PHB Genes Research Articles

Characterization and functional analysis of the proteins Prohibitin 1 and 2 in Trypanosoma cruzi.

BackgroundChagas disease is the third most important neglected tropical disease. There is no vaccine available, and only two drugs are generally prescribed for the treatment, both of which with a wide range of side effects. Our study of T. cruzi PHBs revealed a pleiotropic function in different stages of the parasite, participating actively in the transformation of the non-infective replicative epimastigote form into metacyclic trypomastigotes and also in the multiplication of intracellular amastigotes.Methodology/principal findingsTo obtain and confirm our results, we applied several tools and techniques such as electron microscopy, immuno-electron microscopy, bioinformatics analysis and molecular biology. We transfected T. cruzi clones with the PHB genes, in order to overexpress the proteins and performed a CRISPR/Cas9 disruption to obtain partially silenced PHB1 parasites or completely silenced PHB2 parasites. The function of these proteins was also studied in the biology of the parasite, specifically in the transformation rate from non-infective forms to the metacyclic infective forms, and in their capacity of intracellular multiplication.Conclusion/significanceThis research expands our understanding of the functions of PHBs in the life cycle of the parasite. It also highlights the protective role of prohibitins against ROS and reveals that the absence of PHB2 has a lethal effect on the parasite, a fact that could support the consideration of this protein as a possible target for therapeutic action.

Genetic determinants of sporadic breast cancer in Sri Lankan women

BackgroundWhile a range of common genetic variants have been identified to be associated with risk of sporadic breast cancer in several Western studies, little is known about their role in South Asian populations. Our objective was to examine the association between common genetic variants in breast cancer related genes and risk of breast cancer in a cohort of Sri Lankan women.MethodsA case-control study of 350 postmenopausal women with breast cancer and 350 healthy postmenopausal women was conducted. Genotyping using the iPLEX GOLD assay was done for 56 haplotype-tagging single nucleotide polymorphisms (SNPs) in 36 breast cancer related genes. Testing for association was done using an additive genetic model. Odds ratios and 95% confidence intervals were calculated using adjusted logistic regression models.ResultsFour SNPs [rs3218550 (XRCC2), rs6917 (PHB), rs1801516 (ATM), and rs13689 (CDH1)] were significantly associated with risk of breast cancer. The rs3218550 T allele and rs6917 A allele increased breast cancer risk by 1.5-fold and 1.4-fold, respectively. The CTC haplotype defined by the SNPs rs3218552|rs3218550|rs3218536 on chromosome 7 (P = 0.0088) and the CA haplotype defined by the SNPs rs1049620|rs6917 on chromosome 17 (P = 0.0067) were significantly associated with increased risk of breast cancer. The rs1801516 A allele and the rs13689 C allele decreased breast cancer risk by 0.6-fold and 0.7-fold, respectively.ConclusionsThese findings suggest that common genetic polymorphisms in the XRCC2, PHB, CDH1 and ATM genes are associated with risk of breast cancer among Sri Lankan postmenopausal women. The exact biological mechanisms of how these variants regulate overall breast cancer risk need further evaluation using functional studies.

Statistical optimization of a culture medium for biomass and poly(3-hydroxybutyrate) production by a recombinant Escherichia coli strain using agroindustrial byproducts.

A statistically based Plackett-Burman screening design identified milk whey and corn steep liquor concentrations as well as ionic strength (based on phosphate buffer concentration) as the three main independent components of the culture medium that significantly (p < 0.05) influenced biomass and poly(3-hydroxybutyrate) (PHB) production in recombinant cells of Escherichia coli. This strain carries a plasmid encoding phb genes from a natural isolate of Azotobacter sp. Response surface methodology, using a central composite rotatable design, demonstrated that the optimal concentrations of the three components, defined as those yielding maximal biomass and PHB production in shaken flasks, were 37.96 g deproteinated milk whey powder/l, 29.39 g corn steep liquor/l, and 23.76 g phosphates/l (r2 = 0.957). The model was validated by culturing the recombinant cells in medium containing these optimal concentrations, which yielded 9.41 g biomass/l and 6.12 g PHB/l in the culture broth. Similar amounts of PHB were obtained following batch fermentations in a bioreactor. These results show that PHB can be produced efficiently by culturing the recombinant strain in medium containing cheap carbon and nitrogen sources.

