Isolation and characterization of polyhydroxyalkanoate-degrading bacteria in seawater at two different depths from Suruga Bay.

IMPORTANCE: Polyhydroxyalkanoate (PHA) is a highly biodegradable microbial polyester, even in marine environments. In this study, we incorporated an enrichment culture-like approach in the process of isolating marine PHA-degrading bacteria. The resulting 91 isolates were suggested to fall into five genera (Alloalcanivorax, Alteromonas, Arenicella, Microbacterium, and Pseudoalteromonas) based on 16S rRNA analysis, including two novel genera (Arenicella and Microbacterium) as marine PHA-degrading bacteria. Microbacterium schleiferi (DSM 20489) and Alteromonas macleodii (NBRC 102226), the type strains closest to the several isolates, have an extracellular poly(3-hydroxybutyrate) [P(3HB)] depolymerase homolog that does not fit a marine-type domain composition. However, A. macleodii exhibited no PHA degradation ability, unlike M. schleiferi. This result demonstrates that the isolated Alteromonas spp. are different species from A. macleodii. P(3HB) depolymerase homologs in the genus Alteromonas should be scrutinized in the future, particularly about which ones work as the depolymerase.

Oxidation of methionine-derived 2-hydroxyalkanoate unit in biosynthesized polyhydroxyalkanoate copolymers.

In this study, 2-hydroxy-4-methylthiobutyrate (2H4MTB)-containing polyhydroxyalkanoate (PHA) copolymers were biosynthesized using methionine as the 2H4MTB precursor. 2H4MTB is a novel monomer unit that contains a sulfur atom in its side chain. The 2H4MTB-containing PHA was biosynthesized by functionalizing the leucine degradation and PHA synthesis pathways in recombinant Escherichia coli. The 2H4MTB fraction in the PHA copolymer was increased up to 30.6 mol% by increasing the methionine concentration in the medium. Using purified polymers containing 10.9 mol% 2H4MTB unit, the sulfide group of the 2H4MTB side chain was oxidized with hydrogen peroxide or peracetic acid, which resulted in the conversion of sulfide to sulfoxide and sulfone groups. The oxidized polymer was relatively hydrophilic, as revealed by water contact angle measurements, and swelled slightly when soaked in water. These results suggest that the 2H4MTB unit can be used as an oxidation site to impart hydrophilicity to the PHA copolymers.

Optimization of Culture Conditions for Secretory Production of 3-Hydroxybutyrate Oligomers Using Recombinant Escherichia coli.

Poly(3-hydroxybutyrate) [P(3HB)] is the most representative polyhydroxyalkanoate (PHA), which is a storage polyester for prokaryotic cells. P(3HB)-producing recombinant Escherichia coli secretes diethylene glycol (DEG)-terminated 3HB oligomers (3HBO-DEG) through a PHA synthase-mediated chain transfer and alcoholysis reactions with externally added DEG. The purpose of this study was to optimize the culture conditions for the secretory production of 3HBO-DEG with jar fermenters. First, the effects of culture conditions, such as agitation speed, culture temperature, culture pH, and medium composition on 3HBO-DEG production, were investigated in a batch culture using 250-ml mini jar fermenters. Based on the best culture conditions, a fed-batch culture was conducted by feeding glucose to further increase the 3HBO-DEG titer. Consequently, the optimized culture conditions were reproduced using a 2-L jar fermenter. This study successfully demonstrates a high titer of 3HBO-DEG, up to 34.8 g/L, by optimizing the culture conditions, showing the feasibility of a new synthetic strategy for PHA-based materials by combining secretory oligomer production and subsequent chemical reaction.

Screening Method for Polyhydroxyalkanoate Synthase Mutants Based on Polyester Degree of Polymerization Using High-Performance Liquid Chromatography.

A high-throughput screening method based on the degree of polymerization (DP) of polyhydroxyalkanoate (PHA) was developed using high-performance liquid chromatography (HPLC). In this method, PHA production was achieved using recombinant Escherichia coli supplemented with benzyl alcohol as a chain terminal compound. The cultured cells containing benzyl alcohol-capped PHA were decomposed by alkaline treatment, and the peaks of the decomposed monomer and benzyl alcohol were detected using HPLC. The DP of PHA could be determined from the peak ratio of the decomposed monomer to terminal benzyl alcohol. The measured DP was validated by other instrumental analyses using purified PHA samples. Using this system, mutants of PHA synthase from Bacillus cereus YB-4 (PhaRCYB4) were screened, and some enzymes capable of producing PHA with higher DP than the wild-type enzyme were obtained. The PHA yields of two of these enzymes were equivalent to the yield of the wild-type enzyme. Therefore, this screening method is suitable for the selection of beneficial mutants that can produce high molecular weight PHAs.

