Sadazo Yoshino

High butanol production by Clostridium saccharoperbutylacetonicum N1-4 in fed-batch culture with pH-Stat continuous butyric acid and glucose feeding method.

A pH-stat fed-batch culture by feeding butyric acid and glucose has been studied in an acetone-butanol-ethanol (ABE) fermentation using Clostridium saccharoperbutylacetonicum N1-4. The specific butanol production rate increased from 0.10 g-butanol/g-cells/h with no feeding of butyric acid to 0.42 g-butanol/g-cells/h with 5.0 g/l butyric acid. The pH value in broth decreases with butyric acid production during acidogenesis, and then butyric acid reutilization and butanol production result in a pH increase during solventogensis. The pH-stat fed-batch culture was performed to maintain a constant pH and butyric acid concentration in the culture broth, but feeding only butyric acid could not support butyric acid utilization and butanol production. Subsequently, when a mixture of butyric acid and glucose was fed, butyric acid was utilized and butanol was produced. To investigate the effect of the feeding ratio of butyric acid to glucose (B/G ratio), several B/G ratio solutions were fed. The maximum butanol production was 16 g/l and the residual glucose concentration in broth was very low at a B/G ratio of 1.4. Moreover, yields of butanol in relation to cell mass and glucose utilization were 54% and 72% higher in pH-stat fed-batch culture with butyric acid than that of conventional batch culture, respectively.

Extractive acetone-butanol-ethanol fermentation using methylated crude palm oil as extractant in batch culture of Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564)

The possibility of employing methylated crude palm oil (CPOE) as an extraction solvent to reduce end-product inhibition and to enhance solvent productivity in acetone-butanol-ethanol (ABE) fermentation was evaluated using oleyl alcohol as the standard butanol extractant. Fermentation was carried out at an initial glucose concentration of 90 g/l. CPOE did not inhibit the growth of the fermentative organism. Without solvent extraction, butanol production ceased after 30 h at a concentration of 15.4 g/l limiting cell growth to 3.98 g/l and glucose consumption to 62%. Applying CPOE as the extraction solvent, about 47% of the total butanol produced was extracted, glucose consumption was increased to 83% and relatively high glucose consumption rates and solvent productivities were obtained. Butanol production increased to 20.9 g/l; total ABE solvents and yield also increased from 21.2 g/l and 38% (in conventional fermentation) to 29.8 g/l and 40.4%, respectively.

Characterization of the sol Operon in Butanol-Hyperproducing Clostridium saccharoperbutylacetonicum Strain N1-4 and Its Degeneration Mechanism

The solventogenic sol operon consisting of bld, ctfA, ctfB, and adc was cloned from Clostridium saccharoperbutylacetonicum strain N1-4. These genes share as high as 95–98% similarity with the corresponding sol genes of Clostridium beijerinckii NCIMB 8052. The N1-4 sol gene cluster was transcribed in a polycistronic manner under the control of two promoters, and its transcription was highly induced during solventogenesis. Strain DGN3-4, the degenerated strain derived from N1-4, maintained the sol genes, but transcription of the DGN3-4 sol operon was hardly induced during solventogenesis. A substance extracted from the culture supernatants of wild-type N1-4 allowed us to induce transcription of the sol operon in DGN3-4. These results suggest that the degeneration is caused by the incompetence of the induction mechanism of the sol operon, and that transcription might be under the control of a quorum-sensing mechanism.

Production of acetone, butanol and ethanol from palm oil waste by Clostridium saccharoperbutylacetonicum N1-4

SummaryA study of the feasibility of utilizing palm oil waste as a substrate for acetonebutanol-ethanol fermentation by Clostridium saccharoperbutylacetonicum N1-4 and, hence, to reduce the biochemical oxygen demand (BOD) of waste disposal was carried out. Among the two types of palm oil waste tested, separator sludge was the better substrate and it was able to support production of solvents by C. saccharoperbutylacetonicum N1-4 without a need for any mineral supplements. Enzymatic hydrolysis catalyzed by cellulase prior to fermentation was found to increase the yield of butanol by 75% (from 2.47g/l to 4.37g/l) and decreased the BOD by 66% (from 26,500 ppm to 8,900 ppm).