Overexpression of miR165 Affects Apical Meristem Formation, Organ Polarity Establishment and Vascular Development in Arabidopsis

The class III homeodomain leucine-zipper (HD-ZIP III) genes are thought to be targets of microRNAs (miRNAs) 165 and 166, but it is not known whether all the developmental processes affected by mutations of the HD-ZIP III genes could be recapitulated by an alteration in the expression of miR165 and miR166. Previous work showed that overexpression of miR166 by activation tagging results in down-regulation of the ATHB-9/PHV, ATHB-14/PHB and ATHB-15 genes, and concomitantly causes an enlargement of shoot apical meristems (SAMs) and an enhancement in vascular development. Here we demonstrated that overexpression of miR165 causes a drastic reduction in the transcript levels of all five HD-ZIP III genes in Arabidopsis. The miR165 overexpressors display prominent phenotypes reminiscent of loss-of-function mutants of rev phb phv and rev/ifl1, including loss of SAM, alteration of organ polarity, abnormal formation of carpels, inhibition of vascular development and aberrant differentiation of interfascicular fibers. Global gene expression analysis revealed a link between miR165 overexpression and altered expression of genes involved in auxin signaling and vascular development. Our results demonstrate that overexpression of miR165 recapitulates the phenotypes caused by loss-of-function mutations of HD-ZIP III genes, such as loss of SAM, altered organ polarity and defects in development of vascular tissues and interfascicular fibers.

Effects of recombinant precursor pathway variations on poly[(R)-3-hydroxybutyrate] synthesis in Saccharomyces cerevisiae

Different recombinant R-3-hydroxybutyryl-CoA (3-HB) synthesis pathways strongly influenced the rate and accumulation of the biopolymer poly[(R)-3-hydroxybutyrate] (PHB) in Saccharomyces cerevisiae. It has been previously shown that expression of the Cupriavidus necator PHB synthase gene leads to PHB accumulation in S. cerevisiae [Leaf, T., Peterson, M., Stoup, S., Somers, D., Srienc, F., 1996. Saccharomyces cerevisiae expressing bacterial polyhydroxybutyrate synthase produces poly-3-hydroxybutyrate. Microbiology 142, 1169-1180]. This finding indicates that native S. cerevisiae expresses genes capable of synthesizing the correct stereochemical substrate for the synthase enzyme. The effects of variations of 3-HB precursor pathways on PHB accumulation were investigated by expressing combinations of C. necator PHB pathway genes. When only the PHB synthase gene was expressed, the cells accumulated biopolymer to approximately 0.2% of their cell dry weight. When the PHB synthase and reductase gene were co-expressed, the PHB levels increased approximately 18 fold to about 3.5% of the cell dry weight. When the beta-ketothiolase, reductase and synthase genes were all expressed, the strain accumulated PHB to approximately 9% of the cell dry weight which is 45 fold higher than in the strain with only the synthase gene. Fluorescent microscopic analysis revealed significant cell-to-cell heterogeneity in biopolymer accumulation. While the population average for the strain expressing three PHB genes was approximately 9% of the cell dry weight, some cells accumulated PHB in excess of 50% of their cell volume. Other cells accumulated no biopolymer. In addition, the recombinant strain was shown to co-produce ethanol and PHB under anaerobic conditions. These results demonstrate that the technologically important organism S. cerevisiae is capable of accumulating PHB aerobically and anaerobically at levels similar to some bacterial systems. The easily assayed PHB system also creates a convenient means of probing in vivo the presence of intracellular metabolites which could be useful for studying the intermediary metabolism of S. cerevisiae.

Gene expression patterns for metabolic pathway in pgi knockout Escherichia coli with and without phb genes based on RT-PCR

Metabolic regulations were investigated from the viewpoint of gene expressions for Escherichia coli JM109 and pgi knockout E. coli with and without phb genes using RT-PCR. One of the main features of pgi knockout E. coli is the overproduction of NADPH produced in pentose phosphate (PP) pathway. NADPH overproduction in PP pathway in pgi mutant causes some reducing power imbalance that ultimately affects the cell growth. It was shown that this reducing power imbalance can be recovered to some extent by introducing NADPH absorbing pathway such as PHB synthetic pathway into pgi mutant E. coli. To get insight into the regulation mechanism of pgi mutant E. coli at the transcriptional level, 87 E. coli genes involved in central metabolic pathways and key regulatory mechanisms were investigated by semi-quantitative RT-PCR analysis. The analysis showed that pentose phosphate pathway genes and part of the glycolysis pathway genes were affected significantly by expression of phb genes in pgi mutant E. coli DF11/pAeKG1 as well as in pgi mutant E. coli DF11 as compared with those in E. coli JM109. In contrast, most of the TCA cycle genes except icdA were downregulated in both pgi mutants E. coli. The upregulation of icdA gene may be due to the positive regulation of fruR. Moreover, it was found that ack gene as well as aceA and aceB genes involved in the glyoxylate shunt were upregulated in pgi mutants while ppc gene was downregulated, indicating that pgi inactivation changes the anaplerotic pathway from ppc pathway to glyoxylate shunt. Enzyme activities of glk, zwf, tpiA, fbaA, ldhA, gltA, aceA, mdh and maeB were also measured and compared with the corresponding gene expressions. Most of them are well correlated except for aceA gene indicating that glyoxylate pathway is regulated on the protein level, not on the gene level.

