Characterization of mutants expressing thermostable D1 and D2 polypeptides of photosystem II in the cyanobacterium Synechococcus elongatus PCC 7942.

Photosystem II complex embedded in thylakoid membrane performs oxygenic photosynthesis where the reaction center D1/D2 heterodimer accommodates all components of the electron transport chain. To express thermostable D1/D2 heterodimer in a cyanobacterium Synechococcus elongatus PCC 7942, we constructed a series of mutant strains whose psbA1 and psbD1 genes encoding, respectively, the most highly expressed D1 and D2 polypeptides were replaced with those of a thermophilic strain, Thermosynechococcus vulcanus. Because the C-terminal 16 amino acid sequences of D1 polypeptides should be processed prior to maturation but diverge from each other, we also constructed the psbA1ΔC-replaced strain expressing a thermostable D1 polypeptide devoid of the C-terminal extension.

Potent L-lactic acid assimilation of the fermentative and heterothallic haploid yeast Saccharomyces cerevisiae NAM34-4C.

We screened an industrial thermotolerant Saccharomyces cerevisiae strain, KF7, as a potent lactic-acid-assimilating yeast. Heterothallic haploid strains KF7-5C and KF7-4B were obtained from the tetrads of the homothallic yeast strain KF7. The inefficient sporulation and poor spore viability of the haploid strains were improved by two strategies.

Competitive advantage and tolerance of selected shochu yeast in barley shochu mash.

A shochu yeast strain, Saccharomyces cerevisiae BAW-6, was previously isolated from Kagoshima yeast strain Ko, and has since been utilized in shochu production. The BAW-6 strain carries pho3/pho3 homozygous genes in contrast to the heterozygous PHO3/pho3 genes in the parental Ko strain. However, absence of the PHO3 gene per se cannot explain the fermentation superiority of BAW-6.

Ethanol production from D-lactic acid by lactic acid-assimilating Saccharomyces cerevisiae NAM34-4C.

The lactic acid-assimilating yeast Saccharomyces cerevisiae NAM34-4C grew rapidly in minimal D-lactate medium (pH 3.5) at 35°C, compared with minimal L-lactate medium. A laboratory strain, S.

Genetic instability of constitutive acid phosphatase in shochu and sake yeast.

Genetic instability of constitutive acid phosphatase (cAPase) activity was observed in a shochu brewer's yeast strain (Ko), which consistently produced 0.3-1% progeny without cAPase when it had been subcultured for a long period of time in barley shochu mash or in conventional complete medium. Genetic analysis showed that the cAPase-negative phenotype was associated with a single mutation in the PHO3 gene and that the Ko strain had heteroallelic PHO3/pho3 genes, while the PHO3⁻ mutants had the homoallelic pho3/pho3 defect.

Functional replacement of yeast flavocytochrome b(2) with bacterial L-lactate dehydrogenase.

Using yeast genetic complementation, we show here that Rhodobacter sphaeroidesl-lactate dehydrogenase can functionally replace flavocytochrome b(2) in Saccharomyces cerevisiae, only when a matrix-targeting signal of flavocytochrome b(2) is fused with the bacterial enzyme. The recombinant l-lactate dehydrogenase may add alternative route of mitochondrial electron transport other than flavocytochrome b(2).

Derivation of consensus sequence for protein binding site in Yarrowia lipolytica centromere.

The transmission of replicative plasmids in the yeast Yarrowia lipolytica is mediated solely by a part of its centromere DNA, in which an essential protein binding site has been analyzed recently. Here, we extended the analysis to other minimized centromeric regions, revealing a consensus sequence of a 17- to 21-bp imperfect palindrome.

Dissection of centromeric DNA from yeast Yarrowia lipolytica and identification of protein-binding site required for plasmid transmission.

In a dimorphic yeast, Yarrowia lipolytica, replicative plasmids can be established only in the coexistence of the replication origin (ORI) and centromere (CEN) from its own chromosomal DNA. Although six CEN sequences so far isolated from this yeast exhibit no similarity with conventional CEN DNA elements from other budding yeasts, they are confined within short regions (approximately 0.2 kb) and contain various conserved sequence blocks.

Construction and analysis of a recombinant cyanobacterium expressing a chromosomally inserted gene for an ethylene-forming enzyme at the psbAI locus.

The coding sequence of a gene for a Pseudomonas syringae ethylene-forming enzyme was inserted at the psbAI locus in a cyanobacterium, Synechococcus elongatus PCC 7942 via rps12-mediated gene replacement. The recombinant strain photoautotrophically produced ethylene at 451 nl ml(-1) h(-1) OD730(-1), but showed a depressed specific growth rate as well as a yellow-green phenotype indicating a severe metabolic stress. The rate of ethylene production in the recombinant culture decreased as a result of competition with faster growing ethylene-non-forming mutants that carried short nucleotide insertions within the coding sequence of the gene for the ethylene-forming enzyme.

Establishment of a pure culture of the hitherto uncultured unicellular cyanobacterium Aphanothece sacrum, and phylogenetic position of the organism.

Aphanothece sacrum, an edible freshwater unicellular cyanobacterium, was isolated by using novel synthetic media (designated AST and AST-5xNP). The media were designed on the basis of the ratio of inorganic elements contained in A. sacrum cells cultured in a natural pond.

High-frequency gene replacement in cyanobacteria using a heterologous rps12 gene.

Multiple targeted gene replacements are often required for functional analyses of cyanobacterial genomes. For this purpose, we previously devised a simple genetic method, termed rps12-mediated gene replacement, in a cyanobacterium Synechococcus elongatus PCC 7942 for construction of mutants free from drug resistance markers. Here, we improved the method by employing a heterologous rps12 gene encoding a ribosomal protein S12 from Synechocystis sp.

Gene replacement in cyanobacteria mediated by a dominant streptomycin-sensitive rps12 gene that allows selection of mutants free from drug resistance markers.

Chromosomal gene replacement in cyanobacteria often relies upon the availability of drug resistance markers, and thus multiple replacements have been restricted. Here, a versatile gene replacement system without this restriction is reported in a unicellular cyanobacterium, Synechococcus sp. PCC 7942.