Functional analysis of HO gene in delayed homothallism in Saccharomyces cerevisiae wy2

Saccharomyces cerevisiae wy2 exhibits a novel life cycle, with delayed homothallism caused by a defective HO gene. In this strain, gradual diploidization occurs during successive subcultures. Three amino acids of wy2 HO were different from those of wild‐type (wt) HO, which included a nonsense mutation (TAG) from Trp‐292 and two amino acid changes of His‐475 to Leu and Glu‐530 to Lys. The ho gene of heterothallic strain CG379 was also sequenced in this study. Four amino acids of ho were different from those of HO. Among different amino acids in wy2 HO and ho, the alteration of His‐475 to Leu was common between them. His‐475 in HO was previously suggested to be involved in the DNA binding. We constructed a variety of chimeric HO genes by exchanging the corresponding restriction fragments generated from the wt HO, wy2 HO and ho genes. These results and the site‐directed mutagenesis studies allowed us to draw the following conclusions: (a) Gly‐223 is essential for HO activity; (b) mutation of His‐475 to Leu significantly reduces the HO activity; (c) amber mutation (TAG) in wy2 HO can be suppressed inefficiently. Copyright

Engineering of Polyploid Saccharomyces cerevisiae for Secretion of Large Amounts of Fungal Glucoamylase

ABSTRACT We engineered Saccharomyces cerevisiae cells that produce large amounts of fungal glucoamylase (GAI) from Aspergillus awamori var. kawachi. To do this, we used the δ-sequence-mediated integration vector system and the heat-induced endomitotic diploidization method. δ-Sequence-mediated integration is known to occur mainly in a particular chromosome, and the copy number of the integration is variable. In order to construct transformants carrying the GAI gene on several chromosomes, haploid cells carrying the GAI gene on different chromosomes were crossed with each other. The cells were then allowed to form spores, which was followed by dissection. Haploid cells containing GAI genes on multiple chromosomes were obtained in this way. One such haploid cell contained the GAI gene on five chromosomes and exhibited the highest GAI activity (5.93 U/ml), which was about sixfold higher than the activity of a cell containing one gene on a single chromosome. Furthermore, we performed heat-induced endomitotic diploidization for haploid transformants to obtain polyploid mater cells carrying multiple GAI genes. The copy number of the GAI gene increased in proportion to the ploidy level, and larger amounts of GAI were secreted.

Effects of UV dose on formation of spontaneously developing pocks in Streptomyces azureus ATCC14921.

Spontaneously developing pocks (S pocks) of Streptomyces azureus ATCC14921 were formed by the both functions of conjugative plasmid pSA1 and lysogenic phage SAt2. The formation was affected by the dose of UV irradiation. The mean pock diameter in cultures treated with UV light at 0, 7.1, 14.2 and 21.3×102 μW·erg/cm, respectively, were 1.3, 0.4, 2.2, and 0.5 mm. The dose affected conjugative plasmid pSA1 related to pock formation. There was UV damage of autonomous pSA1 replicon and UV induction of the chromosomal integrated sequence. Increases and decreases in the amount of autonomous pSA1 replicon corresponded to increases and decreases, respectively, in the diameter of the pocks. Both pSA1 and SAt2 syntheses were developed in the large pocks (1.3 and 2.2 mm), but only SAt2 synthesis was developed in the pinhole pocks (0.4 and 0.5 mm).