The pea ( Pisum sativum L.) rbcS transit peptide directs the Alcaligenes eutrophus polyhydroxybutyrate enzymes into the maize ( Zea mays L.) chloroplasts

With a concept of producing polyhydroxybutyrate (PHB), a biodegradable thermoplastic polymer in maize chloroplasts, over 200 independent transgenic maize plants were produced. Five different constructs, pPHBA, pPHBB, pPHBC and pBY520 or pJS 101, were used in transformation experiments. PHB constructs contained phb genes from Alcaligenes eutrophus fused with the pea Rubisco small subunit (rbcS) transit peptide (TP) plus the first 24 amino acids of the mature rbcS protein. Plasmids BY520 and JS 101 contained the selectable marker bar gene linked with abiotic stress-related gene either hva1 or mtlD. Southern blots on putative transgenic plants confirmed that the transgenic plants harbor transgenes from all five constructs. Transcription of transgenes was confirmed by Northern blot analyses. Western blot analyses using total cellular protein and protein from isolated chloroplasts confirmed that all the three genes produced their corresponding enzymes, and all PHB enzymes were targeted into maize chloroplasts. Immunofluorescent localization on both transgenic and non-transgenic maize chloroplasts treated with polyhydroxybutyrate synthase (PHBC)-antiserum also confirmed the targeting of PHBC enzyme to the chloroplasts of transgenic maize.

Yeasts as Producers of Polyhydroxyalkanoates: Genetic Engineering of Saccharomyces cerevisiae

The genes of the poly(β-hydroxybutyrate) (PHB) synthesis pathway in Ralstonia eutropha and Methylobacterium extorquens were successfully established in the yeast Saccharomyces cerevisiae. Expression of just the polyhydroxyalkanoate (PHA) synthase gene in some experiments, and all three PHB genes (i.e., the genes encoding β-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase) in others, were detected in S. cerevisiae. Thus, it can be used as a “cell factory” for the production of PHB. The maximum amount of polyester accumulated was 6.7% (wt./wt.) when all three genes were expressed. The amount of polymer accumulated in the transgenic yeast harboring just the PHA synthase gene was similar (5.2%), but slightly lower, indicating the necessity of expressing all three genes for high PHB contents in the cells. For viable production of the polymer in yeasts, more needs to be learned about the metabolism of the yeast, especially about the pathways and intermediates competing with formation of the biopolymer. Another host probably needs to be chosen. Bacteria (on the top) with PHB inclusions and yeasts with storage compounds (on the bottom).

Expression of bacterial poly(3-hydroxybutyrate) synthesis genes in hairy roots of sugar beet (Beta vulgaris L.).

Three genes from Ralstonia eutropha necessary for poly(3-hydroxybutyrate) (PHB) synthesis were introduced into the hairy roots of sugar beet. Transformation of a vector construct harbouring the PHB genes, each fused to the coding region of the pea ribulose-bisphosphate carboxylase plastid targeting sequence, resulted in 20 transgenic hairy-root clones, producing up to 55 mg high molecular PHB/g dry weight, as identified by gas chromatography, gel permeation chromatography and HPLC. Accumulation of PHB polymer in sugar beet root leucoplasts was confirmed by transmission electron microscopy. Thus, for the first time, plastidic PHB production was demonstrated for roots of a carbohydrate-storing crop plant.