The influence of medium composition on the microbial secretory production of hydroxyalkanoate oligomers.

With the aid of a chain transfer (CT) reaction, hydroxyalkanoate (HA) oligomers can be secreted by recombinant Escherichia coli carrying the gene encoding a lactate-polymerizing enzyme (PhaC1PsSTQK) in Luria-Bertani (LB) medium supplemented with a carbon source and CT agent. In this study, HA oligomers were produced through microbial secretion using a mineral-based medium instead of LB medium, and the impact of medium composition on HA oligomer secretion was investigated. The focused targets were medium composition and NaCl concentration related to osmotic conditions. It was observed that 4.21 g/L HA oligomer was secreted by recombinant E. coli in LB medium, but the amount secreted in the mineral-based modified R (MR) medium was negligible. However, when the MR medium was supplemented with 5 g/L yeast extract, 3.75 g/L HA oligomer was secreted. This can be accounted for by the enhanced expression and activity of PhaC1PsSTQK upon supplementation with growth-activated nutrients as supplementation with yeast extract also promoted cell growth and intracellular growth-associated polymer accumulation. Furthermore, upon adding 10 g/L NaCl to the yeast extract-supplemented MR medium, HA oligomer secretion increased to 6.86 g/L, implying that NaCl-induced osmotic pressure promotes HA oligomer secretion. These findings may facilitate the secretory production of HA oligomers using an inexpensive medium.

Microbial oversecretion of (R)-3-hydroxybutyrate oligomer with diethylene glycol terminal as a macromonomer for polyurethane synthesis.

Poly((R)-3-hydroxybutyrate) (P(3HB)) is a polyester that is synthesized and accumulated in many prokaryotic cells. Recently, a new culture method for the secretion of the intracellularly synthesized (R)-3-hydroxybutyrate oligomer (3HBO) from recombinant Escherichia coli cells was developed. In this study, we attempted to produce microbial 3HBO capped with a diethylene glycol terminal (3HBO-DEG) as a macromonomer for polymeric materials. First, we prepared recombinant E. coli strains harboring genes encoding various polyhydroxyalkanoate (PHA) synthases (PhaC, PhaEC or PhaRC) that can incorporate chain transfer (CT) agents such as DEG into the polymer's terminal and generate CT end-capped oligomers. To this end, each strain was cultivated under DEG supplemental conditions, and the synthesis of 3HBO-DEG was confirmed. As a result, the highest secretory production of 3HBO-DEG was observed for the PHA synthase derived from Bacillus cereus YB-4 (PhaRCYB4). To evaluate the usability of the secreted 3HBO-DEG as a macromonomer, 3HBO-DEG was purified from the culture medium and polymerized with 4,4'-diphenylmethane diisocyanate as a spacer compound. Characterization of the polymeric products revealed that 3HBO-based polyurethane was successfully obtained and was a flexible and transparent noncrystalline polymer, unlike P(3HB). These results suggested that microbial 3HBO-DEG is a promising platform building block for synthesizing polyurethane and various other polymers.

Unilateral renal cortical necrosis with contralateral hydronephrosis after surgery for uterus carcinoma.

Renal cortical necrosis (RCN) represents a rare cause of acute renal failure that is characterized by necrosis of the renal cortex with sparing of the medulla. Most previous RCN cases reported have been bilateral and have occurred in pregnancy. Unilateral RCN is a quite rare disorder. Here, we report a case of unilateral RCN with contralateral hydronephrosis after surgery for uterus carcinoma. In this patient it seems that hydronephrosis had been present before RCN occurrence. It is suggested that ureteric obstruction protects against RCN development.

Pulmonary hyalinizing granuloma with hydronephrosis.

A 49-year-old man was admitted for the evaluation of a bilateral mass shadow in his chest X-ray film. No definitive diagnosis was established either by brushing cytology or biopsy through bronchoscopy. No malignancies were suggested by general work-up. Both masses were surgically removed, and were diagnosed as pulmonary hyalinizing granuloma (PHG). Fifteen months later, low grade fever continued and the renal function decreased. Laboratory examinations revealed bilateral hydronephrosis with polyclonal hypergammaglobulinemia. The findings of abdominal CT and urography were compatible with retroperitoneal fibrosis. Steroid treatment completely reversed the initial abnormality in laboratory data and the symptoms disappeared.