Production of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from Gluconate and Glucose by Recombinant Aeromonas hydrophila and Pseudomonas putida

Aeromonas hydrophila 4AK4 and Pseudomonas putida GPp104 were genetically engineered to synthesize poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) using gluconate and glucose rather than fatty acids. A truncated tesA gene, encoding cytosolic thioesterase I of Escherichia coli which catalyzes the conversion of acyl-ACP into free fatty acids, was introduced into A. hydrophila 4AK4. When grown in gluconate, the recombinant A. hydrophila 4AK4 synthesized 10% (w/w) PHBHHx containing 14% (mol/mol) 3-hydroxyhexanoate. If additional PHBHHx synthesis genes, phaPCJ, were over-expressed with the truncated tesA in A. hydrophila 4AK4, the PHBHHx content increased to 15% (w/w) and contained 19% (mol/mol) 3-hydroxyhexanoate. Recombinant P. putida GPp104 harboring phaC encoding PHBHHx synthase of A. hydrophila, phaB encoding acetoacetyl-CoA reductase of Wautersia eutropha and phaG encoding 3-hydroxyacyl-ACP-CoA transferase of P. putida, synthesized 19% (w/w) PHBHHx containing 5% (mol/mol) 3-hydroxyhexanoate from glucose. The results suggest that the engineered pathways were applicable to synthesize PHBHHx from unrelated carbon sources such as gluconate and glucose.     

A Pseudomonas strain accumulating polyesters of 3-hydroxybutyric acid and medium-chain-length 3-hydroxyalkanoic acids

Summary A citronellol-utilizing bacterium was isolated that accumulated a polyester consisting of 3-hydroxybutyric acid (3HB) and of medium-chain-length 3-hydroxyalkanoic acids (3HA_MCL) from various carbon sources up to approximately 70% of the cellular dry matter if the cells were cultivated in ammineral salts medium under nitrogen limitation. In octanoate-grown cells, for instance, the polyester consisted of 87.5 mol% 3HB and 12.5 mol% 3-hydroxyoctanoic acid (3HO), whereas it consisted of 10.3 mol% 3HB, 16.7 mol% 3HO and 73.0 mol% 3-hydroxydecanoic acid (3HD) in gluconate-grown cells. However, the results of various experiments indicated that a blend rather than a copolyester was synthesized in the cell. It was the only strain among 45 different recently isolated citronellol-utilizing bacteria that accumulated such a polyester. All other citronellol-utilizing bacteria behaved like Pseudomonas aeruginosa with respect to their polyhydroxyalkanoic acid (PHA) biosynthetic capabilities and accumulated PHA consisting of 3HA_MCL with 3HO and 3HD as the main constituents from octanoate or gluconate, respectively, whereas 3HB was never present. None of 232 different heavy-metal-resistant bacteria was able to accumulate PHA composed of 3HB plus, for example, 3HO. Only 20.3% did not accumulate any PHA at all, 44.8% accumulated PHB from gluconate, and 34.9% behaved like P. aeruginosa. Many bacteria belonging to the latter group were distinguished from the other by rapid growth in nutrient broth and in gluconate mineral salts medium and by their ability to grow in the presence of a high concentration (up to 1.5%, w/v) of octanoate. Correspondence to: A. Steinbüchel     

Studies on polyhydroxyalkanoate (PHA) accumulation in a PHA synthase I-negative mutant of Burkholderia cepacia generated by homogenotization

In the genome of Burkholderia cepacia strain IPT64, which accumulates a blend of the two homopolyesters poly(3-hydroxybutyrate), poly(3HB), and poly(3-hydroxy-4-pentenoic acid), poly(3H4PE), from sucrose or gluconate as single carbon source, the polyhydroxyalkanoate (PHA) synthase structural gene was disrupted by the insertion of a chloramphenicol-resistant gene cassette (phaC1::Cm). The suicide vector pSUP202 harboring phaC1::Cm was transferred to B. cepacia by conjugation. The inactivated gene was integrated into the chromosome of B. cepacia by homologous recombination. This mutant and also 15 N-methyl-N'-nitrosoguanidine (NMG)-induced mutants still accumulated low amounts of PHAs and expressed low PHA synthase activity. The analysis of the mutant phaC1::Cm showed that it accumulated about 1% of PHA consisting of 68.2 mol% 3HB and 31.8 mol% 3H4PE from gluconate. The wild-type, in contrast, accumulated 49.3% of PHA consisting of 96.5 mol% 3HB and 3. 5 mol% 3H4PE. Our results indicated that the genome of B. cepacia possesses at least two PHA synthase genes, which probably have different substrate specificities.     

