In vitro synthesis of poly(3-hydroxydecanoate): purification and enzymatic characterization of type II polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa

For the first time, the purification has been achieved of the type II polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa applying N-terminal His₆-tag fusions and metal chelate affinity chromatography. In vivo His₆-tagged PHA synthase activity was confirmed by functional expression of the corresponding genes in Escherichia coli, and PHA synthase activity could also be measured in vitro with the enzymes. The specific enzyme activity of PHA synthases PhaC1 and PhaC2 was 0.039 U mg⁻¹ and 0.035 U mg⁻¹ protein, respectively. Kinetic studies showed a lag phase for both PHA synthases using (R,S)-3-hydroxydecanoyl-CoA as substrate. Specific enzyme activity was increased to 0.055 U mg⁻¹ when the phasin GA24 from Ralstonia eutropha was added to the assay. CoA inhibited PHA synthase activity, and a K _i of 85 μM was determined. A two-enzyme system was established, employing commercially available acyl-CoA synthetase and PHA synthase, which allowed the in vitro de novo PHA granule formation and the in vitro synthesis of poly(3-hydroxydecanoate) exhibiting a weight average molar mass of 9.8 × 10⁴ g mol⁻¹, and which occurred independently of pre-existing PHA granules. Received: 3 December 1999 / Revision received: 10 January 2000 / Accepted: 14 January 2000     

Polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas stutzeri 1317 had different substrate specificities

The whole polyhydroxyalkanoate (PHA) synthesis gene locus of Pseudomonas stutzeri strain 1317 containing PHA synthase genes phaC1Ps, phaC2Ps and PHA depolymerase gene phaZPs was cloned using a PCR cloning strategy. The sequence analysis results of the phaC1Ps, phaC2Ps and phaZPs showed high homology to the corresponding pha loci of the known Pseudomonas strains, respectively. PhaC1Ps and PhaC2Ps were functionally expressed in recombinant Escherichia coli strains and their substrate specificity was compared. The results demonstrated that PhaC1Ps and PhaC2Ps from P. stutzeri 1317 had different substrate specificities when expressed in E. coli. In details, PhaC2Ps could incorporate both short-chain-length 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates (mcl 3HA) into PHA, while PhaC1Ps only favored mcl 3HA for polymerization.     

Purification and characterization of two extracellular polyhydroxyalkanoate depolymerases from Pseudomonas mendocina

Two polyhydroxyalkanoate depolymerases, PHAase I and PHAase II, were purified to homogeneity from the culture supernatant of an effective PHA-degrading bacterium, Pseudomonas mendocina DS04-T. The molecular masses of PHAase I and PHAase II were determined by SDS-PAGE as 59.4 and 33.8 kDa, respectively. Their optimum pH values were 8.5 and 8, respectively. Enzymatic activity was optimal at 50 °C. Both purified enzymes could degrade PHB, PHBV, and P(3HB-co-4HB). Addition of Na⁺ and K⁺ slightly increased the rate of PHAase II. EDTA significantly inhibited PHAase II but not PHAase I. Mercaptoethanol and H₂O₂ also inhibited the activities of both enzymes.     

Biochemical observations on medium-chain-length polyhydroxyalkanoate biosynthesis and accumulation in Pseudomonas mendocina

Certain Pseudomonads are capable of accumulating high levels of medium-chain-length polyhydroxyalkanates (PHAmcl) when grown with carbohydrates as the main carbon source. 3-OH acyl components of PHAmcl are derived from fatty acid synthase (FAS) and these components are accessed by action of 3-hydroxyacyl-acyl carrier protein (ACP)-coenzyme A (CoA) transferase (transacylase). However, little is known with regard to the time courses of 3-OH acyl component occurrence and of transacylase activity during PHAmcl induction. Also, little is known with regard to the coupling mechanism between FAS and PHAmcl synthesis or whether the FAS pathway itself is specialized in PHAmcl-producing cells. Our results with regard to the time course of formation of 3-OH acids, 3-OH acyl-ACPs, and PHAmcl are consistent with the view that transacylase provides the key link between FAS and PHAmcl synthase. They also suggest that FAS specialization is not a feature of the mechanism. Further, we observed the formation of a 3-OH 10:0 homopolymer early in the induction phase followed by later formation of a mixed polymer containing 3-OH 8:0 and 3-OH 12:0 in addition to 3-OH 10:0. Early occurrence of 3-OH 10:0-CoA transacylase activity was coincident with homopolymer formation.     

