Takahide Kishimoto

A Gene Cluster for Biosynthesis of Mannosylerythritol Lipids Consisted of 4-O-β-D-Mannopyranosyl-(2R,3S)-Erythritol as the Sugar Moiety in a Basidiomycetous Yeast Pseudozyma tsukubaensis.

Mannosylerythritol lipids (MELs) belong to the glycolipid biosurfactants and are produced by various fungi. The basidiomycetous yeast Pseudozyma tsukubaensis produces diastereomer type of MEL-B, which contains 4-O-β-D-mannopyranosyl-(2R,3S)-erythritol (R-form) as the sugar moiety. In this respect it differs from conventional type of MELs, which contain 4-O-β-D-mannopyranosyl-(2S,3R)-erythritol (S-form) as the sugar moiety. While the biosynthetic gene cluster for conventional type of MELs has been previously identified in Ustilago maydis and Pseudozyma antarctica, the genetic basis for MEL biosynthesis in P. tsukubaensis is unknown. Here, we identified a gene cluster involved in MEL biosynthesis in P. tsukubaensis. Among these genes, PtEMT1, which encodes erythritol/mannose transferase, had greater than 69% identity with homologs from strains in the genera Ustilago, Melanopsichium, Sporisorium and Pseudozyma. However, phylogenetic analysis placed PtEMT1p in a separate clade from the other proteins. To investigate the function of PtEMT1, we introduced the gene into a P. antarctica mutant strain, ΔPaEMT1, which lacks MEL biosynthesis ability owing to the deletion of PaEMT1. Using NMR spectroscopy, we identified the biosynthetic product as MEL-A with altered sugar conformation. These results indicate that PtEMT1p catalyzes the sugar conformation of MELs. This is the first report of a gene cluster for the biosynthesis of diastereomer type of MEL.

Epistasis effects of multiple ancestral-consensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp. NT3060

Thermostable variants of the Cellulomonas sp. NT3060 glycerol kinase have been constructed by through the introduction of ancestral-consensus mutations. We produced seven mutants, each having an ancestral-consensus amino acid residue that might be present in the common ancestors of both bacteria and of archaea, and that appeared most frequently at the position of 17 glycerol kinase sequences in the multiple sequence alignment. The thermal stabilities of the resulting mutants were assessed by determining their melting temperatures (Tm), which was defined as the temperature at which 50% of the initial catalytic activity is lost after 15 min of incubation, as well as when the half-life of the catalytic activity occurs at a temperature of 60°C (t1/2). Three mutants showed increased stabilities compared to the wild-type protein. We then produced five more mutants with multiple amino acid substitutions. Some of the resulting mutants showed thermal stabilities much greater than those expected given the stabilities of the respective mutants with single mutations. Therefore, the effects of mutations are not always simply additive and some amino acid substitutions, which do not affect or only slightly improve stability when individually introduced into the protein, show substantial stabilizing effects in combination with other mutations.

Comparison of Direct and Mediated Electron Transfer in Electrodes with Novel Fungal Flavin Adenine Dinucleotide Glucose Dehydrogenase

Direct and mediated electron transfer (DET and MET) in enzyme electrodes with a novel flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) from fungi are compared for the first time. DET is achieved by placing a single-walled carbon nanotube (CNT) between GDH and a flat gold electrode where the CNT is close to FAD within the distance for DET. MET is induced by using a free electron transfer mediator, potassium hexacyanoferrate, and shuttles electrons from FAD to the gold electrode. Cyclic voltammetry shows that the onset potential for glucose response current in DET is smaller than in MET, and that the distinct redox current peak pairs in MET are observed whereas no peaks are found in DET. The chronoamperometry with respect to a glucose biosensor shows that (i) the response in DET is more rapid than in MET; (ii) the current at more than +0.45V in DET is larger than the current at the current-peak potential in MET; (iii) a DET electrode covers the glucose concentration range for clinical requirements and is not susceptible to interfering agents at +0.45 V; and (iv) a DET electrode with the novel fungal FAD-GDH does not affect sensing accuracy in the presence of up to 5 mM xylose, while it often shows a similar response level to glucose with other conventionally used fungus-derived FAD-GDHs. It is concluded that our DET system overcomes the disadvantage of MET.