ISSN:0008-4166    

DOI:10.1139/m95-161

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

The biosynthesis of the 3-hydroxyvalerate (3HV) monomer of the polyhydroxyalkanoate produced from glucose byRhodococcus ruberwas investigated using nuclear magnetic resonance analysis. Spectra obtained for polymer produced from [6-13C]-, [2-13C]-, and [1-13C]glucose were compatible with carboxylation of pyruvate followed by TCA cycle reactions to yield succinate, which is a precursor of propionyl-CoA in the biosynthesis of 3HV in this organism. Carboxylation of pyruvate may occur directly or involve methylmalonyl-CoA transcarboxylase but the latter process is more probable as it provides a route for recycling oxaloacetate, which is essential for the pathway to operate. The observed incorporation of label from NaH13CO3into C-3 of 3HV monomers cannot occur via forward reactions of the TCA cycle but may be explained by direct carboxylation of pyruvate, followed by reverse reactions of the TCA cycle to yield succinyl-CoA. This minor route accounts for at least 12% of the carbon flux in the synthesis of 3HV monomer units. Inhibitors of key enzymes of possible pathways for polyhydroxyalkanoate biosynthesis inR.ruberwere tested. Addition of monofluoroacetate, an inhibitor of aconitase, to cultures decreased production of 3HV, supporting the proposed major route for 3HV synthesis.Key words:Rhodococcus ruber, polyhydroxyalkanoate, PHA, biosynthesis.

ISSN:0008-4166    

DOI:10.1139/m95-162

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

Poly(hydroxyalkanoates) (PHAs) were isolated fromPseudomonas aeruginosa44T1 cultivated on euphorbia oil and castor oil. With the aid of 2-D proton NMR spectra and proton-detected multiple bond coherence NMR spectra the structures of the PHAs were determined. In addition to the usual PHA constituents (C6–C143-hydroxy fatty acids), PHAs formed from euphorbia oil contained Δ8,9-epoxy-3-hydroxy-5c-tetradecenoate, and probably Δ6,7-epoxy-3-hydroxydodecanoate and Δ4,5-epoxy-3-hydroxydecanoate. These novel constituents account for approximately 15% of the total amount of monomers and are clearly generated via β-oxidation of vernolic acid (Δ12,13-epoxy-9c-octadecenoic acid), the main component of euphorbia oil. In PHAs formed from castor oil, 7% of the monomers found were derived from ricinoleic acid (12-hydroxy-9c-octadecenoic acid). The presence of 3,8-dihydroxy-5ctetradecenoate was clearly demonstrated. Furthermore, NMR analysis strongly suggested the presence of 3,6-dihydroxydodecanoate, 6-hydroxy-3c-dodecenoate, and 4-hydroxydecanoate.Key words: poly(hydroxyalkanoates), pseudomonads, ricinoleic acid, vernolic acid.

ISSN:0008-4166    

DOI:10.1139/m95-163

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

Azotobacter vinelandiiUWD cells fill with up to 80% (per dry mass) poly(β-hydroxybutyrate) (PHB) after 24 h growth in medium containing sugars and fish peptone. However, peptones were not usually added toAzotobacterculture as they induced pleomorphism and compromised cell wall strength. This study examines the morphology of these PHB-producing pleomorphic cells in the transmission electron microscope. PHB-producing cells incubated for 18–24 h were most frequently 2–3 μm diameter spheres containing up to 20 PHB inclusions/cross section, or a calculated ≈ 100 inclusions/cell volume. These inclusions tended to be of small size (≈ 0.5 μm diameter) and became fewer and larger in older cells. The most striking feature of these pleomorphic cells was the apparent extrusion of polymer from the cells. It is unlikely that PHB extrusion is an active process from a viable cell as there was considerable cell wall damage at the point of polymer extrusion. The results suggest that the extrusion of PHB may be the result of polymer expansion, caused by the dehydration of the specimen for transmission electron microscopy, coupled with the inability of the pleomorphic cell wall to retain the expanding polymer. Thus, freeze-substituted sections of similar cells that were prepared without chemical dehydration did not extrude PHB. However, lysed cells prepared for transmission electron microscopy by chemical dehydration also did not extrude PHB, which suggests differences in the fluidity of the PHB in intact cell inclusions and lysed cell granules.Key words: poly(β-hydroxybutyrate), inclusions, polymer expansion, dehydration artif