Polyhydroxyalkanoate accumulation in Burkholderia sp.: a molecular approach to elucidate the genes involved in the formation of two homopolymers consisting of short-chain-length 3-hydroxyalkanoic acids

Burkholderia sp. accumulates polyhydroxyalkanoates (PHAs) containing 3-hydroxybutyrate and 3-hydroxy-4-pentenoic acid when grown on mineral media under limited phosphate or nitrogen, and using sucrose or gluconate as a carbon and energy source. Solvent fractionation and NMR spectroscopic characterization of these polyesters revealed the simultaneous accumulation of two homopolyesters rather than a co-polyester with random sequence distribution of the monomers [Valentin HE, Berger PA, Gruys KJ, Rodrigues MFA, Steinbüchel A, Tran M, Asrar J (1999) Macromolecules 32: 7389–7395]. To understand the genetic requirements for such unusual polyester accumulation, we probed total genomic DNA from Burkholderia sp. by Southern hybridization experiments using phaC-specific probes. These experiments indicated the presence of more than one PHA synthase gene within the genome of Burkholderia sp. However, when total genomic DNA from Burkholderia sp. was used to complement a PHA-negative mutant of Ralstonia eutropha for PHA accumulation, only one PHA synthase gene was obtained resembling the R. eutropha type of PHA synthases, based on amino acid sequence similarity. In addition to the PHA synthase gene, based on high sequence homology, genes encoding a β-ketothiolase and acetoacetyl-CoA reductase were identified in a gene cluster with the PHA synthase gene. The arrangement of the three genes is quite similar to the R. eutropha poly-β-hydroxybutyrate biosynthesis operon. Received: 3 September 1999 / Received revision: 29 October 1999 / Accepted: 5 November 1999     

Identification of 4-hydroxyvaleric acid as a constituent of biosynthetic polyhydroxyalkanoic acids from bacteria

Summary Twenty-four different strains of aerobic Gram-negative bacteria, mainly belonging to the genera Alcaligenes, Paracoccus, Pseudomonas and Methylobacterium, were examined with respect to their ability to utilize 4-hydroxyvaleric acid (4HV), 4-valerolactone (4VL) and 3-hydroxypropionic acid (3HP) as carbon sources for growth and for accumulation of polyhydroxyalkanoic acid (PHA). A gas chromatographic (GC) method for the detection of 3-hydroxyalkanoic acid methyl esters has been extended for the detection of derivatives obtained from the methanolysis of 4-hydroxybutyric acid (4HB) and 4HV. Most of the Alcaligenes species and P. oxalaticus Ox1 accumulated a terpolyester consisting of 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV) and 4HV as constituents from 4HV or 4VL as sole carbon sources in batch, fed-batch or two-stage fed-batch cultures. Poly(3HB-co-3HV-co-4HV) accumulated from 4HV by A. eutrophus strain NCIB 11599 amounted to approximately 50% of the cell dry matter and was composed of 42.0 mol % 3HB, 52.2 mol % 3HV and 5.6 mol % 4HV, respectively. Pseudomonads, which belong to the rRNA homology group I, were not able to incorporate 4HV. With 3HP as carbon source, the GC analysis provided evidence for the presence of 3HP in the PHA of many bacteria. Nuclear magnetic resonance spectroscopic analysis confirmed that, for example, A. eutrophus strain TF93 accumulated poly(3HB-co-3HP) with 98 mol % 3HB and 2 mol % 3HP if the cells were cultivated in the presence of 0.5% (w/v) 3HP. Offprint requests to: A. Steinbüchel     

Synthesis and production of poly(3-hydroxyvaleric acid) homopolyester by Chromobacterium violaceum

