The atf2 gene is involved in triacylglycerol biosynthesis and accumulation in the oleaginous Rhodococcus opacus PD630

Rhodococcus opacus PD630 is an oleaginous bacterium able to accumulate large amounts of triacylglycerols (TAG) in different carbon sources. The last reaction for TAG biosynthesis is catalyzed by the bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) enzymes encoded by atf genes. R. opacus PD630 possesses at least 17 putative atf homologous genes in its genome, but only atf1 and atf2 exhibited a significant DGAT activity when expressed in E. coli, as revealed in a previous study. The contribution of atf1 gene to TAG accumulation by strain PD630 has been demonstrated previously, although additional Atfs may also contribute to lipid accumulation, since the atf1-disrupted mutant is still able to produce significant amounts of TAG (Alvarez et al., Microbiology 154:2327–2335, 2008). In this study, we investigated the in vivo role of atf2 gene in TAG accumulation by R. opacus PD630 by using different genetic strategies. The atf2-disrupted mutant exhibited a decrease in TAG accumulation (up to 25–30 %, w/w) and an approximately tenfold increase in glycogen formation in comparison with the wild-type strain. Surprisingly, in contrast to single mutants, a double mutant generated by the disruption of atf1 and atf2 genes only showed a very low effect in TAG and in glycogen accumulation under lipid storage conditions. Overexpression of atf1 and atf2 genes in strain PD630 promoted an increase of approximately 10 % (w/w) in TAG accumulation, while heterologous expression of atf2 gene in Mycobacterium smegmatis caused an increase in TAG accumulation during cultivation in nitrogen-rich media. This study demonstrated that, in addition to atf1 gene, atf2 is actively involved in TAG accumulation by the oleaginous R. opacus PD630.     

Comparative and functional genomics of Rhodococcus opacus PD630 for biofuels development

The Actinomycetales bacteria Rhodococcus opacus PD630 and Rhodococcus jostii RHA1 bioconvert a diverse range of organic substrates through lipid biosynthesis into large quantities of energy-rich triacylglycerols (TAGs). To describe the genetic basis of the Rhodococcus oleaginous metabolism, we sequenced and performed comparative analysis of the 9.27 Mb R. opacus PD630 genome. Metabolic-reconstruction assigned 2017 enzymatic reactions to the 8632 R. opacus PD630 genes we identified. Of these, 261 genes were implicated in the R. opacus PD630 TAGs cycle by metabolic reconstruction and gene family analysis. Rhodococcus synthesizes uncommon straight-chain odd-carbon fatty acids in high abundance and stores them as TAGs. We have identified these to be pentadecanoic, heptadecanoic, and cis-heptadecenoic acids. To identify bioconversion pathways, we screened R. opacus PD630, R. jostii RHA1, Ralstonia eutropha H16, and C. glutamicum 13032 for growth on 190 compounds. The results of the catabolic screen, phylogenetic analysis of the TAGs cycle enzymes, and metabolic product characterizations were integrated into a working model of prokaryotic oleaginy.     

Glycogenformation by Rhodococcus species and the effect of inhibition of lipid biosynthesis on glycogen accumulation in Rhodococcus opacus PD630

Members of the genus Rhodococcus were investigated for their ability to produce glycogen during cultivation on gluconate or glucose. Strains belonging to Rhodococcus ruber, Rhodococcus opacus, Rhodococcus fascians, Rhodococcus erythropolis and Rhodococcus equi were able to produce glycogen up to 0.2–5.6% of cellular dry weight (CDW). The glycogen content varied from 0.8% to 3.2% of CDW in cells of R. opacus PD630, which is a well-known oleaginous bacterium, during the exponential growth phase, when cultivated on diverse carbon sources. Maltose and pyruvate promoted glycogen accumulation by cells of strain PD630 to a greater extent than glucose, gluconate, lactose, sucrose or acetate. This strain was able to produce triacylglycerols, polyhydroxyalkanoates and glycogen as storage compounds during growth on gluconate, although triacylglycerols were always the main product under the conditions of this study. Cerulenin, an inhibitor of de novo fatty acid synthesis, inhibited the accumulation of triacylglycerols from gluconate and increased the content of polyhydroxyalkanoates (from 2.0% to 4.2%, CDW) and glycogen (from 0.1% to 3.0%, CDW). An increase of the polyhydroxyalkanoates and glycogen content was also observed in two mutants of R. opacus PD630, which produced reduced amounts of triacylglycerols during cultivation of cells on gluconate.     

