Tatsuji Seki

Asaia bogorensis gen. nov., sp. nov., an unusual acetic acid bacterium in the alpha-Proteobacteria.

Eight Gram-negative, aerobic, rod-shaped and peritrichously flagellated strains were isolated from flowers of the orchid tree (Bauhinia purpurea) and of plumbago (Plumbago auriculata), and from fermented glutinous rice, all collected in Indonesia. The enrichment culture approach for acetic acid bacteria was employed, involving use of sorbitol medium at pH 3.5. All isolates grew well at pH 3.0 and 30 degrees C. They did not oxidize ethanol to acetic acid except for one strain that oxidized ethanol weakly, and 0.35% acetic acid inhibited their growth completely. However, they oxidized acetate and lactate to carbon dioxide and water. The isolates grew well on mannitol agar and on glutamate agar, and assimilated ammonium sulfate for growth on vitamin-free glucose medium. The isolates produced acid from D-glucose, D-fructose, L-sorbose, dulcitol and glycerol. The quinone system was Q-10. DNA base composition ranged from 59.3 to 61.0 mol% G + C. Studies of DNA relatedness showed that the isolates constitute a single species. Phylogenetic analysis based on their 16S rRNA gene sequences indicated that the isolates are located in the acetic acid bacteria lineage, but distant from the genera Acetobacter, Gluconobacter, Acidomonas and Gluconacetobacter. On the basis of the above characteristics, the name Asaia bogorensis gen. nov., sp. nov. is proposed for these isolates. The type strain is isolate 71T (= NRIC 0311T = JCM 10569T).

Construction of xylose-assimilating Saccharomyces cerevisiae

Abstract The xylose reductase gene originating from Pichia stipitis was subcloned on an expression vector with the enolase promoter and terminator from Saccharomyces cerevisiae . The transformants of S. cerevisiae harboring the resultant plasmids produced xylose reductase constitutively at a rate about 3 times higher than P. stipitis , but could not assimilate xylose due to the deficient conversion of xylitol to xylulose. The xylitol dehydrogenase gene was also isolated from the gene library of P. stipitis by plaque hybridization using a probe specific for its N-terminal amino acid sequence. The gene transferred into S. cerevisiae was well expressed. Furthermore, high expressions of the xylose reductase and xylitol dehydrogenase genes in S. cerevisiae were achieved by introducing both genes on the same or coexisting plasmids. The transformants could grow on a medium containing xylose as the sole carbon source, but ethanol production from xylose was less than that by P. stipitis and a significant amount of xylitol was excreted into the culture broth.

Deoxyribonucleic Acid Homology and Taxonomy of the Genus Bacillus

The taxonomic relationships among 56 strains of 16 species of the genus Bacillus were studied by deoxyribonucleic acid (DNA)-DNA hybridization. In general, no significant DNA homology was detected between two strains of different species, except for a group of species consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, and B. pumilus and for another group of species including B. cereus and B. thuringiensis. Species of the former group were related, but they were independent of each other as their DNA homologies were 19% or less. The DNA homology indexes of three strains of B. thuringiensis to B. cereus T, so far tested, showed high DNA homologies (54 to 80%). This fact indicates that B. thuringiensis should be an identical species to B. cereus. The intraspecific DNA homology indexes of 16 strains of B. pumilus were 51% or more to strain IFO 12092 as the standard, and those of 10 strains of B. coagulans were 76 to 113% to strain ATCC 7050 as the standard. Thus, the species identification of B. pumilus and B. coagulans by the conventional taxonomic method was well in accord with the DNA homology data. On the other hand, significant heterogeneities were suggested among the strains of B. circulans and among those of B. sphaericus by the DNA homology data of the three and five strains so far tested, respectively. Although B. lentus was described to be closely related to B. firmus in Bergeys Manual (8th ed., 1974), the interspecific DNA homology index between these two species was 3%. It was concluded that these two species are independent.

Asaia siamensis sp. nov., an acetic acid bacterium in the alpha-proteobacteria.