Chromobacterium violaceum DSM 30191 accumulated a homopolyester of 3-hydroxyvaleric acid (3HV) up to 65% of the cellular dry matter during cultivation in fed-batch cultures with valeric acid as sole carbon source and during cell starvation of the nitrogen source. From fructose, gluconate, propionate or hexanoate a homopolyester of 3-hydroxybutyrate (3HB) was accumulated. Poly(3HV) homopolyster was also accumulated by two different strains of C. violaceum, whereas two other strains of C. violaceum and three strains of Janthinobacterium lividum accumulated poly(3HB-co-3HV) copolyesters from valerate. The composition of the biosynthetic poly(3HV) was confirmed by various nuclear magnetic resonance spectroscopic methods. Differential scanning calorimetry analysis of four poly(3HV) samples that were isolated from different batches of cells revealed glass transition temperatures between –10 and –12°C and melting points between 107 and 112°C. Viscosity measurements gave intrinsic viscosities between 62.5 and 124.8 × 10^–2 dl/g for these samples, indicating approximate relative molecular masses between 60 000 and 145 000 of the biosynthetic poly(3HV). Correspondence to: A. Steinbüchel     

Formation of new polyhydroxyalkanoate containing 3-hydroxy-4-methylvalerate monomer in Burkholderia sp

Burkholderia sp. synthase has been shown to polymerize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate, and 3-hydroxy-4-pentenoic acid monomers. This study was carried out to evaluate the ability of Burkholderia sp. USM (JCM 15050) and its transformant harboring the polyhydroxyalkanoate (PHA) synthase gene of Aeromonas caviae to incorporate the newly reported 3-hydroxy-4-methylvalerate (3H4MV) monomer. Various culture parameters such as concentrations of nutrient rich medium, fructose and 4-methylvaleric acid as well as harvesting time were manipulated to produce P(3HB-co-3H4MV) with different 3H4MV compositions. The structural properties of PHA containing 3H4MV monomer were investigated by using nuclear magnetic resonance and Fourier transform infrared spectroscopy (FTIR). The relative intensities of the bands at 1,183 and 1,228 cm⁻¹ in the FTIR spectra enabled the rapid detection and differentiation of P(3HB-co-3H4MV) from other types of PHA. In addition, the presence of 3H4MV units in the copolymer was found to considerably lower the melting temperature and enthalpy of fusion values compared with poly(3-hydroxybutyrate) (P(3HB)). The copolymer exhibited higher thermo-degradation temperature but similar molecular weight and polydispersity compared with P(3HB).     

Biosynthesis of copolyesters consisting of 3-hydroxybutyric acid and medium-chain-length 3-hydroxyalkanoic acids from 1,3-butanediol or from 3-hydroxybutyrate by Pseudomonas sp. A33

Pseudomonas sp. A33 and other isolates of aerobic bacteria accumulated a complex copolyester containing 3-hydroxybutyric acid (3HB) and various medium-chain-length 3-hydroxyalkanoic acids (3HA_MCL) from 3-hydroxybutyric acid or from 1,3-butanediol under nitrogen-limitated culture conditions. 3HB contributed to 15.1 mol/100 mol of the constituents of the polyester depending on the strain and on the cultivation conditions. The accumulated polymer was a copolyester of 3HB and 3HA_MCL rather than a blend of poly(3HB) and poly(3HA_MCL) on the basis of multiple evidence. 3-Hydroxyhexadecenoic acid and 3-hydroxyhexadecanoic acid were detected as constituents of polyhydroxyalkanoates, which have hitherto not been described, by¹³C nuclear magnetic resonance or by gas chromatography/mass spectrometric analysis. In total, ten different constituents were detected in the polymer synthesized from 1,3-butanediol by Pseudomonas sp. A33:besides seven saturated (3HB, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydrohexadecanoate) three unsaturated (3-hydroxydodecenoate, 3-hydroxytetradecenoate and 3-hydrohexadecanoate) hydroxyalkanoic acid constituents occured. The polyhydroxyalkanoate synthase of Pseudomonas sp. A33 was cloned, and its substrate specificity was evaluated by heterologous expression in various strains of P. putida, P. oleovorans and Alcaligenes eutrophus.     

Biosynthesis of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyvalerate) copolyesters with a high molar fraction of 3-hydroxyvalerate by an insect-symbiotic <Emphasis Type="Italic">Burkholderia</Emphasis> sp. IS-01

Burkholderia sp. IS-01 capable of biosynthesizing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)] copolyesters with a high molar fraction of 3HV was isolated from the gut of the adult longicorn beetle, Moechotypa diphysis. The strain IS-01 was relatively tolerant to high concentrations of levulinic acid and accumulated a poly(13.5 mol% 3HB-co-86.5 mol% 3HV) copolyester when cultivated on a mixture of gluconate (20 g/L) and levulinic acid (12.5 g/L). In this case, the content of the copolyester in the cells was approximately 60.0%. The compositions of the copolyesters were easily regulated by altering the molar ratio of gluconate and levulinic acid in the medium. The organism was found to possess a class I PHA synthase (PhaC) gene (1,881 bp) that encodes a protein with a deduced molecular mass of 68,538 Da that consists of 626 amino acids. The PhaC of this organism was most similar to that of B. cenocepacia PC184 (92% similarity).     

