To identify gene candidates related to the morphological and physiological differences between the gametophytic and sporophytic generations of a marine red alga, Porphyra yezoensis Ueda, large‐scale expressed sequence tag (EST) analysis was conducted. A total of 10,625 5′‐end EST sequences was generated from the sporophyte cDNA library, in addition to the previously published 10,154 ESTs from the gametophyte. A total of 20,779 ESTs was clustered into 4496 nonredundant groups at the criterion of 95% identity for 50 bases. Among the EST groups, only 1013 (22.5%) groups were classified as ESTs that commonly occurred in both generations, whereas a large proportion of EST groups were identified as being unique to either the gametophyte (1940 EST groups = 43.1%) or the sporophyte (1543 EST groups = 34.3%). A statistical significance test revealed 89 and 112 highly expressed gene candidates in the gametophyte and the sporophyte, respectively. Twelve candidate genes were subjected to RT‐PCR analysis, and the differential expression was confirmed. Generation‐specific regulation of alternative splicing of a transcript was demonstrated by RT‐PCR analyses.

was metabolically engineered to produce 4-hydroxybenzoic acid (4-HBA), a valuable aromatic compound used as a raw material for the production of liquid crystal polymers and paraben. was found to have a higher tolerance to 4-HBA toxicity than previously reported hosts used for the production of genetically engineered 4-HBA. To obtain higher titers of 4-HBA, we employed a stepwise overexpression of all seven target genes in the shikimate pathway in Specifically, multiple chromosomal integrations of a mutated gene from, encoding a 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthase, and wild-type from, encoding chorismate synthase, shikimate kinase, and 3-dehydroquinate synthase, were effective in increasing product titers. The last step of the 4-HBA biosynthesis pathway was recreated in by expressing a highly 4-HBA-resistant chorismate pyruvate-lyase (UbiC) from the intestinal bacterium To enhance the yield of 4-HBA, we reduced the formation of by-products, such as 1,3-dihydroxyacetone and pyruvate, by deleting , a gene coding for a haloacid dehalogenase superfamily phosphatase, and, a gene coding for a pyruvate kinase, from the bacterial chromosome. The maximum concentration of 4-HBA produced by the resultant strain was 36.6 g/liter, with a yield of 41% (mol/mol) glucose after incubation for 24 h in minimal medium in an aerobic growth-arrested bioprocess using a jar fermentor. To our knowledge, this is the highest concentration of 4-HBA produced by a metabolically engineered microorganism ever reported. Since aromatic compound 4-HBA has been chemically produced from petroleum-derived phenol for a long time, eco-friendly bioproduction of 4-HBA from biomass resources is desired in order to address environmental issues. In microbial chemical production, product toxicity often causes problems, but we confirmed that wild-type has high tolerance to the target 4-HBA. A growth-arrested bioprocess using this microorganism has been successfully used for the production of various compounds, such as biofuels, organic acids, and amino acids. However, no production method has been applied for aromatic compounds to date. In this study, we screened for a novel final reaction enzyme possessing characteristics superior to those in previously employed microbial 4-HBA production. We demonstrated that the use of the highly 4-HBA-resistant UbiC from the intestinal bacterium is very effective in increasing 4-HBA production.

Manuscript 2 AbsrtactThe marine red alga Porphyra yezoensis has been proposed as a model plant for physiological and genetic studies in seaweeds because of its biological and economical importance. However, the progress of molecular biological studies using gene transfection and genetic transformation systems has been hindered by difficulties in the expression of foreign genes in P. yezoensis cells. To overcome this situation, we developed a transient gene expression system to monitor gene expression in P. yezoensis cells. An artificial -glucuronidase (GUS) coding region was synthesized to adapt it to the codon usage of P. yezoensis (PyGUS) and then evaluated for efficiency as a reporter of transient gene expression by particle bombardment. We also demonstrated the importance of using the promoter of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene from P. yezoensis for efficient expression of PyGUS, because the cauliflower mosaic virus (CaMV) 35S promoter, which has been successfully used for monitoring gene expression in nuclei and chloroplasts of higher plants, was less active in P. yezoensis cells. Therefore, the lack of knowledge about differences in the regulatory machinery of gene expression between P. yezoensis and terrestrial plants seems to be why experimental systems for monitoring gene expression were previously not developed in P. yezoensis. Establishment of the transient gene expression system in P. yezoensis could facilitate biotechnological developments in this organism.3

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We investigated genetic diversity in 10 strains of Porphyra yezoensis and related species, collected from Japan and Korea, in order to identify an appropriate crossing partner for use in various genetic analyses. Firstly, the phylogenetic relationships among the 10 strains were examined using SSU rDNA sequences. All five Japanese strains (TU-1, TU-2, TUH-25, JHS and JHU) and two of the Korean strains (KGJ and KPH) were identified as P. yezoensis while the other three Korean strains (KTY1, KTY2 and KTY3) were identified as P. tenera. Using cleaved amplified polymorphic sequence analysis, genetic polymorphisms lying in five regions of four genes (ß-tubulin, TOP2, EF-1 and V-ATPase) were examined. Twenty out of 34 restriction endonucleases revealed genetic polymorphisms between the seven strains of P. yezoensis and the three strains of P. tenera in all five regions tested. Furthermore, when they were employed to digest the fragments of TOP2 and V-ATPase, 14 enzymes discriminated the two Korean strains of P. yezoensis (KGJ and KPH) from the five Japanese strains of this species. The level of polymorphism was much higher in the amplified fragment of V-ATPase (including four introns) than in that of TOP2 without an intron. The gene regions with detected polymorphisms would be useful molecular markers for confirmation of cross-fertilization in sporophytic thalli and for construction of a linkage map. A suitable combination of the strains in the cross experiment is discussed.