Cloning, expression, and characterization of sialic acid synthases

The most commonly occurring sialic acid, N-acetylneuraminic acid, is the repeating unit in polysialic acid chain of human neuronal cell adhesion molecule as well as in capsular polysialic acid of neuroinvasive bacteria, Escherichia coli K1 and Neisseria meningitidis. Sialic acid synthesis and polymerization occur in slightly different pathways in animals and bacteria. N-Acetylneuraminic acid (NeuNAc) is synthesized by the condensation of phosphoenolpyruvate and N-acetylmannosamine by NeuNAc synthase in bacteria. The mammalian homologue N-acetylneuraminic acid-9-phosphate (NeuNAc-9-P) synthase uses N-acetylmannosamine-6-phosphate in the condensation reaction to produce NeuNAc-9-P. Both subfamilies of sialic acid synthases possess N-terminal triosephosphate isomerase barrel domain and C-terminal antifreeze protein domain. We report cloning of the genes, expression, purification, and characterization of human NeuNAc-9-P synthase and N. meningitidis NeuNAc synthase. Stability of the purified enzymes and effects of pH and temperature on their activities were evaluated. Enzyme kinetics and preliminary mutagenesis experiments reveal the importance of C-terminal antifreeze protein domain and a conserved cysteine residue for the enzyme activities.     

Comparison of medium-chain-length polyhydroxyalkanoates synthases from Pseudomonas mendocina NK-01 with the same substrate specificity

The medium-chain-length polyhydroxyalkanoate (PHA_MCL) synthase genes phaC1 and phaC2 of Pseudomonas mendocina NK-01 were cloned and inserted into expression plasmid pBBR1MCS-2 to form pBBR1MCS-C1 and pBBR1MCS-C2 which were expressed respectively in the PHA_MCL-negative strain P. mendocina C7 whose PHA_MCL synthesis operon was defined knock out. P. mendocina C7 derivatives P. mendocina C7C1 and C7C2 carrying pBBR1MCS-C1 and pBBR1MCS-C2 respectively were constructed. Fermentation and gel permeation chromatography (GPC) revealed that P. mendocina C7C1 had higher PHA_MCL production rate but its PHA_MCL had lower molecular weight than that of P. mendocina C7C2. Gas chromatograph/mass spectrometry (GC/MS) analysis revealed that the two PHA_MCL had similarity in monomer composition with 3HD as the favorite monomer i.e. PhaC1 and PhaC2 had the same substrate specificity. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and X-ray diffraction (XRD) also revealed that the two PHA_MCL had the same physical properties. P. mendocina NK-01was the first reported strain whose PHA_MCL synthases PhaC1 and PhaC2 had the same substrate specificity.     

Biotransformation of N-nitrosodimethylamine by Pseudomonas mendocina KR1

N-Nitrosodimethylamine (NDMA) is a potent carcinogen and an emerging contaminant in groundwater and drinking water. The metabolism of NDMA in mammalian cells has been widely studied, but little information is available concerning the microbial transformation of this compound. The objective of this study was to elucidate the pathway(s) of NDMA biotransformation by Pseudomonas mendocina KR1, a strain that possesses toluene-4-monooxygenase (T4MO). P. mendocina KR1 was observed to initially oxidize NDMA to N-nitrodimethylamine (NTDMA), a novel metabolite. The use of 18O2 and H(2)18O revealed that the oxygen added to NDMA to produce NTDMA was derived from atmospheric O2. Experiments performed with a pseudomonad expressing cloned T4MO confirmed that T4MO catalyzes this initial reaction. The NTDMA produced by P. mendocina KR1 did not accumulate, but rather it was metabolized further to produce N-nitromethylamine (88 to 94% recovery) and a trace amount of formaldehyde (HCHO). Small quantities of methanol (CH3OH) were also detected when the strain was incubated with NDMA but not during incubation with either NTDMA or HCHO. The formation of methanol is hypothesized to occur via a second, minor pathway mediated by an initial alpha-hydroxylation of the nitrosamine. Strain KR1 did not grow on NDMA or mineralize significant quantities of the compound to carbon dioxide, suggesting that the degradation process is cometabolic.     