Production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by <Emphasis Type="Italic">Rhodopseudomonas palustris</Emphasis> SP5212

Summary Production of poly(3-hydroxybutyric acid) [P(3HB)] by Rhodopseudomonas palustris SP5212 isolated in this laboratory has been optimized under phototrophic microaerophilic conditions. Cells grown in malate medium accumulated 7.7% (w/w) P(3HB) of cellular dry weight at the early stationary phase of growth. The accumulated P(3HB) however, attained 15% (w/w) of cellular dry weight when acetate (1.0%, w/v) was used as the sole carbon source under nitrogen-limiting conditions. Synthesis and accumulation of polymer was favoured by sulphate-free conditions and at a phosphate concentration sub-optimal for growth. The polymer content of cells was increased drastically (34% of cellular dry weight) when the acetate containing medium was supplemented with n-alkanoic acids. Compositional analysis by H¹ NMR revealed that these accumulated polymers were composed of 3-hydroxybutyric acid and 3-hydroxyvaleric acid (3HV). The contents of 3HV in these copolymers ranged from 14 to 38 mol%.     

Effect of 4-Pentenoic Acid on Intermediate Metabolism of Tetrahymena*

SYNOPSIS. The growth of Tetrahymena pyriformis strain HSM was strongly inhibited by 4-pentenoic acid. Supplementing the medium with acetate reversed the growth inhibition, but pyruvate was ineffective. Glycogen content was much lower in cells grown with 4-pentenoic acid than in controls; this effect was not reversed by acetate or by pyruvate. There was little effect of 4-pentenoic acid on the incorporation of label from [1-¹⁴C]acetate, [2-¹⁴C]glycerol, [1-³⁴]ribose, [U-¹⁴C]fructose, or [1-¹⁴C]glucose into CO₂, but incorporation of label into glycogen was inhibited, the strongest inhibition being on acetate and the weakest (～ 20%) on ribose, fructose, and glucose. A 3-compartment model for quantitation of labeled acetyl CoA fluxes was shown to be applicable to Tetrahymena grown in the presence of 4-pentenoic acid, and experiments were performed to establish the flux of [1-¹⁴C]acetyl CoA into glycogen, lipids, CO₂, glutamate, and alanine. It was evident from the results of these experiments that 4-pentenoic acid did not appreciably inhibit β-oxidation or lipogenesis, but markedly decreased the glyconeogenic flux of labeled acetyl-CoA from the peroxismal and outer mitochondrial compartments. At least 2 mechanisms have been proposed for the action of 4-pentenoic acid: (a) reduction of the levels of acetyl CoA or free CoA and (b) direct inhibition of enzymes by 4-pentenoyl CoA or its metabolites. Although 4-pentenoic acid has little effect on acetyl-CoA metabolism in the inner mitochondrial compartment, the present data suggest that the flux through the outer mitochondrial compartment of acetyl-CoA derived from pyruvate is inhibited largely by the first, and that the glyconeogenic flux of acetyl-CoA is inhibited largely by the 2nd mechanism.     

3β-hydroxysteroid dehydrogenase activity in tissues of the human fetus determined with 5α-androstane-3β,17β-diol and dehydroepiandrosterone as substrates