ISSN:0008-4166    

DOI:10.1139/m95-164

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

Novel functional poly(β-hydroxyalkanoates), PHAs, with hyperpolarizable side groups were prepared.Pseudomonas oleovoransATCC 29347 andPseudomonas putidaKT 2442 were selected as biocatalysts. These organisms were first grown on citrate (40 mM) before polymer formation was studied in a second stage. The carbon sources in second-stage cultivations were mixtures (total of 15 mM) of octanoate (OA) with either 7.5 or 10 mMpara-cyanophenoxyhexanoate (CPH),para-cyanophenoxyvalerate (CPV),para-cyanophenoxybutyrate (CPB), orpara-nitrophenoxyhexanoate (NPH). For both organisms, only small deviations in the number of colony-forming units per millilitre were observed when second-stage cultivations containing only 15 mM octanoate were compared with those using mixtures of OA with either CPB, CPV, CPH, or NPH. PHA volumetric yields as a function of organism, carbon source mixture, and culture time are reported. The percent incorporations of substituted phenoxy side groups in the polymer products were determined by1H NMR spectroscopy.Pseudomonas putidaformed PHAs with up to 24.2% 3-hydroxy-6-(para-cyanophenoxy)hexanoate side groups when 5 mM OA and 10 mM CPH were used. A dramatic decrease in the percent incorporation ofpara-cyanophenoxy (CP) byP.putidawas observed when the relatively shorter CP-substitutedn-alkanoate chain CPV and CPB cosubstrates were used. Use of NPH in place of CPH had deleterious effects on both polymer formation and percent incorporation of substituted phenoxy side groups inP.putida.Pseudomonas oleovoransformed PHAs with only up to ~2% CP side chains when a combination of OA and CPH was used and little to no CP side groups when a combination of OA and CPV or CPB was used. Substitution of NPH in place of CPH led to a modest increase (up to ~5%) in substituted phenoxy side groups. Thus, a new route to first-generation chiral polymer structures for nonlinear optical applications was demonstrated.Key words:Pseudomonas oleovorans,Pseudomonas putida, poly(β-hydroxyalkanoate), poly(3-hydroxy-6-para-cyanophenoxyhexanoate), poly(3-hydroxy-6-para-nitrophenoxyhexanoate), poly(β-hydroxy-para-cyanophenoxyvalerate).

ISSN:0008-4166    

DOI:10.1139/m95-165

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

Metabolites associated with the poly(3-hydroxybutyrate) (PHB) biosynthetic pathway inAlcaligenes eutrophuswere measured to gain an insight into the regulation of PHB synthesis in vivo.Alcaligenes eutrophuswas grown in carbon-limited chemostat culture to provide bacteria producing negligible PHB, and in nitrogen-limited chemostat culture to yield PHB-synthesizing bacteria. 3-Hydroxybutyryl-CoA (3HBCoA) was detected only in polymer-accumulating bacteria. The level of coenzyme A (CoASH) was approximately three times higher in the absence of PHB synthesis, in accord with the putative role of this metabolite in the regulation of 3-ketothiolase. The level of acetoacetyl-CoA was, however, similar in PHB-accumulating and nonaccumulating bacteria, suggesting that NADPH-acetoacetyl-CoA reductase may regulate PHB synthesis in bacteria grown under carbon limitation. Immediately after nitrogen exhaustion in batch culture ofA.eutrophus, there was an initial large decrease in the weight-average molecular weight, which corresponded to the rapid disappearance of CoASH and the maximum level of 3HBCoA. The decrease in the rate of PHB synthesis in batch culture was consistent with regulation involving NADPH-acetoacetyl-CoA reductase. The disappearance of 3HBCoA coincided with the cessation of PHB synthesis and the maximum level of acetyl-CoA.Key words: metabolites, PHB biosynthesis, regulation,Alcaligenes eutrophus, molecular weight.

ISSN:0008-4166    

DOI:10.1139/m95-166

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

It is increasingly clear that poly(3-hydroxybutyrate) (PHB) is not just an inert storage polymer, confined to certain bacteria, but a ubiquitous, interactive, solvating biopolymer involved in important physiological functions. Low molecular weight PHB, complexed to other macromolecules (c-PHB), is widely distributed in biological cells, being found in representative organisms of nearly all phyla. Complexation modifies the physical and chemical properties of c-PHB, allowing it to pervade aqueous as well as hydrophobic regions of the cell, and as a result c-PHB can be found in cytoplasm and intracellular fluids as well as in membranes and lipoproteins. The lipidic homopolymer associates with other macromolecules primarily via its ester carbonyl oxygens, which can act as hydrogen-bond acceptors or as ligands for coordinate bonds to cations. The spacing of the electron-donating groups along the flexible backbone allows for multiple bonding interactions, and forms the basis for the ability of c-PHB to bind to proteins, or to form ion-conducting complexes with salts. The singular ability of c-PHB to dissolve salts and facilitate their transfer across hydrophobic barriers defines a potential physiological niche for c-PHB in cell metabolism.Key words: polyhydroxybutyrate, polyphosphate, polymer electrolyte, ion transport.

