A Mutant D‐Fructose‐6‐Phosphate Aldolase (Ala129Ser) with Improved Affinity towards Dihydroxyacetone for the Synthesis of Polyhydroxylated Compounds

A mutant of D ‐fructose‐6‐phosphate aldolase (FSA) of Escherichia coli, FSA A129S, with improved catalytic efficiency towards dihydroxyacetone (DHA), the donor substrate in aldol addition reactions, was explored for synthetic applications. The k_cat/K_M value for DHA was 17‐fold higher with FSA A129S than that with FSA wild type (FSA wt). On the other hand, for hydroxyacetone as donor substrate FSA A129S was found to be 3.5‐fold less efficient than FSA wt. Furthermore, FSA A129S also accepted glycolaldehyde (GA) as donor substrate with 3.3‐fold lower affinity than FSA wt. This differential selectivity of both FSA wt and FSA A129S for GA makes them complementary biocatalysts allowing a control over donor and acceptor roles, which is particularly useful in carboligation multi‐step cascade synthesis of polyhydroxylated complex compounds. Production of the mutant protein was also improved for its convenient use in synthesis. Several carbohydrates and nitrocyclitols were efficiently prepared, demonstrating the versatile potential of FSA A129S as biocatalyst in organic synthesis.     

Construction of functional aliphatic polycarbonates for biomedical applications

Aliphatic polycarbonates are one important kind of biodegradable polymers and have been commonly used as integral components of engineered tissues, medical devices and drug delivery systems. As far as the biomedical application is concerned, traditional aliphatic polycarbonates usually suffer from the strong hydrophobicity, deficient functionality, and insufficient compatibility with cell/organs. Consequently, the application is quite limited in scope. Due to the imparted appealing properties, aliphatic polycarbonates bearing specifically designed functional/reactive groups attract great interest from researchers in the recent years. The present review outlines the development up to date concerning the design and biomedical application of functional aliphatic polycarbonates, with an emphasis on their ring-opening (co)polymerization preparation.     

The Pichia pastoris dihydroxyacetone kinase is a PTS1-containing,but cytosolic,protein that is essential for growth on methanol

Dihydroxyacetone kinase (DAK) is essential for methanol assimilation in methylotrophic yeasts. We have cloned the DAK gene from Pichia pastoris by functional complementation of a mutant that was unable to grow on methanol. An open reading frame of 1824 bp was identified that encodes a 65·3 kDa protein with high homology to DAK from Saccharomyces cerevisiae. Although DAK from P. pastoris contained a C-terminal tripeptide, TKL, which we showed can act as a peroxisomal targeting signal when fused to the green fluorescent protein, the enzyme was primarily cytosolic. The TKL tripeptide was not required for the biochemical function of DAK because a deletion construct lacking the DNA encoding this tripeptide was able to complement the P. pastoris dakΔ mutant. Peroxisomes, which are essential for growth of P. pastoris on methanol, were present in the dakΔ mutant and the import of peroxisomal proteins was not disturbed. The dakΔ mutant grew at normal rates on glycerol and oleate media. However, unlike the wild-type cells, the dakΔ mutant was unable to grow on methanol as the sole carbon source but was able to grow on dihydroxyacetone at a much slower rate. The metabolic pathway explaining the reduced growth rate of the dakΔ mutant on dihydroxyacetone is discussed. The nucleotide sequence reported in this paper has been submitted to GenBank with Accession Number AF019198. © 1998 John Wiley & Sons, Ltd.     

Cloning,sequencing and characterization of a gene encoding dihydroxyacetone kinase from Zygosaccharomyces rouxii NRRL2547

The dihydroxyacetone pathway, an alternative pathway for the dissimilation of glycerol via reduction by glycerol dehydrogenase and subsequent phosphorylation by dihydroxyacetone (DHA) kinase, is activated in the yeasts Saccharomyces cerevisiae and Zygosaccharomyces rouxii during osmotic stress. In experiments aimed at investigating the physiological function of the DHA pathway in Z. rouxii, a typical osmotolerant yeast, we cloned and characterized a DAK gene encoding dihydroxyacetone kinase from Z. rouxii NRRL 2547. Sequence analysis revealed a 1761 bp open reading frame, encoding a peptide composed of 587 deduced amino acids with the predicted molecular weight of 61 664 Da. As the amino acid sequence was most closely homologous (68% identity) to the S. cerevisiae Dak1p, we named the gene and protein ZrDAK1 and ZrDak1p, respectively. A putative ATP binding site was also found but no consensus element associated with osmoregulation was found in the upstream region of the ZrDAK1 gene. The ZrDAK1 gene complemented a S. cerevisiae W303-1A dak1delta dak2 delta strain by improving the growth of the mutant on 50 mmol/l dihydroxyacetone and by increasing the tolerance to dihydroxyacetone in a medium containing 5% sodium chloride, suggesting that it is a functional homologue of the S. cerevisiae DAK1. However, expression of the ZrDAK1 gene in the S. cerevisiae dak1delta dak2 delta strain had no significant effect on glycerol levels during osmotic stress. The ZrDAK1 sequence has been deposited in the public data bases under Accession No. AJ294719; regions upstream and downstream of ZrDAK1are deposited as Accession Nos AJ294739 and AJ294720, respectively.     

