Human Fidgetin is a microtubule severing the enzyme and minus-end depolymerase that regulates mitosis

Fidgetin is a member of the AAA protein superfamily with important roles in mammalian development. Here we show that human Fidgetin is a potent microtubule severing and depolymerizing the enzyme used to regulate mitotic spindle architecture, dynamics and anaphase A. In vitro, recombinant human Fidgetin severs taxol-stabilized microtubules along their length and promotes depolymerization, primarily from their minus-ends. In cells, human Fidgetin targets to centrosomes, and its depletion with siRNA significantly reduces the velocity of poleward tubulin flux and anaphase A chromatid-to-pole motion. In addition, the loss of Fidgetin induces a microtubule-dependent enlargement of mitotic centrosomes and an increase in the number and length of astral microtubules. Based on these data, we propose that human Fidgetin actively suppresses microtubule growth from and attachment to centrosomes.     

Enhanced polyhydroxybutyrate (PHB) production via the coexpressed phaCAB and vgb genes controlled by arabinose PBAD promoter in Escherichia coli

Aim: To develop an approach to enhance polyhydroxybutyrate (PHB) production via the coexpressed phaCAB and vgb genes controlled by arabinose P_BAD promoter in Escherichia coli. Method and Results: The polyhydroxyalkanoates (PHAs) synthesis operon, (phaCAB), from Ralstonia eutropha was overexpressed under the regulation of the arabinose P_BAD promoter in Escherichia coli, and the vgb gene encoding bacterial haemoglobin from Vitreoscilla stercoraria (VHb) was further cloned at downstream of phaCAB to form an artificial operon. The cell dry weight (CDW), PHB content and PHB concentration were enhanced around 1·23‐, 1·57‐, and 1·93‐fold in the engineered cell harbouring phaCAB–vgb (SY‐2) upon 1% arabinose induction compared with noninduction (0% arabinose). Furthermore, by using a recombinant strain harbouring P_BAD promoter‐vgb along with native promoter‐phaCAB construction, the effect of vgb expression level on PHB biosynthesis was positive correlation. Conclusions: The results exploit the possibility to improve the PHB production by fusing the genes phaCAB–vgb from different species under the arabinose regulation system in E. coli. It also demonstrates that increase in VHb level enhances the PHB production. Significance and Impact of the Study: We were successful in providing a new coexpressed system for PHB synthesis in E. coli. This coexpressed system could be regulated by arabinose inducer, and is more stable and cheaper than other induced systems (e.g. IPTG). Furthermore, it could be applied in many biotechnology or fermentation processes.     

Characterization of an extracellular medium-chain-length poly(3-hydroxyalkanoate) depolymerase from Streptomyces sp. KJ-72

A bacterial strain capable of degrading medium-chain-length polyhydroxyalkanoates (MCL-PHAs) was isolated from a soil sample. This organism, which was identified as Streptomyces sp. KJ-72, secreted MCL-PHA depolymerase into the culture fluid only when it was cultivated on MCL-PHAs. The extracellular MCL-PHA depolymerase of the organism was purified to electrophoretic homogeneity by ion exchange column chromatography and gel filtration. The enzyme consisted of a monomeric subunit having a molecular mass of 27.1 kDa and isoelectric point of 4.7. The maximum activity was observed at pH 8.7 and 50 °C. The enzyme was sensitive to N-bromosuccinimide and acetic anhydride, indicating the presence of tryptophan and lysine residues in the catalytic domain. The enzyme was able to hydrolyze various chain-length p-nitrophenyl esters of fatty acids and polycaprolactone as well as various types of MCL-PHAs. However, lipase activity of the enzyme was not detected. The main hydrolysis product of poly(3-hydroxyheptanoate) was identified to be the dimer of 3-hydroxyheptanoate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enhanced polyhydroxybutyrate production in transgenic sugarcane