Formation of blends of various poly(3-hydroxyalkanoic acids) by a recombinant strain of Pseudomonas oleovorans

Summary Recombinant strains of Pseudomonas oleovorans, which harbour the poly(3-hydroxybutyrate)-biosynthetic genes of Alcaligenes eutrophus, accumulated poly(hydroxyalkanoates), composed of 3-hydroxybutyrate(3HB), 3-hydroxyhexanoate (3HHx) and 3-hydroxyactanoate (3HO), up to 70% of the cell dry weight if the cells were cultivated with sodium octanoate as the carbon source. Physiological and chemical analysis revealed multiple evidence that this polymer is a blend of the homopolyester poly(3HB) and of the copolyester poly(3HHx-co-3HO) rather than a random or a block copolyester of 3HB, 3HHx and 3HO. The molar ratio between poly(3HHx-co-3HO) and poly(3HB) varied drastically during the process of fermentation. Whereas synthesis of poly(3HHx-co-3HO) started immediately after ammonium was exhausted in the medium, synthesis of poly(3HB) occurred only after a lag-phase. From freeze-dried cells poly(3HHx-co-3HO) was much more readily extracted with chloroform than was poly(3HB). The blend was fractionated into petrol-ether-insoluble poly(3HB) and petrol-ether-soluble poly(3HHx-co-3HO). The molecular weight values of these polyesters measured by gel permeation chromatography were 2.96 × 10⁶ and 0.35 × 10⁶ and were similar of those polymers accumulated by A. eutrophus or by wild-type P. oleovorans, respectively. Offprint requests to: A. Steinbüchel     

In vitro effects of sterculic acid on lipid biosynthesis in Rhodococcus opacus strain PD630 and isolation of mutants defective in fatty acid desaturation

The in vivo effects of sterculic acid methyl ester on triacylglycerol fatty acid composition in the oleaginous, hydrocarbon-degrading bacterium R. opacus strain PD630 was investigated. Sterculic acid, a cyclopropene fatty acid and an inhibitor of the stearoyl-CoA desaturase system, strongly inhibited the synthesis of monoenic fatty acids, of saturated fatty acids with more than 16 carbon atoms and of odd-numbered fatty acids when added to the culture medium. In addition, chemical mutagenesis and the application of the penicillin enrichment technique provided mutants, which were more or less completely impaired in the desaturation of long-chain fatty acids and exhibited in some cases a similar fatty acid composition like the wild-type in the presence of sterculic acid methyl ester. The implications of these findings for fatty acid metabolism in R. opacus strain PD630 are discussed.     

Identification, cloning and sequence analysis of the poly(3-hydroxyalkanoic acid) synthase gene of the Gram-positive bacterium Rhodococcus ruber

The first polyhydroxyalkanoic acid (PHA) synthase gene (phbCRr) of a Gram-positive bacterium was cloned from a genomic library of Rhodococcus ruber in the broad-host-range plasmid vector pRK404. The hybrid plasmid harboring phbCRr allowed the expression of polyhydroxybutyric acid (PHB) synthase activity and restored the ability of PHB synthesis in a PHB-negative mutant of Alcaligenes eutrophus. Nucleotide sequence analysis of phbCRr revealed an open reading frame of 1686 bp starting with the rare codon TTG and encoding a protein of relative molecular mass 61,371. The deduced amino acid sequence of phbCRr exhibited homologies to the primary structures of the PHA synthases of A. eutrophus and Pseudomonas oleovorans. Preparation of PHA granules by discontinuous density gradient centrifugation of crude cellular extracts revealed four major bands in an SDS polyacrylamide gel. A Mr 61,000 protein was identified as the PHA synthase of R. ruber by N-terminal amino acid sequence determination.     