Five bacterial strains were isolated from tropical flowers collected in Thailand and Indonesia by the enrichment culture approach for acetic acid bacteria. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates were located within the cluster of the genus Asaia. The isolates constituted a group separate from Asaia bogorensis on the basis of DNA relatedness values. Their DNA G+C contents were 58.6-59.7 mol%, with a range of 1.1 mol%, which were slightly lower than that of A. bogorensis (59.3-61.0 mol%), the type species of the genus Asaia. The isolates had morphological, physiological and biochemical characteristics similar to A. bogorensis strains, but the isolates did not produce acid from dulcitol. On the basis of the results obtained, the name Asaia siamensis sp. nov. is proposed for these isolates. Strain S60-1T, isolated from a flower of crown flower (dok rak, Calotropis gigantea) collected in Bangkok, Thailand, was designated the type strain ( = NRIC 0323T = JCM 10715T = IFO 16457T).

Isolation of xylose reductase gene of Pichia stipitis and its expression in Saccharomyces cerevisiae.

A NADPH/NADH-dependent xylose reductase gene was isolated from the xylose-assimilating yeast,Pichia stipitis. DNA sequence analysis showed that the gene consists of 951 bp. The gene introduced inSaccharomyces cerevisiae was transcribed to mRNA, and a considerable amount of enzyme activity was observed constitutively, whereas transcription and translation inP stipitis were inducible.S. cerevisiae carrying the xylose reductase gene could not, however, grow on xylose medium, and could not produce ethanol from xylose. Since xylose uptake and accumulation of xylitol byS. cerevisiae were observed, the conversion of xylitol to xylulose seemed to be limited.

Physiologically motivated strategies for control of the fed-batch cultivation of recombinant Escherichia coli for phenylalanine production

Abstract The efficiency of the fed-batch cultivation of recombinant Escherichia coli AT2471 for phenylalanine production is highly dependent on the distribution of the carbon flow between the main process products — biomass, phenylalanine, acetic acid and carbon dioxide. In order to enhance the process performance, the effects of several factors, namely glucose feeding, tyrosine feeding and oxygen supply, were investigated experimentally. As a result, a set of control strategies was developed, designed to tolerate phenylalanine synthesis at the expense of the remaining products. The DO was controlled to prevent acetic acid excretion due to oxygen limitation. The total amount of tyrosine fed was used to provide an optimal balance between biomass synthesis and that of phenylalanine. Special algorithms for control of the glucose feed rate were applied to eliminate the threat of acetic acid excretion due to overfeeding, and at the same time, to reduce excessive CO 2 evolution caused by unnecessarily severe glucose limitation. The joint application of these strategies resulted in greatly improved efficiency in the phenylalanine production process: the final phenylalanine concentration reached 46 g/ l , the yield was above 17%, and the productivity-0.85 g/ l ·h. In combination, these data exceed the results reported by others, and are much higher than those obtained by use before the implementation of the proposed complex of techniques.

Bradyrhizobium iriomotense sp. nov., isolated from a tumor-like root of the legume Entada koshunensis from Iriomote Island in Japan.

A polyphasic study was performed to determine the taxonomic position of strain EK05T isolated from a root-outgrowth of Entada koshunensis, a legume available in Okinawa, Japan. Phylogenetic analysis of the 16S rRNA gene showed that the strain belongs to the genus Bradyrhizobium. Subsequent multilocus sequence analysis with ITS, glnII, recA, gyrB, and atpD sequences revealed that the isolate represents a distinct evolutionary lineage within the genus Bradyrhizobium. DNA-DNA hybridization indicated that strain EK05T shares <61% DNA relatedness with the type strains of all six recognized species of Bradyrhizobium, confirming that this strain is a novel species within the genus. Phylogenetic trees based on symbiotic loci, nifH and nodC, also placed strain EK05T clearly in a novel branch. On the basis of its phylogenetic distinctiveness, we propose Bradyrhizobium iriomotense sp. nov. for strain EK05T. The type strain is EK05T (= NBRC 102520T = LMG 24129T).

