Comparison of the efficacy and safety of once-daily insulin degludec/insulin aspart (IDegAsp) and long-acting second-generation basal insulin (insulin degludec and insulin glargine 300 units/mL) in insulin-naïve Japanese adults with type 2 diabetes: a pilot, randomized, controlled study.

To examine the efficacy and safety of once-daily insulin degludec/insulin aspart (IDegAsp) or once-daily second-generation basal insulin analogs (insulin degludec and insulin glargine 300 units/mL) in insulin-naïve Japanese adults with type 2 diabetes in routine clinical practice. A 12-week multicenter, open-label, randomized, pilot study was performed in 52 subjects with type 2 diabetes treated with oral antidiabetic drugs (OADs). Subjects were randomized to once-daily IDegAsp (n = 26) or basal insulin (n = 26). The primary endpoint was percent change in HbA1c from baseline to week 12. Furthermore, it was analyzed post hoc in subgroups stratified by baseline HbA1c. During a follow-up period, percent change in HbA1c was not significantly different between the two groups (p = 0.161). Daily insulin doses and frequency of overall hypoglycemia were also similar in the two groups. In post hoc analyses, once-daily basal insulin was more effective than IDegAsp in subjects with HbA1c more than or equal to 8.5% (p < 0.05); however, in subjects with HbA1c less than 8.5%, once-daily IDegAsp showed a significant improvement in percent change in HbA1c at week 12, compared with basal insulin (p < 0.01). Although there was no apparent difference in the HbA1c-lowering effects between two groups, when compared in subjects with HbA1c less than 8.5%, once-daily IDegAsp showed a significant effect in comparison with once-daily basal insulin. These findings suggest that the baseline HbA1c level might provide the important information for choosing IDegAsp or basal insulin in patients insufficiently controlled with OADs. This trial was registered with UMIN (no. UMIN000035431).

Biosynthesis of novel lactate-based polymers containing medium-chain-length 3-hydroxyalkanoates by recombinant Escherichia coli strains from glucose.

Novel lactate (LA)-based polymers containing medium-chain-length 3-hydroxyalkanoates (MCL-3HA) were produced in fadR-deficient Escherichia coli strains from glucose as the sole carbon source. The genes encoding LA and 3-hydroxybutyrate (3HB) monomers supplying enzymes [propionyl-CoA transferase (PCT), d-lactate dehydrogenase (D-LDH), ß-ketothiolase (PhaA), and NADPH-dependent acetoacetyl-CoA reductase (PhaB)], MCL-3HA monomers supplying enzymes [(R)-3-hydroxyacyl-ACP thioesterase (PhaG) and (R)-3-hydroxyacyl (3HA)-CoA ligase] via fatty acid biosynthesis pathway, and modified polyhydroxyalkanoate (PHA) synthase [PhaC1(STQK)] of Pseudomonas sp. 61-3 were introduced into E. coli LS5218. This resulted in the synthesis of a novel LA-based copolymer, P(LA-co-3HB-co-3HA). 1H-nuclear magnetic resonance (NMR) analysis revealed the composition of P(LA-co-3HB-co-3HA) to be 19.7 mol% LA (C3), 74.9 mol% 3HB (C4), and 5.4 mol% MCL-3HA units of C8 and C10. Furthermore, the recombinant E. coli CAG18497 strain carrying these genes, excluding the phaAB genes, accumulated P(92.0% LA-co-3HA) with a novel monomer composition containing C3, C8, C10, and C12. 13C-NMR analysis showed the existence of LA-3HA sequence in the polymer. The solvent cast film of P(92.0% LA-co-3HA) exhibited transparency similar to poly(lactic acid).

Biosynthesis of Polyhydroxyalkanoate from Steamed Soybean Wastewater by a Recombinant Strain of Pseudomonas sp. 61-3.