Staining method of poly(3-hydroxyalkanoic acids) producing bacteria by nile blue

Summary Using an ethanol solution of nile blue, we have developed an efficient method to detect the colonies of poly(3-hydroxyalkanoic acids) (PHA) producing bacteria on the agar plate. When the bacterial colonies with PHA granules were stained with nile blue, the stained colonies fluoresced bright orange on the irradiation of UV light. In the fluoresce emission spectra, fluorescence intensity increased with an increase in the PHA content of bacterial cells.Alcaligenes eutrophus andA.latus colonies with poly(3-hydroxybutyric acid) (PHB) homopolymer exhibited an emission maximum at 580nm on the excitation at 490nm. On the other hand,Pseudomonas oleovorans andP.putida with medium-chain-length (mcl-) PHA copolymers of C6, C8 and C10 units exhibited an emission maximum at 570nm.     

Isolation and identification of granule-associated proteins relevant for poly(3-hydroxyalkanoic acid) biosynthesis in Chromatium vinosum D

Abstract Poly(3-hydroxybutyric acid) granules, which harbored only four major granule-associated proteins as revealed by SDS polyacrylamide gel electrophoresis, were isolated from crude cellular extracts of Chromatium vinosum D by centrifugation in a linear sucrose gradient. N-Terminal amino acid sequence determination identified two proteins of M _r 41 000 and M _{r 40 000} as the phaE _Cv and phaC _Cv translational products, respectively, of C. vinosum D. In a previous study it was shown that both proteins are required for the expression opf poly(3-hydroxyalkanoic acid) synthase activity. The N-terminus of the third protein ( M _r 17 000) exhibited no homology to other proteins. Lysozyme, which was during purification of the granules, exhibited a strong affinity to PHB granules and was identified as the fourth protein enriched with the granules.     

Establishment of a gene transfer system for Rhodothermus marinus

Genetic manipulation of Rhodothermus marinus has been hampered by the lack of a selection system for gene transfer. We report construction of a Rhodothermus/Escherichia coli shuttle plasmid, containing the R. marinus trpB gene, based on pUC18 and the cryptic R. marinus plasmid pRM21. A plasmid-less R. marinus recipient strain was selected on the basis of growth characteristics and absence of restriction activity. The shuttle plasmid, pRM100, was successfully introduced into a TrpB^– mutant of the recipient strain using electroporation and was found to transform it to prototrophy. No loss or rearrangement of pRM100 was observed after growth for 80 generations in non-selective medium. The relative copy numbers of pRM100 and of the parental plasmid, pRM21, were determined as 7±1 and 42±4, respectively. The shuttle plasmid was used to optimize an electroporation protocol, and the maximal number of transformants obtained was 4.3±0.7×10⁶ cfu/g DNA at 22.5 kV/cm, 200 and 25 F. Transformation failed, however, after chemical preparation of cells according to several protocols. This is the first report of genetic transformation in the genus Rhodothermus.     

Recent advances in rational gene transfer vector design based on poly(ethylene imine) and its derivatives

The continually increasing wealth of knowledge about the role of genes involved in acquired or hereditary diseases renders the delivery of regulatory genes or nucleic acids into affected cells a potentially promising strategy. Apart from viral vectors, non-viral gene delivery systems have recently received increasing interest, due to safety concerns associated with insertional mutagenesis of retro-viral vectors. Especially cationic polymers may be particularly attractive for the delivery of nucleic acids, since they allow a vast synthetic modification of their structure enabling the investigation of structure-function relationships. Successful clinical application of synthetic polycations for gene delivery will depend primarily on three factors, namely (1) an enhancement of the transfection efficiency, (2) a reduction in toxicity and (3) an ability of the vectors to overcome numerous biological barriers after systemic or local administration. Among the polycations presently used for gene delivery, poly(ethylene imine), PEI, takes a prominent position, due to its potential for endosomal escape. PEI as well as derivatives of PEI currently under investigation for DNA and RNA delivery will be discussed.This review focuses on structure-function relationships and the physicochemical aspects of polyplexes which influence basic characteristics, such as complex formation, stability or in vitro cytotoxicity, to provide a basis for their application under in vivo conditions. Rational design of optimized polycations is an objective for further research and may provide the basis for a successful cationic polymer-based gene delivery system in the future.     