3β-Hydroxysteroid dehydrogenase (3β-HSD)/Δ^5→4-isomerase activity in steroidogenic tissues is required for the synthesis of biologically active steroids. Previously, by use of dehydroepiandrosterone (3β-hydroxy-5-androsten-17-one, DHEA) as substrate, it was established that in addition to steroidogenic tissues 3β-HSD/Δ^5→4-isomerase activity also is expressed in extraglandular tissues of the human fetus. In the present study, we attempted to determine whether the C-5,C-6-double bond of DHEA serves to influence 3β-HSD activity. For this purpose, we compared the efficiencies of a 3β-hydroxy-5-ene steroid (DHEA) and a 3β-hydroxy-5α-reduced steroid (5α-androstane-3β,17β-diol, 5α-A-diol) as substrates for the enzyme. The apparent Michaelis constant (K_m) for 5α-A-diol in midtrimester placenta, fetal liver, and fetal skin tissues was at least one order of magnitude higher than that for DHEA, viz the apparent K_m of placental 3β-HSD for 5α-A-diol was in the range of 18 to 40 μmol/l (n = 3) vs 0.45 to 4 μmol/l for DHEA (n = 3); for the liver enzyme, 17 μmol/l for 5α-A-diol and 0.60 μmol/l for DHEA, and for the skin enzyme 14 and 0.18 μmol/l, respectively. Moreover, in 13 human fetal tissues evaluated the maximal velocities obtained with 5α-A-diol as substrate were higher than those obtained with DHEA. A similar finding in regard to K_ms and rates of product formation was obtained by use of purified placental 3β-HSD with DHEA, pregnenolone, and 3β-hydroxy-5α-androstan-17-one (epiandrosterone) as substrates: the K_m of 3β-HSD for DHEA was 2.8 μmol/l, for pregnenolone 1.9 μmol/l, and for epiandrosterone 25 μmol/l. The specific activity of the purified enzyme with pregnenolone as substrate was 27 nmol/mg protein·min and, with epiandrosterone, 127 nmol/mg protein·min. With placental homogenate as the source of 3β-HSD, DHEA at a constant level of 5 μmol/l behaved as a competitive inhibitor when the radiolabeled substrate, [³H]5α-A-diol, was present in concentrations of 20 to 60 μmol/l, but a lower substrate concentrations the inhibition was of the mixed type; similar results were obtained with [³H]DHEA as the substrate at variable concentrations in the presence of a fixed concentration of 5α-A-diol (40 μmol/l). These findings are indicative that both steroids bind to a common site on the enzyme, however, the binding affinity for these steroids appear to differ markedly as suggested by the respective K_ms. Studies of inactivation of purified placental 3β-HSD/Δ^5→4-isomerase by an irreversible inhibitor, viz 5,10-secoestr-4-yne-3,10,17-trione, were suggestive that the placental protein adopts different conformations depending on whether the steroidal substrate has a 5α-configuration, e.g. epiandrosterone, or a C-5,C-6-double bond e.g. DHEA or pregnenolone. The lower rates of product formation obtained with placenta and fetal tissues by use of 3β-hydroxy-5-ene steroids as substrates when compared with those obtained with 3β-hydroxy-5α-reduced steroids may be explained by a combination of factors, including: (i) inhibition of 3β-HSD activity by end products of metabolism of 3β-hydroxy-5-ene steroids, e.g. 4-androstene-3,17-dione formed with DHEA as substrate; (ii) higher binding affinity of the enzyme for 3β-hydroxy-5-ene steroids—and possibly for their 3-oxo-5-ene metabolites; (iii) lack of a requirement for the isomerization step with 5α-reduced steroids as substrates, and (iv) the possible presence in fetal tissues of an enzyme with 3β-HSD activity only (i.e. no Δ^5→4-isomerase).     

Inactivation of Escherichia coli threonine synthase by dl-Z-2-amino-5-phosphono-3-pentenoic acid

The rhizocticines and plumbemicines are two groups of di- and tripeptid antibiotics thought to interfere with threonine or threonine-related metabolism. Z-2-amino-5-phosphono-3-pentenoic acid, the common unusual amino acid constituent of the rhizocticines and plumbemicines, was found to irreversibly inhibit Escherichia coli threonine synthase in a time-dependent reaction that followed pseudo-first order and saturation kinetics. These data provide evidence that the toxicity of the rhizocticines and plumbemicines is due to the inhibition of threonine synthase by Z-2-amino-5-phosphone-3-pentenoic acid, which is liberated by peptidases after uptake into the target cell. Additionally, methods for the purification of threonine synthase from an overproducing E. coli strain and for the enzymatic synthesis of l-homoserine phosphate are described.Abbreviations APPA Z-2-amino-5-phosphono-3-pentenoic acid - HSerP l-homoserine phosphate - PEP phosphoenolpyruvate - PLP pyndoxal 5-phosphate - TS threonine synthase     

Continuous preparation of (S)-3-hydroxy-3-phenylpropionate by asymmetric reduction of 3-oxo-3-phenylpropionic acid ethyl ester with <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> CGMCC No.2266 in a membrane reactor