Pseudomonas sp. 61-3 accumulates a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer, poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HB-co-3HA)], consisting of 3HA units of 4-12 carbon atoms. Pseudomonas sp. 61-3 possesses two types of PHA synthases, PHB synthase (PhbC) and PHA synthases (PhaC1 and PhaC2), encoded by the phb and pha loci, respectively. The P(94 mol% 3HB-co-6 mol% 3HA) copolymer synthesized by the recombinant strain of Pseudomonas sp. 61-3 (phbC::tet) harboring additional copies of phaC1 gene is known to have desirable physical properties and to be a flexible material with moderate toughness, similar to low-density polyethylene. In this study, we focused on the production of the P(3HB-co-3HA) copolymer using steamed soybean wastewater, a by-product in brewing miso, which is a traditional Japanese seasoning. The steamed soybean wastewater was spray-dried to produce a powder (SWP) and used as the sole nitrogen source for the synthesis of P(3HB-co-3HA) by the Pseudomonas sp. 61-3 recombinant strain. Hydrolyzed SWP (HSWP) was also used as a carbon and nitrogen source. P(3HB-co-3HA)s with relatively high 3HB fractions could be synthesized by a recombinant strain of Pseudomonas sp. 61-3 (phbC::tet) harboring additional copies of the phaC1 gene in the presence of 2% glucose and 10-20 g/L SWP as the sole nitrogen source, producing a PHA concentration of 1.0-1.4 g/L. When HSWP was added to a nitrogen- and carbon-free medium, the recombinant strain could synthesize PHA without glucose as a carbon source. The recombinant strain accumulated 32 wt% P(3HB-co-3HA) containing 80 mol% 3HB and 20 mol% medium-chain-length 3HA with a PHA concentration of 1.0 g/L when 50 g/L of HSWP was used. The PHA production yield was estimated as 20 mg-PHA/g-HSWP, which equates to approximately 1.0 g-PHA per liter of soybean wastewater.

Characterization and identification of the proteins bound to two types of polyhydroxyalkanoate granules in Pseudomonas sp. 61-3.

Pseudomonas sp. 61-3 accumulates two types of polyhydroxyalkanoates (PHAs), poly(3-hydroxybutyrate) [P(3HB)], and poly(3HB-co-3-hydroxyalkanoates) [P(3HB-co-3HA)], and some proteins associated with their PHA granules have been identified. To date, PhaFPs (GA36) and PhaIPs (GA18) were identified from P(3HB-co-3HA) granules. In this study, the gene encoding GA24 associated with P(3HB) granule was identified as phbPPs. PhbPPs was composed of 192 amino acids with a calculated molecular mass of 20.4 kDa and was assumed to be a phasin. phbFPs gene and unknown ORF were also found on phb locus. PhbFPs was anticipated to be the transcriptional repressor of phbPPs gene. PhbPPs was bound to the P(3HB-co-3HA) granules with 3HB composition of more than 87 mol%, and PhaIPs and PhaFPs were bound to the P(3HB-co-3HA) granules with 3HA (C6-C12) composition of more than 13 mol% in the producing cells, suggesting that localization of these proteins is attributed to the monomer compositions of the copolymers.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant Escherichia coli from glucose.

The polyhydroxyalkanoate (PHA) copolymers consisting of short-chain-length (scl) and medium-chain-length (mcl) monomers have various properties ranging from stiff to flexible depending on the molar fraction of the monomer compositions. It has been reported that PhaG, which is first known as a (R)-3-hydroxyacyl-acyl carrier protein (ACP)-CoA transferase, actually functions as a 3-hydroxyacyl-ACP thioesterase, and the product of PP0763 gene from Pseudomonas putida KT2440 has a (R)-3-hydroxyacyl (3HA)-CoA ligase activity (Wang et al., Appl. Environ. Microbiol., 78, 519-527, 2012). In this study, we found a new (R)-3HA-CoA ligase (the product of PA3924 gene) from Pseudomonas aeruginosa PAO. The PA3924 gene was coexpressed with PHA synthase 1 gene (phaC1Ps) and phaGPs gene from Pseudomonas sp. 61-3, and ß-ketothiolase gene (phbARe) and acetoacetyl-CoA reductase gene (phbBRe) from Ralstonia eutropha in Escherichia coli LS5218 at 25°C. As a result, the copolymer containing 94.6 mol% 3-hydroxybutyrate (3HB) and 5.4 mol% mcl-3-hydroxyalkanoates (3HA) consisting of C8, C10, C12 and C14 was synthesized by recombinant E. coli LS5218 from glucose as the sole carbon source. The concentration of P(3HB-co-3HA) (scl-co-mcl-PHA) synthesized by the recombinant E. coli LS5218 harboring phaC1Ps, phaGPs, phbABRe and the PA3924 genes was approximately 7-fold higher than that of the recombinant LS5218 harboring phaC1Ps, phaGPs, phbABRe and the PP0763 genes. The number-average molecular weight of the P(3HB-co-5.4% 3HA) copolymer was 233 × 10(3), which was relatively high molecular weight. In addition, the physical and the mechanical properties of the copolymer were demonstrated to improve the brittleness of P(3HB) homopolymer.