Synthesis of cyclic ß-1,6-oligosaccharides from glucosamine monomers by electrochemical polyglycosylation.

The synthesis of protected precursors of cyclic ß-1,6-oligoglucosamines from thioglycosides as monomers is performed by electrochemical polyglycosylation. The monomer with a 2,3-oxazolidinone protecting group afforded the cyclic disaccharide exclusively. Cyclic oligosaccharides up to the trisaccharide were obtained using the monomer with a 2-azido-2-deoxy group.

Efficient pathway-driven scyllo-inositol production from myo-inositol using thermophilic cells and mesophilic inositol dehydrogenases: a novel strategy for pathway control.

Mesophilic enzymes, which are active at moderate temperatures, may dominate enzymatic reactions even in the presence of thermophilic crude enzymes. This study was conducted to investigate this hypothesis with mesophilic inositol dehydrogenases (IolG and IolX) produced in Geobacillus kaustophilus HTA426. To ensure the efficient production of mesophilic enzymes, we first screened for promoters induced at moderate temperatures using transcriptome analysis and identified four genes highly expressed at 30°C in the thermophile. We further characterized these promoters using fluorescent reporter assays to determine that the mti3 promoter could direct efficient gene expression at 40°C. We cloned the promoter into an Escherichia coli-Geobacillus shuttle plasmid and confirmed that the resulting vector functioned in G. kaustophilus and other thermophiles. We then used this vector for the cooperative expression of the iolG and iolX genes from Bacillus subtilis 168. G. kaustophilus cells carrying the expression vector were incubated at 60°C for cellular propagation and then at 40°C for the production of IolG and IolX. When the cells were permeabilized, IolG and IolX acted as catalysts to convert exogenous myo-inositol into scyllo-inositol at 30°C. In a scaled-up reaction, 10 g of myo-inositol was converted to 1.8 g of scyllo-inositol, which was further purified to yield 970 mg of pure powder. Notably, myo-inositol was degraded by intrinsic enzymes of G. kaustophilus at 60°C but not at 30°C, supporting our initial hypothesis. We indicate that this approach is useful for preparing enzyme cocktails without the need for purification. IMPORTANCE: Enzyme cocktails are commonly employed for cell-free chemical synthesis; however, their preparation involves cumbersome processes. This study affirms that mesophilic enzymes in thermophilic crude extracts can function as specific catalysts at moderate temperatures, akin to enzyme cocktails. The catalyst was prepared by permeabilizing cells without the need for concentration, extraction, or purification processes; hence, its preparation was considerably simpler compared with conventional methods for enzyme cocktails. This approach was employed to produce pure scyllo-inositol from an economical substrate. Notably, this marks the first large-scale preparation of pure scyllo-inositol, holding potential pharmaceutical significance as scyllo-inositol serves as a promising agent for certain diseases but is currently expensive. Moreover, this approach holds promise for application in pathway engineering within living cells. The envisioned pathway is designed without chromosomal modification and is simply regulated by switching culture temperatures. Consequently, this study introduces a novel platform for both whole-cell and cell-free synthetic systems.

Simplified Modular Access to Enantiopure 1,2-Aminoalcohols via Ni-Electrocatalytic Decarboxylative Arylation.

Chiral aminoalcohols are omnipresent in bioactive compounds. Conventional strategies to access this motif involve multiple-step reactions to install the requisite functionalities stereoselectively using conventional polar bond analysis. This study reveals that a simple chiral oxazolidine-based carboxylic acid can be readily transformed to substituted chiral aminoalcohols with high stereochemical control by Ni-electrocatalytic decarboxylative arylation. This general, robust, and scalable coupling can be used to synthesize a variety of medicinally important compounds, avoiding protecting and functional group manipulations, thereby dramatically simplifying their preparation.

Synthesis of protected precursors of chitin oligosaccharides by electrochemical polyglycosylation of thioglycosides.

The synthesis of protected precursors of chitin oligosaccharides by electrochemical polyglycosylation of thioglycosides as monomer is described. Oligosaccharides up to the hexasaccharide were synthesized under optimized reaction conditions. Further, a modified method enabled the synthesis of oligosaccharides up to the octasaccharide by repeating electrolysis with additional monomers. The mechanism of the electrochemical polyglycosylation is also discussed, based on the oxidation potential of the monomer and oligosaccharides.

Synthesis of cyclic α-1,4-oligo-N-acetylglucosamine 'cyclokasaodorin' via a one-pot electrochemical polyglycosylation-isomerization-cyclization process.