Structural identification of polyhydroxyalkanoic acid (PHA) containing 4-hydroxyalkanoic acids by gas chromatography-mass spectrometry (GC-MS) and its application to bacteria screening

The methanolysis products of polyhydroxyalkanoic acids (PHAs) containing 4-hydroxybutyric acid (4HB), 4-hydroxyvaleric acid (4HV), and 4-hydroxyhexanoic acid (4HHx), when analyzed by GC-MS, showed two major chromatographic peaks with characteristic retention times of each methyl ester of 4-hydroxyalkanoic acid and the corresponding g-lactone (-butyrolactone, -valerolactone, -caprolactone, respectively). The method and results of GC-MS could be incorporated into an efficient screening procedure for isolation of bacterial strains which could accumulate a PHA containing 4-hydroxyalkanoic acid as the principal monomer from structurally related carbon substrates.     

Metabolic engineering of Escherichia coli for enhanced biosynthesis of poly(3-hydroxybutyrate) based on proteome analysis

We have previously analyzed the proteome of recombinant Escherichia coli producing poly(3-hydroxybutyrate) [P(3HB)] and revealed that the expression level of several enzymes in central metabolism are proportional to the amount of P(3HB) accumulated in the cells. Based on these results, the amplification effects of triosephosphate isomerase (TpiA) and fructose-bisphosphate aldolase (FbaA) on P(3HB) synthesis were examined in recombinant E. coli W3110, XL1-Blue, and W lacI mutant strains using glucose, sucrose and xylose as carbon sources. Amplification of TpiA and FbaA significantly increased the P(3HB) contents and concentrations in the three E. coli strains. TpiA amplification in E. coli XL1-Blue lacI increased P(3HB) from 0.4 to 1.6 to g/l from glucose. Thus amplification of glycolytic pathway enzymes is a good strategy for efficient production of P(3HB) by allowing increased glycolytic pathway flux to make more acetyl-CoA available for P(3HB) biosynthesis.     

重组角质酶的发酵制备及其对涤纶纤维的表面改性

对大肠杆菌表达嗜热子囊菌Thermobifida fusca角质酶的摇瓶诱导条件及3 L发酵罐扩大培养进行了研究,并探讨了角质酶对涤纶纤维的改性作用。结果表明,在摇瓶培养中,采用工业级TB培养基,用2 g/L乳糖诱导,菌体培养至对数生长前期添加0.5％甘氨酸,角质酶产量可达到128 U/mL。在3 L发酵罐扩大培养中,补料培养生物量 (OD600) 最大达到35,角质酶酶活最高达506 U/mL,是迄今国内外报道细菌来源角质酶的最高水平。紫外分光光度法分析初步表明涤纶纤维经角质酶水解产生了对苯二甲酸类物质     

Stimulatory effects of amino acids and oleic acid on poly(3-hydroxybutyric acid) synthesis by recombinant Escherichia coli

Addition of cysteine, isoleucine, methionine, or proline promoted poly(3-hydroxybutyric acid) [PHB] synthesis by recombinant Escherichia coli more than two-fold. Oleic acid also enhanced PHB synthesis more than three-fold. A PHB concentration of 70 g/l could be obtained by fed-batch culture of recombinant E. coli in a defined medium supplemented with small amounts of isoleucine, methionine, and proline. The stimulatory effects of amino acids and oleic acid on PHB synthesis seems to be due to the availability of more acetyl-CoA and/or NADPH.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by recombinant Escherichia coli harboring propionyl-CoA synthase gene (prpE) or propionate permease gene (prpP)