In this study, (S)-3-hydroxy-3-phenylpropionate was prepared continuously by coupling microbial transformation and membrane separation. The effect of several factors on membrane flux, reactor capacity, and reaction conversion were investigated. A kinetic model of the continuous reduction process was also developed. The appropriate molecular weight cut-off of the ultrafiltration membrane was 30 kDa. The reactor capacity reached a maximum of 0.136/h at a biomass concentration and membrane flux of 86 g/L (dry weight/reaction volume) and 20 mL/h, respectively. The (S)-3-hydroxy-3-phenylpropionate yield was 3.68 mmol/L/day after continuous reduction over seven days. The enantiometric excess of (S)-3-hydroxy-3-phenylpropionate reached above 99.5%. The kinetic constants of continuous reduction were as follows: r _m = 3.00 × 10⁻³ mol/L/h, k _cat = 3.49 × 10⁻⁴ mol/L/h, k ₁ = 3.09 × 10⁻² mol/L, and k ₂ = 5.00 × 10⁻⁷ mol/L. The kinetic model was in good agreement with the experimental data obtained during continuous reduction. Compared with batch reduction, continuous reduction can significantly improve the catalytic efficiency of microbial cells and increase the reactor capacity.     

Production of a copolyester of 3-hydroxybutyric acid and 3-hydroxyvaleric acid from single unrelated carbon sources by a mutant of Alcaligenes eutrophus

Summary Alcaligenes eutrophus mutant strain R3, which is a spontaneous revertant to prototrophy of an isoleucine-auxotrophic mutant of the wild-type strain H16, accumulated a copolyester consisting of 3-hydroxybutyric acid (3HB) as main constituent and of 3-hydroxyvaleric acid (3HV), i.e. poly(3HB-co-3HV), as the only other constituent from various single unrelated substrates, which were provided in excess, after a nutrient essential for growth was depleted in the medium. Poly(SHB-co-3HV) was produced from fructose, gluconate, succinate, acetate or lactate during cell starvation of the nitrogen, sulphur or magnesium source. Although 3HV amounted to only 8 mol% of the constituents of the polyester, this study provides a general rationale for construction and utilization of mutants of poly(3HB)-accumulating bacteria that are altered in the metabolism of branched-chain amino acids for the production of poly(3HB-co-3HV) from single unrelated substrates. Offprint requests to: A. Steinbüchel     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant bacteria expressing the PHA synthase gene phaC1 from Pseudomonas sp. 61-3

Pseudomonas sp. 61-3 accumulated a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer consisting of 3-hydroxyalkanoate (3HA) units of 4–12 carbon atoms. The genes encoding β-ketothiolase (PhbA_Re) and NADPH-dependent acetoacetyl-CoA reductase (PhbB_Re) from Ralstoniaeutropha were expressed under the control of promoters for Pseudomonas sp. 61-3 pha locus or R. eutropha phb operon together with phaC1 _Ps gene (PHA synthase 1 gene) from Pseudomonas sp. 61-3 in PHA-negative mutants P. putida GPp104 and R. eutropha PHB⁻4 to produce copolyesters [P(3HB-co-3HA)] consisting of 3HB and medium-chain-length 3HA units of 6–12 carbon atoms. The introduction of the three genes into GPp104 strain conferred the ability to synthesize P(3HB-co-3HA) with relatively high 3HB compositions (up to 49 mol%) from gluconate and alkanoates, although 3HB units were not incorporated at all or at a very low fraction (3 mol%) into copolyesters by the strain carrying phaC1 _Ps gene only. In addition, recombinant strains of R. eutropha PHB⁻4 produced P(3HB-co-3HA) with higher 3HB fractions from alkanoates and plant oils than those from recombinant GPp104 strains. One of the recombinant strains, R. eutropha PHB⁻4/pJKSc46-pha, in which all the genes introduced were expressed under the control of the native promoter for Pseudomonas sp. 61-3 pha locus, accumulated P(3HB-co-3HA) copolyester with a very high 3HB fraction (85 mol%) from palm oil. The nuclear magnetic resonance analyses showed that the copolyesters obtained here were random copolymers of 3HB and 3HA units. Received: 12 July 1999 / Received revision: 1 October 1999 / Accepted: 2 October 1999     

Quantitative aspects of glucose metabolism by Escherichia coli B/r,grown in the presence of pyrroloquinoline quinone