Electrochemical synthesis of unnatural cyclic oligosaccharides composed of N-acetylglucosamine with α-1,4-glycosidic linkages has been accomplished. A thioglycoside monomer equipped with the 2,3-oxazolidinone protecting group was used to prepare linear oligosaccharides by electrochemical polyglycosylation. In the same pot, isomerization of the linear oligosaccharides and intramolecular electrochemical glycosylation for cyclization were also conducted sequentially to obtain the precursor of the cyclic α-1,4-oligo-N-acetylglucosamine 'cyclokasaodorin'.

Production of l-Theanine by Escherichia coli in the Absence of Supplemental Ethylamine.

l-Theanine is a nonproteinogenic amino acid present almost exclusively in tea plants and is beneficial for human health. For industrial production, l-theanine is enzymatically or chemically synthesized from glutamine/glutamate (or a glutamine/glutamate derivative) and ethylamine. Ethylamine is extremely flammable and toxic, which complicates and increases the cost of operational procedures. To solve these problems, we developed an artificial biosynthetic pathway to produce l-theanine in the absence of supplemental ethylamine. For this purpose, we identified and selected a novel transaminase (NCBI:protein accession number AAN70747) from Pseudomonas putida KT2440, which catalyzes the transamination of acetaldehyde to produce ethylamine, as well as γ-glutamylmethylamide synthetase (NCBI:protein accession number AAY37316) from Pseudomonas syringae pv. syringae B728a, which catalyzes the condensation of l-glutamate and ethylamine to produce l-theanine. Expressing these genes in Escherichia coli W3110S3GK and enhancing the production capacity of acetaldehyde and l-alanine achieved successful production of l-theanine without ethylamine supplementation. Furthermore, the deletion of ggt, which encodes γ-glutamyltranspeptidase (EC 2.3.2.2), achieved large-scale production of l-theanine by attenuating its decomposition. We show that an alanine decarboxylase-utilizing pathway represents a promising route for the fermentative production of l-theanine. Our study reports efficient methods to produce l-theanine in the absence of supplemental ethylamine.IMPORTANCE l-Theanine is widely used in food additives and dietary supplements. Industrial production of l-theanine uses the toxic and highly flammable precursor ethylamine, raising production costs. In this study, we used Escherichia coli to engineer two biosynthetic pathways that produce l-theanine from glucose and ammonia in the absence of supplemental ethylamine. This study establishes a foundation for safely and economically producing l-theanine.

A case of suicide by helium gas.

Helium is a colorless, tasteless, and odorless gas that is used as a lifting gas for air balloons and small airships due to its high level of safety. In recent years, it has become easy to search the Internet for methods to commit suicide, and the use of helium gas for this purpose has recently been garnering attention. We report a case of a male in his 30's in which helium gas was used to commit suicide. He was found unconscious on a bed with a plastic bag covering his head. When emergency personnel arrived at the scene, he was in cardiac arrest and remained in cardiac arrest at the time of his arrival at the hospital. Continued cardiopulmonary resuscitation resulted in a return of spontaneous circulation. Head computed tomography showed the corticomedullary border was indistinct. Fifteen hours at the hospital, the patient died. Recently, there has been an increasing trend for suicide being performed according to methods obtained through the Internet or mass media. Although there are only a few cases of suicide by helium gas in Japan, attention must be paid to this issue as the number of such cases may increase in the future.

Identification and cloning of the gene involved in the final step of chlortetracycline biosynthesis in Streptomyces aureofaciens.

For chlortetracycline biosynthesis in Streptomyces aureofaciens, the final reduction step is essential to give an antibiotic activity to its intermediate, which is catalyzed by tetracycline dehydrogenase with 7,8-dedimethyl-8-hydroxy-5-deazariboflavin (FO) as a cofactor. We identified and cloned the gene, which is essential for the biosynthesis of 6-demethyltetracycline and participates in the final step of its biosynthesis, from the genomic DNA of the 6-demethyltetracycline producer S. aureofaciens HP77. DNA sequence analysis revealed that the gene (tchA) had an open reading frame of 455 amino acids with an estimated molecular mass of 48.1 kDa. Southern hybridization analysis revealed that the tchA gene was located external to the chlortetracycline biosynthetic gene cluster in the genome. A conserved domain search of protein sequence databases indicated that TchA showed a similarity to FbiB, which is involved in the modification of FO in Mycobacterium bovis.