Periodic change in DO concentration for efficient poly-β-hydroxy-butyrate production using temperature-inducible recombinantEscherichia coli with proteome analysis

Recombinant Escherichia coli strain harboring the λpR-pL promotor and heterologus poly-β- hydroxybutyrate (PHB) biosynthesis genes was used to investigate the effect of culture conditions on the efficient PHB production. The expression of phb genes was induced by a temperature upshift from 33°C to 38°C. The protein expression levels were measured by using two-dimensional electrophoresis, and the enzyme activities were also measured to understand the effect of culture temperature, carbon sources, and the dissolved oxygen (DO) concentration on the metabolic regulations. AcetylCoA is an important branch point for PHB production. The decrease in DO concentration lowers the citrate synthase activity, thus limit the flux toward the TCA cycle, and increase the flux for PHB production. Since NADPH is required for PHB production, the PHB production does not continue leading the overproduction of acetate and lac- tate. Based on these observations, a new operation was considered where DO concentration was changed periodically, and it was verified its usefulness for the efficient PHB production by experiments.

Proteome analysis of a temperature-inducible recombinant Escherichia coli for poly-β-hydroxybutyrate production

A recombinant Escherichia coli strain harboring the λ p R- p L promoter and heterologous poly-β-hydroxybutyrate (PHB) biosynthesis genes was shown to accumulate PHB when the incubation temperature was changed from 34°C to temperatures higher than 37°C. In the present research, total gene expression patterns of the recombinant E. coli before and after induction were investigated by two-dimensional gel electrophoresis. Proteins encoded by serS, sucC, trpA, and alaS were found to be expressed before induction of phb genes at a culture temperature of 34°C. On the other hand, proteins encoded by metG, rplI, and carA were found to be expressed after induction achieved by increasing the temperature to 40°C. In the case of plasmid-free cells, all the selected genes have been shown to be expressed except metG, and ibpA and ibpB among the heat-shock proteins. The heat-shock proteins were found to be upregulated upon induction of phb genes, which may be due to the stress caused by the accumulation of PHB granules as well as by the temperature upshift. The changes in the expression of some of the metabolic pathway-related proteins before and after induction were interpreted in relation to the consumption of NADPH and acetyl-CoA for PHB synthesis.

Temperature-Induced Expression of phb Genes in Escherichia coli and the Effect of Temperature Patterns on the Production of Poly-3-Hydroxybutyrate.

The plasmid pNDTM2 was constructed to contain the p(R)-p(L) promoter and the phbC, A, and B genes which code for PHB synthase, beta-ketothiolase, and acetoacetyl-CoA reductase, respectively. This plasmid was transformed into Escherichia coli XL1-Blue. The effect of several thermal induction patterns on the production of poly-3-hydroxybutyric acid (PHB) was investigated based on fermentor experiments. Based on the experimental results with different induction patterns, it was found that the temperature should be controlled at 34 degrees C during the initial 10 h of cultivation to promote cell growth, and then it should be increased to 40 degrees C for induction. Then the temperature should be lowered to 37 degrees C after 5 h to relieve the effect of the heat shock.

Temperature-induced expression of phb genes in Escherichia coli and the effect of temperature patterns on the production of poly-3-hydroxybutyrate

The plasmid pNDTM2 was constructed to contain the p R- p L promoter and the phbC, A, and B genes which code for PHB synthase, β-ketothiolase, and acetoacetyl-CoA reductase, respectively. This plasmid was transformed into Escherichia coli XL1-Blue. The effect of several thermal induction patterns on the production of poly-3-hydroxybutyric acid (PHB) was investigated based on fermentor experiments. Based on the experimental results with different induction patterns, it was found that the temperature should be controlled at 34°C during the initial 10 h of cultivation to promote cell growth, and then it should be increased to 40°C for induction. Then the temperature should be lowered to 37°C after 5 h to relieve the effect of the heat shock.

Proteome Analysis of a Temperature-Inducible Recombinant Escherichia coli for Poly-.BETA.-Hydroxybutyrate Production.

A recombinant Escherichia coli strain harboring the lambdap(R)-p(L) promoter and heterologous poly-beta-hydroxybutyrate (PHB) biosynthesis genes was shown to accumulate PHB when the incubation temperature was changed from 34 degrees C to temperatures higher than 37 degrees C. In the present research, total gene expression patterns of the recombinant E. coli before and after induction were investigated by two-dimensional gel electrophoresis. Proteins encoded by serS, sucC, trpA, and alaS were found to be expressed before induction of phb genes at a culture temperature of 34 degrees C. On the other hand, proteins encoded by metG, rplI, and carA were found to be expressed after induction achieved by increasing the temperature to 40 degrees C. In the case of plasmid-free cells, all the selected genes have been shown to be expressed except metG, and ibpA and ibpB among the heat-shock proteins. The heat-shock proteins were found to be upregulated upon induction of phb genes, which may be due to the stress caused by the accumulation of PHB granules as well as by the temperature upshift. The changes in the expression of some of the metabolic pathway-related proteins before and after induction were interpreted in relation to the consumption of NADPH and acetyl-CoA for PHB synthesis.

