Production of 4-Hydroxybenzoic Acid by an Aerobic Growth-Arrested Bioprocess Using Metabolically Engineered Corynebacterium glutamicum.

Corynebacterium glutamicum 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. C. glutamicum 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 C. glutamicum Specifically, multiple chromosomal integrations of a mutated aroG gene from Escherichia coli, encoding a 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthase, and wild-type aroCKB from C. glutamicum, 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 C. glutamicum by expressing a highly 4-HBA-resistant chorismate pyruvate-lyase (UbiC) from the intestinal bacterium Providencia rustigianii To enhance the yield of 4-HBA, we reduced the formation of by-products, such as 1,3-dihydroxyacetone and pyruvate, by deleting hdpA, a gene coding for a haloacid dehalogenase superfamily phosphatase, and pyk, 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.IMPORTANCE 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 C. glutamicum 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 P. rustigianii is very effective in increasing 4-HBA production.

Identification of a gene involved in plasmid structural instability in Corynebacterium glutamicum.

Expression plasmids that facilitate production of bio-based products are susceptible to toxic effects that frequently affect plasmid structural stability in recombinant microbial cells. In order to enhance plasmid stability in recombinant Corynebacterium glutamicum, an expression plasmid containing genes of the Clostridium acetobutylicum butyryl-CoA synthesis operon with high structural instability within wild-type C. glutamicum was employed. From a total of 133 mutants exhibiting disruptions in 265 suspect genes, only cgR_0322-deficient mutant was able to maintain the expression plasmid intact. The mutant exhibited normal growth under standard laboratory conditions but its transformation efficiency was about one order of magnitude lower than that of wild-type strain. The cgR_0322 gene encodes an endonuclease that is active against single- as well as double-stranded DNA substrates in the presence of Mg(2+). The cgR_0322-deficient strain should therefore facilitate the development of more robust C. glutamicum strains to be used as microbial production hosts.

Structural features and gene-expression profiles of actin homologs in Porphyra yezoensis (Rhodophyta).

The marine red alga Porphyra yezoensis contains an actin gene family consisting of at least four isoforms (PyACT1, 2, 3 and 4). The amino acid identity between isoforms exceeds 83%, and each contains a putative nuclear export signal (NES). We scanned the sequences for amino acids in regions homologous to the intermonomeric interface of actin filaments. Few residues expected to engage in cross-linking were conserved between the four isoforms. The results of the sequence analyses suggest that PyACT2 probably functions in the nucleus as a monomer (G-actin) or in other unconventional forms. In addition, the distribution and position of the introns were different from those in florideophycean actin genes. The expression level of PyACT3 in matured gametophytes was significantly higher than in those in a vegetative state, although the mRNA was detected at similar levels in both apical and basal parts of thalli. The expression levels of PyACT2 and 4, on the other hand, did not change significantly between the matured and vegetative gametophytes. The PyACT3 may serve as a molecular marker for monitoring thallus maturation in this species.

IDENTIFICATION OF GENES PREFERENTIALLY EXPRESSED DURING ASEXUAL SPORULATION IN PORPHYRA YEZOENSIS GAMETOPHYTES (BANGIALES, RHODOPHYTA)(1).

Asexual reproduction via archeospores in Porphyra yezoensis Ueda gametophytes is a very valuable character to nori farming; however, there is little information available on the molecular basis of the developmental process. To identify genes involved in the Porphyra asexual sporulation, we compared the gene expression profiles derived from four developmental stages of the life cycle (three from gametophytes; one from sporophytes) using cDNA macroarray, which includes 4,896 nonredundant expressed sequence tag (EST) groups. Candidate genes were screened by two different macroarray data analyses combined with reverse transcription-PCR (RT-PCR) analysis or Northern analysis. RT-PCR analysis revealed that nine genes (one: similarity to 5'-3' exoribonuclease; the other eight: no sequence similarity to known proteins) were expressed with a gametophyte (G)-specific manner, and two genes (named ASPO2608, ASPO1527) were expressed only in gametophytes that formed archeospores. The deduced amino acid sequences for the latter two genes are predicted to contain signal peptides for secretion at their N-termini. Northern analysis revealed that expression levels of Calvin cycle genes in the gametophytic stage that formed archeospores (G-A stage) were higher than those of the gametophyte blade with no archeospores (G-NA stage). In the macroarray analysis based on the rank data of G-preferentially expressed genes, which were detected in the previous P. yezoensis EST analysis, one gene encoding the cyclase associated protein (CAP) exhibited a change upwardly in the G-A stage >1,000 ranks to the G-NA stage. We propose that ASPO2608 and CAP may function in a signaling pathway of asexual sporulation.

