Takashi Ohtsuki

Production of l‐2,3‐butanediol by a new pathway constructed in Escherichia coli

Aims:  A metabolic pathway for l‐2,3‐butanediol (BD) as the main product has not yet been found. To rectify this situation, we attempted to produce l‐BD from diacetyl (DA) by producing simultaneous expression of diacetyl reductase (DAR) and l‐2,3‐butanediol dehydrogenase (BDH) using transgenic bacteria, Escherichia coli JM109/pBUD‐comb.

Good or evil: CD26 and HIV infection

Acquired immune deficiency syndrome (AIDS) is an incurable disease at present and so many efforts to conquer this disease are being made around the world. In studies of human immunodeficiency virus (HIV) infection and the disease progression, it has been reported that T cells expressing CD26 are preferentially infected and depleted in HIV-infected individuals. CD26 is a widely distributed 110 kDa cell-surface glycoprotein with known dipeptidyl peptidase IV (DPPIV) activity in its extracellular domain. This ectoenzyme is capable of cleaving N-terminal dipeptides from polypeptides with either proline or alanine residues in the penultimate position. On human T cells, CD26 exhibits the co-stimulatory function and plays an important role in immune response via its ability to bind adenosine deaminase (ADA) and association with CD45. Recent studies have been stripping the veil from over the relationship between CD26 and HIV infection. Susceptibility of cells to HIV infection is correlated with CD26 expression, and HIV transactivator Tat and envelope protein gp120 are reported to interact with CD26. These observations indicate that CD26 is closely involved in HIV cell entry and that CD26-mediated T cell immune response is suppressed. In addition, it has been demonstrated that the anti-HIV and chemotactic activities of RANTES (regulated on activation, normal T cell expressed and secreted) and stromal cell-derived factor-1 (SDF-1) are controlled with the DPPIV activity of CD26. Thus, the regulation of the function of chemokines by CD26/DPPIV appears to be essential for lymphocyte trafficking and infectivity of HIV strains.

Production of large multienzyme complex by aerobic thermophilic fungus Chaetomium sp. nov. MS-017 grown on palm oil mill fibre.

Aims:  A novel xylanolytic multienzyme complex of the aerobic thermophilic fungus Chaetomium sp. nov. MS‐017 was produced on palm oil mill fibre (POMF) and partially characterized.

Structural basis for chiral substrate recognition by two 2,3-butanediol dehydrogenases

2,3‐Butanediol dehydrogenase (BDH) catalyzes the NAD‐dependent redox reaction between acetoin and 2,3‐butanediol. There are three types of homologous BDH, each stereospecific for both substrate and product. To establish how these homologous enzymes possess differential stereospecificities, we determined the crystal structure of l‐BDH with a bound inhibitor at 2.0 Å. Comparison with the inhibitor binding mode of meso‐BDH highlights the role of a hydrogen‐bond from a conserved Trp residue192. Site‐directed mutagenesis of three active site residues of meso‐BDH, including Trp190, which corresponds to Trp192 of l‐BDH, converted its stereospecificity to that of l‐BDH. This result confirms the importance of conserved residues in modifying the stereospecificity of homologous enzymes.

Increased production of antioxidative sesaminol glucosides from sesame oil cake through fermentation by Bacillus circulans strain YUS-2.

Bacillus circulans strain YUS-2 was isolated as the strongest antioxidant-producer in fermentation of sesame oil cake (SOC, defatted residue yielded from sesame seed oil production). Two major strong antioxidants from fermented SOC were purified and identified as known sesaminol triglucoside and sesaminol diglucoside, however, our results demonstrated that the fermentation process with B. circulans YUS-2 was highly effective to gain the extraction efficiency of the sesaminol glucosides.

Acetylacetoin synthase as a marker enzyme for detecting the 2,3-butanediol cycle.

Acetylacetoin synthase (AACSase) and acetylacetoin reductase (AACRase) are representative enzymes of the 2,3-butanediol cycle. After examining their induction conditions in various bacteria, the former was induced by acetoin and the latter by glucose. All strains carrying AACSase also had AACRase, but the reverse was not true. Therefore, AACSase indicates the existence of the cycle. Acetylacetoin (AAC) accumulation or the ratio of 2,3-butanediol isomer formed also indicated the presence of the cycle in bacteria. This cycle is present in some strains and not in others even for those belonging to the same species. The cycle was not always associated with the representative 2,3-butanediol-producing bacteria or bacterial sporogenesis as reported previously.