Escherichia coli B/r was grown in chemostat cultures under various limitations with glucose as carbon source. Since E. coli only synthesized the glucose dehydrogenase (GDH) apo-enzyme and not the appropriate cofactor, pyrroloquinoline quinone (PQQ), no gluconate production could be observed. However, when cell-saturating amounts of PQQ (nmol to mol range) were pulsed into steady state glucose-excess cultures of E. coli, the organisms responded with an instantaneous formation of gluconate and an increased oxygen consumption rate. This showed that reconstitution of GDH in situ was possible.Hence, in order to examine the influence on glucose metabolism of an active GDH, E. coli was grown aerobically in chemostat cultures under various limitations in the presence of PQQ. It was found that the presence of PQQ indeed had a sizable effect: at pH 5.5 under phosphate- or sulphate- limited conditions more than 60% of the glucose consumed was converted to gluconate, which resulted in steady state gluconate concentrations up to 80 mmol/l. The specific rate of gluconate production (0.3–7.6 mmol·h^-1·(g dry wt cells)^-1) was dependent on the growth rate and the nature of the limitation. The production rate of other overflow metabolites such as acetate, pyruvate, and 2-oxoglutarate, was only slightly altered in the presence of PQQ. The fact that the cells were now able to use an active GDH apparently did not affect apo-enzyme synthesis.Abbreviations HEPES N-2-hydroxy-ethylpiperazine-N-2-ethane sulphonic acid - MES 2-morpholinoethane sulphonic acid - PQQ pyrroloquinoline quinone (systematic name: 2,7,9-tricarboxy-1H-pyrrolo-(2,3-f)-quinoline-4,5-dione) - WB Wurster's Blue (systematic name: 1,4-bis-(dimethylamino)-benzene perchlorate     

A lower specificity PhaC2 synthase from Pseudomonas stutzeri catalyses the production of copolyesters consisting of short-chain-length and medium-chain-length 3-hydroxyalkanoates

A polyhydroxyalkanoate (PHA) synthase gene phaC2 _Ps from Pseudomonas stutzeri strain 1317 was introduced into a PHA synthase gene phbC _Re negative mutant, Ralstonia eutropha PHB⁻4. It conferred on the host strain the ability to synthesize PHA, the monomer compositions of which varied widely when grown on different carbon sources. During cultivation on gluconate, the presence of phaC2 _Ps in R. eutropha PHB⁻4 led to the accumulation of polyhydroxybutyrate (PHB) homopolymer in an amount of 40.9 wt% in dry cells. With fatty acids, the recombinant successfully produced PHA copolyesters containing both short-chain-length and medium-chain-length 3-hydroxyalkanoate (3HA) of 4–12 carbon atoms in length. When cultivated on a mixture of gluconate and fatty acid, the monomer composition of accumulated PHA was greatly affected and the monomer content was easily regulated by the addition of fatty acids in the cultivation medium. After the (R)-3-hydroxydecanol-ACP:CoA transacylase gene phaG _Pp from Pseudomonas putida was introduced into phaC2 _Ps-containing R. eutropha PHB⁻4, poly(3HB-co-3HA) copolyester with a very high 3-hydroxybutyrate (3HB) fraction (97.3 mol%) was produced from gluconate and the monomer compositions of PHA synthesized from fatty acids were also altered. This study clearly demonstrated that PhaC2_Ps cloned from P. stutzeri 1317 has extraordinarily low substrate specificity in vivo, though it has only 54% identity in comparison to a previously described low-substrate-specificity PHA synthase PhaC1_Ps from Pseudomonas sp. 61–3. This study also indicated that the monomer composition and content of the synthesized PHA can be effectively modulated by controlling the addition of carbon sources or by modifying metabolic pathways in the hosts.     

Dehalogenation and Deamination of l-2-Amino-4-chloro-4-pentenoic Acid by Proteus mirabilis

Eighty-one strains of bacteria were tested for their ability to catalyze the release of chloride ion from Dl-2-amino-4-chloro-4-pentenoic acid. A dehalogenating enzyme was obtained from the cells of Proteus mirabilis IFO 3849, which can use the l-isomer. The enzyme was constitutively produced. The conversion of l-2-amino-4-chloro-4-pentenoic acid to 2-keto-4-pentenoic acid, ammonia, and chloride ion was demonstrated. The reaction product, 2-keto-4-pentenoic acid, was isolated as its 2,4-dinitrophenylhydrazone and identified by catalytic hydrogenolysis of the hydrazone to the corresponding amino acid, norvaline.