Takashi Shimamura

A microbial factory for lactate-based polyesters using a lactate-polymerizing enzyme

Polylactate (PLA) is synthesized as a representative bio-based polyester by the chemo-bio process on the basis of metal catalyst-mediated chemical polymerization of lactate (LA) supplied by microbial fermentation. To establish the one-step microbial process for synthesis of LA-based polyesters, we explored whether polyhydroxyalkanoate (PHA) synthase would exhibit polymerizing activity toward a LA-coenzyme A (CoA), based on the fact that PHA monomeric constituents, especially 3-hydroxybutyrate (3HB), are structurally analogous to LA. An engineered PHA synthase was discovered as a candidate by a two-phase in vitro polymerization system previously developed. An LA-CoA producing Escherichia coli strain with a CoA transferase gene was constructed, and the generation of LA-CoA was demonstrated by capillary electrophoresis/MS analysis. Next, when the engineered PHA synthase gene was introduced into the resultant recombinant strain, we confirmed the one-step biosynthesis of the LA-incorporated copolyester, P(6 mol% LA-co-94 mol% 3HB), with a number-average molecular weight of 1.9 × 105, as revealed by gel permeation chromatography, gas chromatography/MS, and NMR.

Transgenic sweet potato expressing thionin from barley gives resistance to black rot disease caused by Ceratocystis fimbriata in leaves and storage roots

Black rot of sweet potato caused by pathogenic fungus Ceratocystis fimbriata severely deteriorates both growth of plants and post-harvest storage. Antimicrobial peptides from various organisms have broad range activities of killing bacteria, mycobacteria, and fungi. Plant thionin peptide exhibited anti-fungal activity against C. fimbriata. A gene for barley α-hordothionin (αHT) was placed downstream of a strong constitutive promoter of E12Ω or the promoter of a sweet potato gene for β-amylase of storage roots, and introduced into sweet potato commercial cultivar Kokei No. 14. Transgenic E12Ω:αHT plants showed high-level expression of αHT mRNA in both leaves and storage roots. Transgenic β-Amy:αHT plants showed sucrose-inducible expression of αHT mRNA in leaves, in addition to expression in storage roots. Leaves of E12Ω:αHT plants exhibited reduced yellowing upon infection by C. fimbriata compared to leaves of non-transgenic Kokei No. 14, although the level of resistance was weaker than resistance cultivar Tamayutaka. Storage roots of both E12Ω:αHT and β-Amy:αHT plants exhibited reduced lesion areas around the site inoculated with C. fimbriata spores compared to Kokei No. 14, and some of the transgenic lines showed resistance level similar to Tamayutaka. Growth of plants and production of storage roots of these transgenic plants were not significantly different from non-transgenic plants. These results highlight the usefulness of transgenic sweet potato expressing antimicrobial peptide to reduce damages of sweet potato from the black rot disease and to reduce the use of agricultural chemicals.

Investigation of utilization of the algal biomass residue after oil extraction to lower the total production cost of biodiesel

In this study, component analysis of a novel biodiesel-producing alga, Pseudochoricystis ellipsoidea, was performed. The component analysis results indicated that proteins and amino acids are abundant in P. ellipsoidea while the sugar content is relatively low. Rather than its use as a carbon source, the use of the algal biomass residue after oil extraction as a nutrient source provided a new way for lowering the total production cost of biodiesel. In both lactic acid and ethanol fermentations, the use of the residue instead of high-cost nutrient yeast extract allowed a significant saving, showing the promise of the algal biomass residue for use as a fermentation nutrient source.

pH-uncontrolled lactic acid fermentation with activated carbon as an adsorbent

In this paper, we presented a novel process involving activated carbon (AC) as an adsorbent for lactic acid fermentation, separation and oligomerization. It was found that pH has a significant effect on the adsorption of lactic acid on AC. The use of AC for in situ removal of lactic acid successfully decreased the inhibitory effect of lactic acid, resulting in significant increases in both productivity and yield. Acetone was used to desorb lactic acid and it was confirmed that the acetone treatment did not decrease the optical purity of the lactic acid, i.e., the optical purity was as high as 99.5% after desorption. Due to the presence of little materials influencing lactic acid oligomerization, oligomers with an optical purity of above 96% and a weight-average molecular weight (M(w)) of 2400 were obtained in the oligomerization process.