Enhancement of anthocyanin production by Perilla frutescens cells in a stirred bioreactor with internal light irradiation

Abstract Oxygen supply and light irradiation exhibited significant influence on the production of anthocyanin (red pigments) by suspended cultures of Perilla frutescens cells in a 2.6- l aerated and agitated bioreactor with a six-flat-bladed turbine. When the initial volumetric oxygen transfer coefficient ( k L a ) value was below 10 h −1 and light was not irradiated, the anthocyanin production was never over 0.6 g/ l . By modification of a gas sparger, the oxygen supply capability of the bioreactor was remarkably improved, and 1.65 g/ l of anthocyanin was obtained at an enhanced k L a value of 15.4 h −1 . Moreover, it was found that anthocyanin accumulation at a 0.2 vvm aeration rate was higher than that at 0.1 or 0.4 vvm in the modified bioreactor, with the other cultivation conditions kept the same. Light irradiation also significantly increased anthocyanin accumulation in the stirred reactor at a low k L a value, i.e. 9.9 h −1 . However, a combination of irradiation with a higher oxygen supply reduced the production of anthocyanin in the bioreactor.

Kozakia baliensis gen. nov., sp. nov., a novel acetic acid bacterium in the alpha-proteobacteria.

Four bacterial strains were isolated from palm brown sugar and ragi collected in Bali and Yogyakarta, Indonesia, by an enrichment culture approach for acetic acid bacteria. Phylogenetic analysis based on 16S rRNA gene sequences showed that the four isolates constituted a cluster separate from the genera Acetobacter, Gluconobacter, Acidomonas, Gluconacetobacter and Asaia with a high bootstrap value in a phylogenetic tree. The isolates had high values of DNA-DNA similarity (78-100%) between one another and low values of the similarity (7-25%) to the type strains of Acetobacter aceti, Gluconobacter oxydans, Gluconacetobacter liquefaciens and Asaia bogorensis. The DNA base composition of the isolates ranged from 56.8 to 57.2 mol% G+C with a range of 0-4 mol%. The major quinone was Q-10. The isolates oxidized acetate and lactate to carbon dioxide and water, but the activity was weak, as with strains of Asaia bogorensis. The isolates differed from Asaia bogorensis strains in phenotypic characteristics. The name Kozakia baliensis gen. nov., sp. nov., is proposed for the four isolates. Strain Yo-3T (= NRIC 0488T = JCM 11301T = IFO 16664T = DSM 14400T) was isolated from palm brown sugar collected in Bali, Indonesia, and was designated as the type strain.

Fed-batch fermentation of xylose by a fast-growing mutant of xylose-assimilating recombinant Saccharomyces cerevisiae

Mutants of xylose-assimilating recombinant Saccharomyces cerevisiae carrying the xylose reductase and xylitol dehydrogenase genes on plasmid pEXGD8 were selected, after ethyl methanesulfonate treatment, for their rapid growth on xylose medium. The fastest growing strain (strain IM2) showed a lower activity of xylose reductase but a higher ratio of xylitol dehydrogenase to xylose reductase activities than the parent strain, as well as high xylulokinase activity. Southern hybridization of the chromosomal DNA indicated that plasmid pEXGD8 was integrated into the chromosome of mutant IM2, resulting in an increase in the stability of the cloned genes. In batch fermentation under O2 limitation, the yield and production rate of ethanol were improved 1.6 and 2.7 times, respectively, compared to the parent strain. In fed-batch culture with slow feeding of xylose and appropriate O2 supply at a low level, xylitol excreted from the cells was limited and the ethanol yield increased 1.5 times over that in the batch culture, with a high initial concentration of xylose, although the production rate was reduced. The results suggested that slow conversion of xylose to xylitol led to a lower level of intracellular xylitol, resulting in less excretion of xylitol, and an increase in the ethanol yield. It was also observed that the oxidation of xylitol was strongly affected by the O2 supply.