Polyhydroxybutyrate (PHB) is a bacterial polyester that has properties similar to some petrochemically produced plastics. Plant-based production has the potential to make this biorenewable plastic highly competitive with petrochemical-based plastics. We previously reported that transgenic sugarcane produced PHB at levels as high as 1.8% leaf dry weight without penalty to biomass accumulation, suggesting scope for improving PHB production in this species. In this study, we used different plant and viral promoters, in combination with multigene or single-gene constructs to increase PHB levels. Promoters tested included the maize and rice polyubiquitin promoters, the maize chlorophyll A/B-binding protein promoter and a Cavendish banana streak badnavirus promoter. At the seedling stage, the highest levels of polymer were produced in sugarcane plants when the Cavendish banana streak badnavirus promoter was used. However, in all cases, this promoter underwent silencing as the plants matured. The rice Ubi promoter enabled the production of PHB at levels similar to the maize Ubi promoter. The maize chlorophyll A/B-binding protein promoter enabled the production of PHB to levels as high as 4.8% of the leaf dry weight, which is approximately 2.5 times higher than previously reported levels in sugarcane. This is the first time that this promoter has been tested in sugarcane. The highest PHB-producing lines showed phenotypic differences to the wild-type parent, including reduced biomass and slight chlorosis.     

Human Fidgetin is a microtubule severing the enzyme and minus-end depolymerase that regulates mitosis

Fidgetin is a member of the AAA protein superfamily with important roles in mammalian development. Here we show that human Fidgetin is a potent microtubule severing and depolymerizing the enzyme used to regulate mitotic spindle architecture, dynamics and anaphase A. In vitro, recombinant human Fidgetin severs taxol-stabilized microtubules along their length and promotes depolymerization, primarily from their minus-ends. In cells, human Fidgetin targets to centrosomes, and its depletion with siRNA significantly reduces the velocity of poleward tubulin flux and anaphase A chromatid-to-pole motion. In addition, the loss of Fidgetin induces a microtubule-dependent enlargement of mitotic centrosomes and an increase in the number and length of astral microtubules. Based on these data, we propose that human Fidgetin actively suppresses microtubule growth from and attachment to centrosomes.     

The production of polyhydroxybutyrate by Methylobacterium rhodesianum and Ralstonia eutropha in media containing glycerol and casein hydrolysates

Methylobacterium rhodesianum and Ralstonia eutropha were cultivated to produce polyhydroxybutyrate (PHB) using media which contained glycerol and casein hydrolysates as C/N-substrates. In these media the pH had not to be regulated during the fermentations. The first strain accumulated an average of 39% PHB during 92 h of cultivation in flasks and 50% PHB during 45 h of cultivation in fermenters. The second one yielded an average of 47% PHB during 67 h of cultivation using casein peptone and 65% PHB during 45 h of cultivation using Casamino acids in the medium. Calculated N-balances showed that about 65% of the supplied nitrogen was used for growth of non-PHB cell dry mass. The conversion of glycerol to PHB was 17% (w/w).     

Over-expression of 3-ketoacyl-ACP synthase III or malonyl-CoA-ACP transacylase gene induces monomer supply for polyhydroxybutyrate production in Escherichia coli HB101

A Pseudomonas sp. 61-3 malonyl-CoA-ACP transacylase gene (fabD _Ps) was cloned by Southern analysis using an equivalent gene of Escherichia coli (fabD _Ec) as a probe. Some recombinant E. coli HB101 strains harboring fabD _Ps, fabD _Ec, or E. coli 3-ketoacyl-ACP synthase III gene (fabH _Ec) with Aeromonas caviae polyhydroxyalkanoate synthase gene (phaC _Ac) were constructed and grown on one-stage cultivation in Luria-Bertani broth containing glucose as carbon source. These strains accumulated 5 to 11 wt% of poly (3-hydroxybutyrate) (PHB) within cells. Over-expression of fabH _Ec, fabD _Ec, or fabD _Ps has been suggested to lead the monomer-supply of (R)-3-hydroxybutyryl-CoA for PHB synthesis in E. coli cells.