Metabolic regulation in the yeast Hansenula polymorpha. Growth of dihydroxyacetone kinase/glycerol kinase-negative mutants on mixtures of methanol and xylose in continuous cultures

The physiological responses of Hansenula polymorpha wild-type and mutant strains 17B (dihydroxyacetone kinase-negative) and 17BG51 (dihydroxyacetone kinase- and glycerol kinase-negative) to growth on mixtures of xylose and methanol in chemostats were investigated. Increasing methanol concentrations (0–110 mM ) in the feed of the wild-type culture resulted in increasing cell densities and a gradual switch towards methanol metabolism. At the lower methanol feed concentrations the mutant cultures used methanol and xylose to completion and changes in enzyme patterns comparable to the wild type were observed. This was not reflected in significant changes in cell densities. Instead, formaldehyde assimilation resulted in dihydroxyacetone (DHA) production, which was proportional to the amount of methanol added. At intermediate methanol concentration the cultures showed a strong variation in DHA levels and cell densities. Further increased in the methanol feed concentrations resulted in a drop in DHA accumulation rates, repression of alcohol oxidase synthesis and accumulation of residual methanol. The phenomena were studied in more detail in transition experiments and with gradients of methanol. The results indicate that xylulose-5-phosphate (Xu5P) generated in xylose metabolism served as acceptor molecule for formaldehyde assimilation by the peroxisomal enzyme DHA synthase. Accumulation of DHA in the mutant cultures, however, further diminished the availability of carbon for growth. The data suggest that with increasing methanol concentrations Xu5P eventually became growth rate limiting. This resulted in an unstable situation but wash-out of the culture did not occur to a significant extent. Instead, DHA accumulation ceased and cell densities, and enzymes specifically involved in xylose metabolism increase, indicating that the organism resumed its xylose metabolism. The molecular mechanisms controlling the partitioning of Xu5P over xylose (pentose phosphate pathway) and methanol (peroxisome) metabolism under these conditions remain to be elucidated.     

Catalytic synthesis ofdl-serine and glycine from glycerol

dl-serine (dl-Ser), glycine (Gly), serinol (SerOH) and related compounds were catalytically derived from glycerol (GLY). Catalytic oxidation in the presence of ammonia of GLY, followed by hydrogen treatment over noble metals, to achieve direct synthesis of amino acids is not practical. Reductive amination over an Ru-Pd/C catalyst of glyceric acid, one of the oxidation products of GLY, was effective fordl-Ser synthesis. Gly was catalytically formed fromdl-Ser by dehydrogenation and decarbonylation in hydrogen atmosphere. Catalytic oxidation of SerOH prepared by reductive amination over an Rh-Pd/C catalyst of dihydroxyacetone, another oxidation product of GLY, was also effective fordl-Ser synthesis. A new route fordl-Ser and Gly from GLY is presented.dl-alanine and γ-aminobutyric acid also formed, depending on reaction conditions.     

二羟基丙酮的合成研究进展

二羟基丙酮是一种重要的生化原料、合成试剂,在精细化工、食品工业、化妆品工业等很多行业发挥着重要的作用。由于二羟基丙酮用途广泛,市场容量大,因此对二羟基丙酮的合成研究具有重要的意义。介绍了二羟基丙酮的主要合成方法及其应用研究的进展,并对其前景进行了展望。     

Development of an in vitro efficacy test for self-tanning formulations

In the last few years, the desire to acquire a tan without sunbathing has grown. In response, many cosmetic companies have produced self-tanning preparations. However, to optimize these formulations, a suitable model to assess the colour induced on the skin remains to be developed. We have developed an in vitro method using a synthetic skin in order to study the efficacy of self-tanning formulations. The in vitro test was then used to study the influence of cosmetic ingredients upon the colour induced by erythrulose- or dihydroxyacetone-containing formulations. Finally, an in vivo study allowed us to relate the results obtained in vitro with those found on human skin. The results show that this in vitro test system is a reliable tool to predict the efficacy of self-tanning products.