Biotransformation of N-Nitrosodimethylamine by Pseudomonas mendocina KR1

N-Nitrosodimethylamine (NDMA) is a potent carcinogen and an emerging contaminant in groundwater and drinking water. The metabolism of NDMA in mammalian cells has been widely studied, but little information is available concerning the microbial transformation of this compound. The objective of this study was to elucidate the pathway(s) of NDMA biotransformation by Pseudomonas mendocina KR1, a strain that possesses toluene-4-monooxygenase (T4MO). P. mendocina KR1 was observed to initially oxidize NDMA to N-nitrodimethylamine (NTDMA), a novel metabolite. The use of ¹⁸O₂ and H₂¹⁸O revealed that the oxygen added to NDMA to produce NTDMA was derived from atmospheric O₂. Experiments performed with a pseudomonad expressing cloned T4MO confirmed that T4MO catalyzes this initial reaction. The NTDMA produced by P. mendocina KR1 did not accumulate, but rather it was metabolized further to produce N-nitromethylamine (88 to 94% recovery) and a trace amount of formaldehyde (HCHO). Small quantities of methanol (CH₃OH) were also detected when the strain was incubated with NDMA but not during incubation with either NTDMA or HCHO. The formation of methanol is hypothesized to occur via a second, minor pathway mediated by an initial α-hydroxylation of the nitrosamine. Strain KR1 did not grow on NDMA or mineralize significant quantities of the compound to carbon dioxide, suggesting that the degradation process is cometabolic.     

Mutations derived from the thermophilic polyhydroxyalkanoate synthase PhaC enhance the thermostability and activity of PhaC from Cupriavidus necator H16

The thermophile Cupriavidus sp. strain S-6 accumulated polyhydroxybutyrate (PHB) from glucose at 50°C. A 9.0-kbp EcoRI fragment cloned from the genomic DNA of Cupriavidus sp. S-6 enabled Escherichia coli XL1-Blue to synthesize PHB at 45°C. Nucleotide sequence analysis showed a pha locus in the clone. The thermophilic polyhydroxyalkanoate (PHA) synthase (PhaC(Csp)) shared 81% identity with mesophilic PhaC of Cupriavidus necator H16. The diversity between these two strains was found dominantly on their N and C termini, while the middle regions were highly homologous (92% identity). We constructed four chimeras of mesophilic and thermophilic phaC genes to explore the mutations related to its thermostability. Among the chimeras, only PhaC(H16β), which was PhaC(H16) bearing 30 point mutations derived from the middle region of PhaC(Csp), accumulated a high content of PHB (65% [dry weight]) at 45°C. The chimera phaC(H16)(β) and two parental PHA synthase genes were overexpressed in E. coli BLR(DE3) cells and purified. At 30°C, the specific activity of the chimera PhaC(H16β) (172 ± 17.8 U/mg) was 3.45-fold higher than that of the parental enzyme PhaC(H16) (50 ± 5.2 U/mg). At 45°C, the half-life of the chimera PhaC(H16β) (11.2 h) was 127-fold longer than that of PhaC(H16) (5.3 min). Furthermore, the chimera PhaC(H16β) accumulated 1.55-fold (59% [dry weight]) more PHA content than the parental enzyme PhaC(H16) (38% [dry weight]) at 37°C. This study reveals a limited number of point mutations which enhance not only thermostability but also PhaC(H16) activity. The highly thermostable and active PHA synthase will provide advantages for its promising applications to in vitro PHA synthesis and recombinant E. coli PHA fermentation.     

Overexpression and characterization of medium-chain-length polyhydroxyalkanoate granule bound polymerases from Pseudomonas putida GPo1

Background  

Polyhydroxyalkanoates (PHA) are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA biosynthesis are PHA polymerases, which catalyze the covalent linkage of 3-hydroxyacyl coenzymeA thioesters by transesterification with concomitant release of CoA. Pseudomonas putida GPo1 and many other Pseudomonas species contain two different class II polymerases, encoded by phaC1 and phaC2. Although numerous studies have been carried out on PHA polymerases and they are well characterized at the molecular level, the biochemical properties of the class II polymerases have not been studied in detail. Previously we and other groups purified the polymerases, however, the activities of the purified enzymes were several magnitude lower than the granule-bound enzymes. It is problematic to study the intrinsic properties of these enzymes with such low activities, although they are pure.     

Isolation of alginate-producing mutants of Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas mendocina

Spontaneous alginate-producing (muc) variants were isolated from strains of Pseudomonas fluorescens, P. putida and P. mendocina at a frequency of 1 in 10(8) by selecting for carbenicillin resistance. The infrared spectrum of the bacterial exopolysaccharide was typical of an acetylated alginate similar to that previously described in Azotobacter vinelandii and in mucoid variants of P. aeruginosa. Mucoid variants were not isolated from P. stutzeri, P. pseudoalcaligenes, P. testosteroni, P. diminuta, P. acidovorans, P. cepacia or P. maltophilia.     