Stereochemical applications of the expression of the L-2,3-butanediol dehydrogenase gene in Escherichia coli

S. UI, Y. TAKUSAGAWA, T. OHTSUKI, A. MIMURA, M. OHKUMA AND T. KUDO. 2001. The L‐2,3‐butanediol dehydrogenase (L‐BDH) gene of Brevibacterium saccharolyticum was strongly expressed in Escherichia coli using the tac promoter. However, the stereospecificity of the resulting L‐BDH was reduced. The region upstream of the meso‐BDH gene of Klebsiella pneumoniae was also involved in the expression of the B. saccharolyticum gene. However, in this case, the resulting L‐BDH exhibited more stable stereospecificity. A stereospecificity recognition region was located within the rear sequence (Hpa I site, carboxy terminal) of the BDH open reading frame. Using a transformed strain of E. coli, the conversion of L‐acetoin (L‐AC), in the commercially available racemic mixture of AC, to L‐2,3‐butanediol (L‐BD) was attempted. As a result, 0·37% L‐BD was formed from 1% AC added to the culture.

Chitin Nanowhiskers Mediate Transformation of Escherichia coli by Exogenous Plasmid DNA

A colloidal solution of chitin nanowhiskers containing pUC18 plasmid DNA and cells of Escherichia coli was placed on an agar hydrogel and stimulated by sliding friction applied between the agar hydrogel and polystyrene stir stick, leading to transformation of the bacteria to antibiotic resistance. The combination of chitin nanowhiskers and sliding friction was necessary for genetic transformation, indicating that the chitin nanowhiskers induced the Yoshida effect, resulting in the formation of E. coli cells penetration-intermediates. The pUC18 adsorbed onto the chitin nanowhiskers is presumably taken up through the penetration-intermediates, leading to transformation. The transformation efficiency changed when the number of recipient cells and amount of chitin nanowhiskers were varied. The maximum number of penetration-intermediates acquiring pUC18 DNA was obtained with concentrations of agar hydrogel, chitin nanowhiskers, and recipient E. coli cells of 2.5%, 5.0 ?g/ml, and 4.8 x 108 cells/ml, respectively. Optimal transformation efficiency with pUC18 was achieved with 2.1 x 1066 colony forming units/?g of pUC18. Induction of the Yoshida effect with chitin nanowhiskers represents a simpler alternative for introducing genes into bacteria.

High-yield production of scutellaria radix flavonoids (baicalein, baicalin and wogonin) by liquid-culture of Scutellaria baicalensis root-derived cells

Production of baicalein, baicalin and wogonin by liquid culture of Scutellaria baicalensis cells derived from the plant root was studied. The maximum production obtained were 119 mg/L of baicalein at two week, 1372 mg/L of baicalin at eight week, and 14 mg/L of wogonin at two week. In addition, the production of baicalin was drastically increased to 1000 mg/L level at 3-week culture, and the extremely high production rate (339 mg/L•week) was obtained. In the comparison of total antioxidative activities among baicalein, baicalin and wogonin, evaluated by thiocyanate method, it was suggested that the location of hydroxyl groups both at 5- and 6-position contributed to enhancement of radical scavenging activity, and/or methoxylation at 8-position diminished the activity. The possibility of utilizing these flavonoids for natural antioxidants and medicine is also discussed.

Construction of a Tailor-Made L (2S,3S)-Butanediol Dehydrogenase by Exchanging Domains Between Native Structural Analogs

The development of a stable L-BDH chimera was attempted by exchanging whole domains between two native structural analogs, L-BDH and meso-BDH, because the S-configuration specificity of L-BDH is valuable from the standpoint of its application but its activity is unstable, whereas meso-BDH is stable. The domain chimeras obtained indicated that the leaf-like structures constituting three domains were likely to be mainly associated with chiral recognition, and the fourth domain, the basic domain, is likely to be mainly associated with enzyme stability. A combination of the leaf domains of L-BDH and the basic domain of meso-BDH attained a sufficient level of practical use as an artificial L-BDH chimera, because the resulting enzyme had both stability and S-configuration specificity. However, the levels of stability and specificity were slightly lower than those of the respective enzymes from which they were derived.