Engineered Aeromonas hydrophila for enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with alterable monomers composition.

Aeromonas hydrophila 4AK4 produces poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) containing 3-hydroxybutyrate (3HB) and about 15 mol% 3-hydroxyhexanoate (3HHx) from dodecanoate. To study the factors affecting the monomer composition and PHBHHx content, genes encoding phasin (phaP), PHA synthase (phaC) and (R)-specific enoyl-CoA hydratase (phaJ) from Aeromonas punctata (formerly named Aeromonas caviae) were introduced individually or jointly into A. hydrophila 4AK4. The phaC gene increased 3HHx fraction more significantly than phaP, while phaJ had little effect. Expression of phaC alone increased the 3HHx fraction from 14 to 22 mol%. When phaC was co-expressed with phaP and phaJ, the 3HHx fraction increased from 14 to 34 mol%. Expression of phaP or phaC alone or with another gene enhanced PHBHHx content up to 64%, cell dry weight (CDW) as much as 4.4 gL(-1) and PHBHHx concentration to 2.7 gL(-1) after 48 h in shake flask culture. The results suggest that a higher PHA synthase activity could lead to a higher 3HHx fraction and PHBHHx content. Co-expression of phaJ with phaC or phaP would favor PHA accumulation, although over-expression of phaJ did not affect PHA synthesis much. In addition, inhibition of beta-oxidation by acrylate in A. hydrophila 4AK4 enhanced PHBHHx content. However, no monomers longer than 3HHx were detected. The results show that genetic modification of A. hydrophila 4AK4 enhanced PHBHHx production and altered monomer composition of the polymer.

[Influence of nutrient sources on Anabaena spiroides growth and odorous compounds production characteristics].

The occurrence of taste and odors, produced by secondary metabolites of cyanobacteria, has been one of the major water quality problems in drinking water. However, the odorous compounds produced by cyanobacteria usually differ significantly with different species. One cyanobacterium isolated from Yanghe reservoir was identified as Anabaena sp., which can produce high level of geosmin consistently during laboratory culture. By culture expanding experiments, the algal growth and geosmin production characteristics of the Anabaena sp. were studied on different conditions of nitrogen and phosphorus sources. The results indicated that geosmin mainly remained in the intracellular algal cells regardless of the nutrient sources, and the extracellular content was only in th range of 0.2% - 9.6%. Compared with ammonia nitrogen conditions, the growth of Anabaena sp. in nitrate nitrogen conditions was much higher, with a 1.4-fold variation in geosmin production. While ammonia nitrogen concentration was 0.5 mg/L, the algal biomass and geosmin production achieved the highest level of 3.8 x 10(4) cells, mL(-1) and 1.1 x 10(4) ngL(-1), respectively. When the nitrate nitrogen concentration was 2.0 mg/L, the algal biomass and geosmin production achieved the highest level of 6.6 x 10(4) cells x mL(-1) and 1.3 x 10(4) ng x L(-1), respectively. Compared with nitrogen sources, the growth of Anabaena sp. could be promoted significantly until phosphorus level attained 0.12 mg/L, indicating that phosphorus is the main limiting nutrient source for Anabaena sp.. For Yanghe reservoir, the nutrient level has already been enough for the growth of Anabaena sp. Therefore, the nutrient source content, especially phosphorus, should be reduced effectively to control the cyanobacterium bloom and taste and odor problems.

Requirements of Src family kinase during meiotic maturation in mouse oocyte.

The Src family kinase (SFK) is important in normal cell cycle control. However, its role in meiotic maturation in mammalian has not been examined. We used confocal microscope immunofluorescence to examine the in vitro dynamics of the subcellular distribution of SFK during the mouse oocyte meiotic maturation and further evaluated the functions of SFK via biochemical analysis using a specific SFK pharmacological inhibitor, PP(2). Our results showed that nonphospho-SFK was absent in oocyte upon its release from follicle. Nonphospho-SFK appeared in cytoplasm 0.5 hr after the release of oocyte and translocated to germinal vesicle (GV) before germinal vesicle breakdown (GVBD). After GVBD, nonphospho-SFK colocated with condensed chromosomes. In occyte at metaphase I (MI) and telophase I, nonphospho-SFK accumulated in the cortex and the cleavage furrow respectively besides its existence in cytoplasm in both stages. In oocyte at metaphase II (MII), nonphospho-SFK concentrated at the aligned chromosomes. In contrast, phospho-SFK was absent in oocyte until 1 hr after its release from the follicle. Phospho-SFK accumulated in the GV, the cortex, and cytoplasm immediately prior to GVBD. After GVBD, phospho-SFK evenly distributed in oocyte. In oocyte at MII, phospho-SFK localized throughout the cytoplasm and under the egg member. When the SFK activity was inhibited, the oocyte failed to initiate GVBD, could not go into MII, and could not extrude the first polar body. Our results demonstrated that SFK is required for meiotic maturation in mouse oocyte.