study, a disturbing nucleotide sequence similar to a Shine-Dalgarno (SD) sequence (SD-like sequence), AGGAG, was identified at the 5'-upstream region near the SD sequence for the second cistron. Silent mutations in the SD-like sequence that lowered the similarity to a typical SD sequence increased the accumulation level of PsCPR. SD-like sequences introduced into mono-cistronic expression plasmids for the Psb5 and PsCPR genes also decreased the accumulation level of these proteins. The SD-like sequence also decreased the accumulation level of the insoluble PsCPR protein. This type of ribosome-binding site interference is useful not only for precise control of protein accumulation but also for increasing the soluble form of recombinant proteins in E. coli cells.

Genetic analysis of the microcystin biosynthesis gene cluster in Microcystis strains from four bodies of eutrophic water in Japan.

The highly conserved organization of microcystin biosynthesis (mcy) gene clusters, which includes nonribosomal peptide synthetase (NRPS) genes, polyketide synthase (PKS) genes, and fused NRPS-PKS genes, has been characterized in the genus Microcystis. In this study, a total of 135 cyanobacterial strains from four different geographical locations in Japan were isolated. Fourteen mcy-possessing (mcy+) strains were identified according to PCR amplification between two genes from domestic mcy+ strains and the mcy gene's organization was classified into five types. Phylogenetic relationships of the 16S-23S internal transcribed spacer region indicated that the five types of mcy gene cluster structure classified into two groups of the genus Microcystis. HPLC of the isolated mcy+ strain containing a partial deletion of mcyI (DeltamcyI) revealed that microcystin production disappeared. A transcriptional analysis of the Delta mcyI-strain and an assay of recombinant McyI dehydrogenase activity showed that McyI is responsible for microcystin biosynthesis. Based on patterns of the PCR amplicons and analyses of nucleotide sequences in the mcy gene cluster of Microcystis, we confirmed the presence of inserts at three specific loci, between mcyA and mcyD, and downstream of mcyC and mcyJ. Our study is the first investigation of the mcy gene cluster structure in the genus Microcystis from environmental samples.

Cloning and characterization of a new hetero-gene cluster of nonribosomal peptide synthetase and polyketide synthase from the cyanobacterium Microcystis aeruginosa K-139.

Two nonribosomal peptide synthetase genes responsible for the biosynthesis of microcystin and micropeptin in Microcystis aeruginosa K-139 have been identified. A new nonribosomal peptide synthetase gene, psm3, was identified in M. aeruginosa K-139. The gene is a cluster extending 30 kb and comprising 13 bidirectionally transcribed open reading frames arranged in two putative operons. psm3 encodes four adenylation proteins, one polyketide synthase, and several unique proteins, especially Psm3L consisting of halogenase, acyl-CoA binding protein-like protein, and acyl carrier protein. Alignment of the binding pocket of the adenylation domain and an ATP-PPi exchange analysis using a recombinant protein with the adenylation domain of Psm3B showed that Psm3G and Psm3B activate aspartic acid and tyrosine, respectively. Although disruption of psm3 did not reveal the product produced by Psm3, we identified microviridin B and aeruginosin K139 in the cells of M. aeruginosa K-139. The above-mentioned results indicated that M. aeruginosa possesses at least five nonribosomal peptide synthetase gene clusters.