ISSN:0008-4166    

DOI:10.1139/m95-167

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

The pink-pigmented facultatively methylotrophic bacteriumMethylobacterium rhodesianumMB 126 is able to grow on methanol as the sole source of carbon and energy. Under certain conditions, e.g., limitation of ammonium, phosphate, or oxygen, carbon from methanol is channeled into poly(3-hydroxybutyric acid) (PHB) whereas other polymers or metabolites are hardly overproduced. A mutant of this strain, which we isolated after chemical mutagenesis, is impaired in its ability to synthesize PHB. Under the conditions mentioned above, the mutant still accumulated PHB, but in the absence of ammonium it simultaneously synthesized PHB and a considerable amount of an exopolysaccharide. This phenomenon was surprising insofar as the wild type did not produce exopolysaccharide in such amounts. An attempt was made to elucidate and discuss the possible reasons for these findings.Key words: methylotrophy, serine pathway bacteria, PHB, exopolysaccharides.

ISSN:0008-4166    

DOI:10.1139/m95-168

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

Hydrogenophaga pseudoflava(formerlyPseudomonas pseudoflava) was able to accumulate a large amount of copolyesters when grown on mixed substrates of glucose and lactones in a batch fermentation. Lactones such as γ-butyrolactone, γ-valerolactone, and higher analogues generally did not support cell growth when used as the sole carbon source. Co-feeding of lactones with glucose enhanced the utilization of lactones for both copolyester accumulation and cell growth. The copolyester from the cells grown on the mixed substrates of glucose (10 g/L) and γ-valerolactone (1–3 mL/L) was poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)), while cells grown with γ-butyrolactone (1–3 mL/L) as a cosubstrate produced poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)). The values of parameterD, calculated from the NMR dyad-sequence data for polymer samples obtained after 72 h of cultivation, showed lactone concentration dependences differing among lactones. The time-course data obtained from growth on the cosubstrates of γ-valerolactone (2 mL/L) and glucose (10 g/L) revealed that 3HV-rich copolymers were synthesized in the early growth phase, and the 3HB-rich fraction steadily increased in the later accumulation phase and then peaked at 80 h when γ-valerolactone was depleted. These polyhydroxyalkanoate accumulation profiles suggested a highDvalue of the final product, whose value was determined to be 3.25. γ-Valerolactone was consumed faster than γ-butyrolactone. The difference between the assimilation behavior of the two lactones was discussed in relation to the heterogeneity of the final copolyester products. A correlation between NMR microstructure and the physiology of polyhydroxyalkanoate accumulation was observed.Key words: microstructural heterogeneity, bacterial copolyesters, lactones,Hydrogenophaga pseudoflava.

ISSN:0008-4166    

DOI:10.1139/m95-169

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

Two distinct crotonyl-CoA hydratases ofMethylobacterium rhodesianumMB 126 were separated by column chromatography on DEAE-Sepharose CL-6B. The two enzymes were further purified by chromatography on red 120 agarose and Mono Q. Enzyme A was specific forL(+)-hydroxybutyryl-CoA. It had an apparent molecular weight of 160 000 with two identical subunits of 43 000 and 34 000. The apparentKmvalues forL(+)-hydroxybutyryl-CoA and crotonyl-CoA were 83 and 90 μM, respectively. Enzyme B was specific forD(−)-hydroxybutyryl-CoA. It had an apparent molecular weight of 39 000 with identical subunits of 12 500. It showed sigmoidal kinetics for crotonyl-CoA, and the Hill coefficient was about 2.5. The apparentKmvalue forD(−)-hydroxybutyryl-CoA was 0.5 mM. The possible contribution of a sequence including β-ketothiolase, NADH-linkedL(+)-specific acetoacetyl-CoA reductase, and two stereospecific crotonyl-CoA hydratases to PHB synthesis in methylotrophic serine-pathway bacteria is discussed.Key words: poly(β-hydroxybutyrate), PHB, crotonyl-CoA hydratase, methylotroph,Methylobacterium rhodesianum.

ISSN:0008-4166    

DOI:10.1139/m95-170

出版商:NRC Research Press    

年代:1995

数据来源: NRC