Cytoplasmic expression of the Aspergillus glucoamylase gene integrated into a linear plasmid in Saccharomyces cerevisiae

Abstract The glaA gene encoding glucoamylase I (GAI) of Aspergillus awamori var. kawachi was successfully expressed in cytoplasm of Saccharomyces cerevisiae , using two cytoplasmic linear plasmids, pGKL1 and pGKL2. In order to integrate the glaA gene, two pGKL1-derived plasmids, designated pKTF951 and pKTF952, were constructed. The glaA gene was placed downstream of the ORF2 promoter (UCS2) between ORF1 and ORF3 of pGKL1 to obtain pKTF951. pKTF952 contained an additional ORF5 promoter (UCS5) from pGKL2, connected downstream of the UCS2 in pKTF951. pKTF951 and pKTF952 were respectively transformed to S. cerevisiae carrying the original pGKL1 and pGKL2. Southern analysis revealed that in vivo homologous recombination took between the indigenous pGKL1 and the recombinant pKTF951 or pKTF952 within the common ORF1 and ORF3 regions, which resulted in the replacement of ORF2 by the glaA gene in pGKL1. Transformants carrying the resultant linear recombinant plasmids, pNS951 or pNS952, produced GAI. S. cerevisiae (pNS952) secreted 2.6-fold more GAI than S. cerevisiae (pNS951).

Distribution of bph Genes Encoding Biphenyl and Polychlorinated Biphenyl Degrading Enzymes in Soil Bacteria

Sixteen biphenyl and polychlorinated biphenyl (PCB)-degrading strains were isolated from soil at various locations and characterized. All the isolates were gram negative bacteria that include eleven species of Pseudomonas, one Achromobacter, one belonging to the CDC Group VE, Biotype I, while three strains were unidentified. Most of these strains grew on basal salts media supplemented with a wide variety of aromatic compounds, which include substituted biphenyls and biphenyl related compounds. Southern blot analysis using two previously cloned bph genes as probes revealed that four (three Pseudomonas, one unidentified) out of the sixteen strains showed significant hybridization with the well studied bph genes of Pseudomonas pseudoalcaligenes KF707. The immunological cross reactivity of the 2,3dihydroxybiphenyl dioxygenases and hydrolases from the four strains corresponded well to the DNA homology. The existence of almost identical bph genes in different soil bacteria indicates that certain chromosomal bph operons have a mechanism of transfer within bacterial populations in the environment. However, the twelve other biphenyl utilizing isolates did not show any significant hybridization with the KF707 bph probe, while all of the sixteen strains did not hybridize with the Moraxella sp. KF704 bph probe. These results suggest the diversity of PCB-degrading genotypes.

Clostridium saccharoperbutylacetonicum のautolysinのアニオン交換体に対する特異的挙動

The autolysin (N-acetylmuramidase) of Clostridium saccharoperbutylaceto nicum (ATCC 13564) strongly adsorbed to anion exchangers such as diethylaminoethyl (DEAE)-cellulose, triethylaminoethyl (TEAE)-cellulose and ECTEOLA-cellulose. Therefore, the adsorbed autolysin was not eluted with buffer containing various concentrations of NaCl or buffer of various pHs. However it could be eluted from them with the buffer containing high concentration of guanidine·HCl, dioxane and ethylene glycol, which were the reagents used for break of hydrogen bonding of proteins or weakening the hydrophobic interaction of proteins. These results indicated that the hydrophobic interaction would be participate in this strong adsorption of autolysin on the anion exchangers. The autolysin could not adsorb to carriers of the anion exchangers (cellulose, agarose and Sephadex), while it adsorbed to aminoalkyl-Sepharose and alkyl-Sepharose prepared. The adsorption rate of autolysin toward those resins increased in proportion of the elongating length of alkyl chain Therefore, its adsorption to the anion exchangers was thought to be caused by affinity for hydrophorbic residues, namely alkyl residues of di-(or tri-)ethylaminoethyl groups of them. These properties of autolysin were applied on its purification, and the specific activity of DEAE-cellulose eluents by guanidine·HCl increased in 20 times more than that of gel filtrated fraction.