门多萨假单胞菌Pseudomonas mendocina NK-01合成褐藻寡糖及其结构鉴定

研究发现门多萨假单胞菌NK-01在菌体内积累中长链聚羟基脂肪酸酯(PHAMCL)的同时也能够合成褐藻寡糖分泌到发酵液中,其产量与培养基的碳氮比有关,高碳氮比有利于褐藻寡糖的合成。本研究利用紫外-可见分光光度法、傅立叶红外光谱分析、1H和13C核磁共振对褐藻寡糖的结构进行了分析鉴定,发现褐藻寡糖的结构是由β-D-甘露糖醛酸、α-L-古洛糖醛酸通过β-(1→4)/α-(1→4)键连接而成的无支化线性多糖,并且在单体的2位或3位羟基上部分乙酰化。凝胶渗透色谱(GPC)对分子量的测定结果为2054。     

Biochemical and molecular characterization of the Pseudomonas lemoignei polyhydroxyalkanoate depolymerase system.

Pseudomonas lemoignei has five different polyhydroxyalkanoate (PHA) depolymerase genes (phaZ1 to phaZ5), which encode the extracellularly localized poly(3-hydroxybutyrate) (PHB) depolymerases C, B, and D, poly(3-hydroxyvalerate) (PHV) depolymerase, and PHB depolymerase A, respectively. Four of the five genes (phaZ1 to phaZ4) have been cloned, and one of them (phaZ1) was studied in detail earlier (D. Jendrossek, B. Müller, and H. G. Schlegel, Eur. J. Biochem. 218:701-710, 1993). The fifth PHA depolymerase gene (phaZ5) was identified by colony hybridization of recombinant Escherichia coli clones with a phaZ5-specific oligonucleotide. The nucleotide sequence of a 3,704-bp EcoRI fragment was determined and found to contain two large open reading frames (ORFs) which coded for a polypeptide with significant similarities to glycerol-3-phosphate dehydrogenases of various sources (313 amino acids; M(r), 32,193) and for the precursor of PHB depolymerase A (PhaZ5; 433 amino acids; M(r), 44,906). The PHV depolymerase gene (phaZ4) was subcloned, and the nucleotide sequence of a 3,109-bp BamHI fragment was determined. Two large ORFs (ORF3 and ORF4) that represent putative coding regions were identified. The deduced amino acid sequence of ORF3 (134 amino acids; M(r), 14,686) revealed significant similarities to the branched-chain amino acid aminotransferase (IlfE) of enterobacteria. ORF4 (1,712 bp) was identified as the precursor of a PHV depolymerase (567 amino acids; M(r), 59,947). Analysis of primary structures of the five PHA depolymerases of P. lemoignei and of the PHB depolymerases of Alcaligenes faecalis and Pseudomonas pickettii revealed homologies of 25 to 83% to each other and a domain structure: at their N termini, they have typical signal peptides of exoenzymes. The adjacent catalytic domains are characterized by several conserved amino acids that constitute putative catalytic triads which consist of the consensus sequence of serine-dependent hydrolases including the pentapeptide G-X-S-X-G, a conserved histidine and aspartate, and a conserved region resembling the oxyanion hole of lipases. C terminal of the catalytic domain an approximately 40-amino-acid-long threonine-rich region (22 to 27 threonine residues) is present in PhaZ1, PhaZ2, PhaZ3, and PhaZ5. Instead of the threonine-rich region PhaZ4 and the PHB depolymerases of A. faecalis and P. pickettii contain an approximately 90-amino-acid-long sequence resembling the fibronectin type III module of eucaryotic extracellular matrix proteins. The function of the fibronectin type III module in PHA depolymerases remains obscure. Two types of C-terminal sequences apparently represent substrate-binding sites; the PHB type is present in the PHB depolymerases of A. faecalis and P. pickettii and in PhaZ2, PhaZ3, and PhaZ5 and the PHV type is present in the PHV-hydrolyzing depolymerases (PhaZ4 and PhaZ1). phaZ1 was transferred to A. eutrophus H16 and JMP222. All transconjugants of both strains were able to grow with extracellular PHB as a carbon source and produced translucent halos on PHB-containing solid media. PhaZ1, PhaZ2, PhaZ4, and PhaZ5 were purified from P. lemoignei and from recombinant E. coli; the processing sites of the precursors in E. coli were the same as in P. lemoignei, and similar substrate specificities were determined for the wild-type and the recombinant proteins. All PHA depolymerases hydrolyzed PHB at high specific activities. PhaZ1 and PhaZ4 additionally cleaved PHV, and PhaZ4 hydrolyzed poly(4-hydroxybutyrate). None of the depolymerases was able to hydrolyze polyactide or PHA consisting of monomers with more than five carbon atoms. While the wild-type depolymerase proteins were glycosylated and found to contain glucose and N-acetylglucosamine, none of the recombinant proteins was glycosylated. PHB hydrolysis was dependent on divalent cations such as Ca2+ and was inhibited by the presence of EDTA.     