IDENTIFICATION OF CROSS-FERTILIZED CONCHOCELIS USING CLEAVED AMPLIFIED POLYMORPHIC SEQUENCE MARKERS IN CROSS-EXPERIMENTS OF PORPHYRA YEZOENSIS (BANGIALES, RHODOPHYTA)(1).

As a part of the construction of a Porphyra yezoensis Ueda genetic linkage map, we conducted intraspecific cross-experiments and subsequent screening of cross-fertilized conchocelis by cleaved amplified polymorphic sequence (CAPS) analysis. The cross-experiments were carried out between males of the wildtype (KGJ) and females of the recessive green mutant (TU-2) using two methods, controlled and random crosses. A total of 42 and 186 wildtype-colored conchocelis colonies were obtained from the former and latter experiments, respectively. Among those, 49 DNA samples (14% and 23% obtained from the former and latter crosses, respectively) showed biparental CAPS patterns in the two gene regions (EF-1α open reading frame [ORF] region and V-ATPase). This study represents the first report in which the cross-fertilized conchocelis of P. yezoensis has been directly confirmed by molecular marker. The combination of the simple DNA extraction and CAPS analysis may be applicable in genetic studies of other macroalgae that are monoecious and/or grow slowly in laboratory culture.

DIURNAL CELL DIVISION REGULATED BY GATING THE G1 /S TRANSITION IN ENTEROMORPHA COMPRESSA (CHLOROPHYTA)(1).

The cell-cycle progression of Enteromorpha compressa (L.) Nees (=Ulva compressa L.) was diurnally regulated by gating the G1 /S transition. When the gate was open, the cells were able to divide if they had attained a sufficient size. However, the cells were not able to divide while the gate was closed, even if the cells had attained sufficient size. The diurnal rhythm of cell division immediately disappeared when the thalli were transferred to continuous light or darkness. When the thalli were transferred to a shifted photoperiod, the rhythm of cell division immediately and accurately synchronized with the shifted photoperiod. These data support a gating-system model regulated by light:dark (L:D) cycles rather than an endogenous circadian clock. A dark phase of 6 h or longer was essential for gate closing, and a light phase of 14 h was required to renew cell division after a dark phase of >6 h.

Inhibition of liver fibrosis in LEC rats by a carotenoid, lycopene, or a herbal medicine, Sho-saiko-to.

We assessed the prevention of hepatic fibrogenesis by a herbal medicine Sho-saiko-to or a carotenoid lycopene in Long-Evans rats with cinnamon coat color (LEC rats). LEC rats were divided into three groups: A (n=40), fed on a basal diet (BD); B (n=25), fed on BD plus 1% Sho-saiko-to; and C (n=40), fed on BD plus 0.005% lycopene. All rats were sacrificed at 76 weeks of age. The liver tissues were stained with Azan--Mallory and alpha-smooth muscle actin (alpha-SMA). The malondialdehyde (MDA) in the liver was measured for the assay of lipoperoxides. The percentage of the total area stained was determined morphometrically. The percentage of the total area involved by fibrosis was 1.35plus minus0.56 in group A, 0.72plus minus0.34 in B (P=0.0020, B vs. A) and 0.78plus minus0.75 in C (P=0.0031, C vs. A). The percentage of the total area that was stained for alpha-SMA was 0.61plus minus0.57 in group A, 0.11plus minus0.05 in B (P=0.0017, B vs. A) and 0.12plus minus0.06 in C (P=0.0021, C vs. A). In group B, MDA in the liver was lower than in group C (P=0.009). In group C, the concentration of iron in the liver was lower than in group A (P=0.0059). In conclusion, Sho-saiko-to suppressed fibrogenesis through reduced generation of lipid peroxides. Hepatic fibrogenesis was also suppressed by lycopene. The mechanisms of this preventive effect of fibrogenesis with Sho-saiko-to and lycopene were suggested to inhibit the stellate cell activity.