Purification of Aspergillus fumigatus (Pdf1) Poly(β-hydroxybutyrate) (PHB) Depolymerase Using a New, Single-Step Substrate Affinity Chromatography Method: Characterization of the PHB Depolymerase Exhibiting Novel Self-Aggregation Behavior

The extracellular poly(β-hydroxybutyrate) (PHB) depolymerase of Aspergillus fumigatus Pdf1 was purified by a new, simple, one-step affinity chromatography method using the substrate PHB. The purified enzyme was glycosylated, with the molecular mass of ≃40 KD, and exhibited a novel self-aggregation behavior by means of hydrophobic interaction that was resolved by Triton X-100 (TX-100) pretreatment of enzyme and also TX-100 incorporation in the native gel. The apparent Km value of purified enzyme for PHB was 119 μg/mL and 3-hydroxybutyrate was detected as the main endproduct of PHB hydrolysis. The depolymerase was insensitive to phenylmethyl sulfonyl fluoride (PMSF), sodium azide, ethylenediaminetetraacetic acid (EDTA), and para-chloromercuric benzoic acid (PCMB), but was inactivated by dithioerythritol (DTT) and showed specificity for short chain-length poly(β-hydroxyalkanoates) (PHAs) such as PHB, poly(hydroxyvalerate) (PHV), and copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). Medium-chain-length PHA failed to get hydrolyzed. The enzyme, however, exhibited strong cross reactivity with the Comamonas sp. PHB depolymerase antibodies, but not with PHV depolymerase antibodies of Pseudomonas lemoignei. Southern hybridization and dot blot analysis of A. fumigatus Pdf1 genomic DNA with alkaline phosphatase labeled probes of P. lemoignei PHB and PHV depolymerase genes revealed no homology, although the enzyme hydrolyzed both PHB and PHV.

Effects of recombinant modulation of the phbCAB operon copy number on PHB synthesis rates in Ralstonia eutropha

We have increased the gene dosage of the poly(3-hydroxybutyrate) biosynthesis operon in Ralstonia eutropha (formerly Alcaligenes eutrophus) to test whether PHB synthesis rates may be increased by recombinant methods. The native R. eutropha phbCAB operon was inserted into the broad-host-range vector pKT230. This PHB operon-containing plasmid, and a control plasmid containing the identical broad-host-range replicon but not the PHB genes, were transferred to R. eutropha H16. Analysis of whole-cell lysates indicated that the strain harboring the operon-containing plasmid possessed β-ketothiolase and acetoacetyl-CoA reductase specific activities that were 6.0 and 6.2 times elevated, respectively, as compared to the control strain with a single operon. After growth on fructose, PHB synthesis rates were sharply dependent on the type of carbon source offered during the PHB accumulation phase under nitrogen limitation. In the case of the strain harboring the control plasmid, and in comparison to fructose as carbon source, PHB accumulation was 2.15, 2.83, and 2.60 times faster when resuspended in nitrogen-free medium with lactate, acetate, or 3-hydroxybutyrate, respectively. The strain harboring the PHB operon-containing plasmid synthesized PHB at a lower specific rate in each case. During exponential growth on fructose, the strain harboring the control plasmid was again more efficient at forming PHB. These results suggest that increasing the intracellular concentration of PHB precursors may be a superior alternative to raising the levels of PHB enzymes for enhancing PHB productivity in R. eutropha.

Poly- -hydroxybutyrate (PHB) biosynthetic genes in Rhizobium meliloti 41

Genes encoding beta-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHB-synthase (phaC) from R. meliloti 41, together with a fourth gene, referred to as ORF1, presumed to be involved in PHB biosynthesis, have been cloned and sequenced. phaA, phaB and ORF1 were identified by heterologous hybridization on a cosmid library, while phaC was isolated by cloning the transposon-tagged fragment from a R. meliloti PHB- Tn5 mutant. phaA and phaB were functionally expressed in Escherichia coli while phaC was able to complement a PHB- strain of R. meliloti 41. The three genes were sufficient to direct the production of polyhydroxyalkanoate in E. coli. The homology of ORF1 with an ORF located near the PHB genes in two phototrophic bacteria suggests its involvement in PHB synthesis.