Separation and partial characterization of the enzymes of the toluene-4-monooxygenase catabolic pathway in Pseudomonas mendocina KR1.

The route of toluene degradation by Pseudomonas mendocina KR1 was studied by separating or purifying from toluene-grown cells the catabolic enzymes responsible for oxidation of p-cresol through the ring cleavage step. Enzymatic transformations corresponding to each of the metabolic steps in the proposed degradative pathway were conducted with cell-free preparations. p-Cresol was metabolized by the enzyme p-cresol methylhydroxylase to p-hydroxybenzaldehyde. p-Hydroxybenzaldehyde was further oxidized by partially purified enzyme preparations to p-hydroxybenzoate and subsequently hydroxylated to form protocatechuate. Protocatechuate was then oxidized by ortho ring cleavage.     

Identification and characterization of two polyhydroxyalkanoate biosynthesis loci in Pseudomonas sp. strain 3Y2

A Pseudomonas strain, 3Y2, that produced polyhydroxyalkanoate (PHA) polymers consisting of 3-hydroxybutyric acid (3HB) and medium-chain-length 3-hydroxyalkanoate (mcl-HA) units, with up to 30% 3HB, was isolated. Two PHA biosynthesis loci (pha _Ps-1 and pha _Ps-2) from 3Y2 were cloned by polymerase chain reaction amplification techniques. The pha _Ps-2 locus was similar to the PHA biosynthesis loci of other PHA-producing Pseudomonas strains, with five tandem open reading frames (ORFs) located in the order ORF1 _Ps-2-phaC1 _Ps-2-phaZ _Ps-2-phaC2 _Ps-2-phaD _Ps-2. The pha _Ps-1 locus that contains phaC1 _Ps-1-phaZ _Ps-1 appears to have arisen by a duplication event that placed it downstream of a gene (ORF1 _Ps-1), encoding a putative glucose-methanol-choline flavoprotein oxidoreductase. The PHA synthases 1 encoded by phaC1 _Ps-1 and phaC1 _Ps-2 were investigated by heterologous expression in Wautersia eutropha PHB⁻4. Both synthases displayed similar substrate specificities for incorporating 3HB and mcl-HA units into PHA. The ability of PhaC1 _Ps-1 to confer PHA synthesis, however, appeared reduced compared to that of PhaC1 _Ps-2, since cells harboring PhaC1 _Ps-1 accumulated 2.5 to 4.6 times less PHA than cells expressing PhaC1 _Ps-2. Primary sequence analysis revealed that PhaC1 _Ps-1 had markedly diverged from the other PHA synthases with a relatively high substitution rate (14.9 vs 2% within PhaC1 _Ps-2). The mutations affected a highly conserved C-terminal region and the surroundings of the essential active site cysteine (Cys296) with a loss of hydrophobicity. This led us to predict that if phaC1 _Ps-1 produces a protein product in the native strain, it is likely that PhaC1 _Ps-1 may be destined for elimination by the accumulation of inactivating mutations, although its specialization to accommodate different substrates cannot be eliminated.     

Metabolic engineering and characterization of phaC1 and phaC2 genes from Pseudomonas putida KCTC1639 for overproduction of medium-chain-length polyhydroxyalkanoate

Two PHA synthase phaC1 and phaC2 genes cloned from the new strain Pseudomonas putida KCTC1639 were metabolically engineered for the overproduction of medium-chain-length polyhydroxyalkanoate (mcl-PHA). The overexpressed phaC1 and phaC2 genes in P. putida KCTC1639 were compared in terms of the biosynthesis of mcl-PHA, fatty acid assimilation, distribution of 3-hydroxylacyl monomer units, granular morphology, and thermophysical properties of the accumulated mcl-PHA. The biosynthesis of mcl-PHA was enhanced only by the overexpressed phaC1 gene up to 2.86-fold, in contrast, the phaC2 gene did not activate the biosynthesis of mcl-PHA. The overexpressed phaC1 gene tended to form enlarged, high molecular weight, and lower crystalline mcl-PHA granules, whereas the amplified phaC2 gene induced the fragmentation of mcl-PHA into a few small-sized granules. The transformant P. putida KCTC1639 overexpressing the phaC1 gene encoding PHA synthase I was cultivated by pH-stat fed-batch cultivation, and the concentration and content of mcl-PHA increased up to 8.91 g L-1 and 70.5%, respectively.