In order to enhance 3-hydroxyvalerate (3HV) fraction in copolyesters of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the propionate permease gene prpP or the propionyl-CoA synthase gene prpE was transformed into Escherichia coli XL10-Gold with co-expression of PHB operon (phaCAB) from Ralstonia eutropha. The recombinant E. coli strains were cultured on mixed carbon sources composed of glucose and propionic acid to promote PHBV accumulation. It was shown that the over-expression of prpE suppressed 3HV incorporation into PHBV copolymer, which led to reduced 3HV fraction. In contrast, the over-expression of prpP improved the 3HV content from 5.6 to 14.3 mol%, followed by an increased PHBV accumulation up to 62 wt%. The results showed that the expression of prpP stimulated the uptake and utilization of propionic acid and increased the 3HV fraction in PHBV. However, the over-expression of prpE in E. coli did not affect 3HV content in PHBV. Surprisingly, co-expression of prpE and prpP did not lead to any 3HV formation. This study showed the possibility to change the PHBV composition without overdose of propionic acid which is expensive and toxic for the cells.     

Highly efficient gene transfer with degradable poly(ester amine) based on poly(ethylene glycol) diacrylate and polyethylenimine in vitro and in vivo

BACKGROUND: Polyethylenimine (PEI) is toxic although it is one of the most successful and widely used gene delivery polymers with the aid of the proton sponge effect. Therefore, development of new novel gene delivery carriers having high efficiency with less toxicity is necessary. METHODS: In this study, a degradable poly(ester amine) carrier based on poly(ethylene glycol) diacrylate (PEGDA) and low molecular weight linear PEI was prepared. Furthermore, we compared the gene expression of the polymer/DNA complexes using two delivery methods: intravenous administration as an invasive method and aerosol as a non-invasive method. RESULTS: The synthesized polymer had a relatively small molecular weight (MW = 7980) with 25 h half-life in vitro. The polymer/DNA complexes were formed at an N/P ratio of 9. The particle sizes and zeta-potentials of the complexes were dependent on N/P ratio. Compared to PEI 25K, the newly synthesized polymer exhibited high transfection efficiency with low toxicity. Poly(ester amine)-mediated gene expression in the lung and liver was higher than that of the conventional PEI carrier. Interestingly, non-invasive aerosol delivery induced higher gene expression in all organs compared to intravenous method in an in vivo mice study. Such an expressed gene via a single aerosol administration in the lung and liver remained unchanged for 7 days. CONCLUSIONS: Our study demonstrates that poly(ester amine) may be applied as an useful gene carrier.     

An amperometric biosensor based on hemoglobin immobilized in poly(epsilon-caprolactone) film and its application

In this study, poly(varepsilon-caprolactone) (PCL) was synthesized using the varepsilon-caprolactone (CL) monomer ring-opening polymerization. We demonstrated that the hemoglobin (Hb) entrapped in PCL film could retain its original conformation by FT-IR spectra. A pair of well-defined redox peaks with a formal potential (E0') of about -0.38V (vs. SCE) in a pH 7.0 phosphate buffer solution was obtained at the Hb-PCL film modified GC electrode. The dependence of [Formula: see text] on the pH of the buffer solution indicated that the conversion of heme Fe(III)/Fe(II) was a reaction of one electron coupled to one proton. The apparent heterogeneous electron transfer rate constants (ks) of Hb confined to PCL was valuated as (18.7+/-0.8)s(-1) according to Laviron's equation. The surface concentration (Gamma*) of the electroactive Hb in the PCL film was estimated to be (7.27+/-0.57)x10(-11)molcm(-2). The Hb-PCL film modified electrode was shown to be an excellent amperometric sensor for the detection of hydrogen peroxide. The linear range is from 2 to 30microM with a detection limit of 6.07x10(-6)M. The sensor was effectively testified by the determination of the hydrogen peroxide in eyedrops as real samples.