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

Various aerobic Gram-negative bacteria were analysed for utilizing 4-hydroxyhexanoic acid (4HHx) as a carbon source for growth and for synthesis of polyhydroxyalkanoic acids (PHA). Although many wild types grew on 4HHx, only recombinant strains of the PHA-negative mutants Pseudomonas putida GPp104 and Alcaligenes eutrophus PHB^–4, which harboured plasmid pHP1014::E156 with the PHA-biosynthesis genes of Thiocapsa pfennigii, incorporated 4HHx up to a molar fraction of 47 or 1.4%, respectively, into PHA if the cells were cultivated in the presence of 4HHx as sole carbon source and under nitrogen starvation. A terpolyester consisting of 3-hydroxybutyric acid (3HB), 3-hydroxyhexanoic acid (3HHx) and 4HHx was synthesized, as revealed by gas chromatographic analysis of the accumulated polyester and as confirmed by nuclear magnetic resonance spectroscopic analysis of the isolated polyester. 4HHx was also detected in PHA accumulated by Rhodococcus ruber if 4HHx was used as a carbon source. However, it occurred at a molar fraction of maximally 1.3 mol% only beside 3HB, 3-hydroxyvaleric acid and 3HHx. Correspondence to: A. Steinbüchel     

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     

Diversity of bacterial polyhydroxyalkanoic acids

Abstract An overview is provided on the diversity of biosynthetic polyhydroxyalkanoic acids, and all hitherto known constituents of these microbial storage compounds are listed. The occurrence of 91 different hydroxyalkanoic acids reflects the low substrate specificity of polyhydroxyalkanoic acid synthases which are the key enzymes of polyhydroxyalkanoic acid biosynthesis. In addition, the importance of bacterial anabolism and catabolism, which provide the coenzyme A thioesters of the respective hydroxyalkanoic acids as substrates to these PHA synthases, is emphasized.     

Quantification of bacterial polyhydroxyalkanoic acids by Nile red staining

The fluorescence properties of one chemically and seven biologically produced polyhydroxyalkanoic acids were investigated as film castings and in living cells respectively after staining with Nile red. All these polyesters show a similar fluorescence behaviour, revealing a clear fluorescence maximum at an excitation wavelength between 540 nm and 560 nm and an emission wavelength between 570 nm and 605 nm. This could be shown by the use of two-dimensional fluorescence spectroscopy and flow cytometry. The examination of native poly(3-hydroxybutyric acid), poly(3HB), granules isolated from cells of Ralstonia eutropha H16 showed that the addition of 6.0 μg Nile red is necessary for total staining of 1.0 mg granules. The fluorescence intensity at an excitation wavelength of 550 nm and an emission wavelength of 600 nm showed high correlation to the poly(3HB) concentration of grana suspensions at different grana concentrations. These results and the staining of cell suspensions during cultivation experiments revealed that Nile red has a high potential for the quantitative determination of hydrophobic bacterial polyhydroxyalkanoic acids. Received: 13 November 1998 / Received revision: 4 February 1999 / Accepted: 12 February 1999     

Evaluation of soil gram-negative bacteria yielding polyhydroxyalkanoic acids from carbohydrates and propionic acid

 A screening programme was developed leading to the isolation of 75 strains of soil gram-negative bacteria which are able to produce polyhydroxyalkanoic acids (PHA) from sugar-cane derivatives. The evaluation of these strains was performed with regard to their efficiency in converting carbohydrates or propionic acid into PHA constituents. Several strains were able to use sucrose as well as glucose and fructose to grow and afterwards to accumulate poly-(3-hydroxybutyric acid) (PHB) with promising yields. Seven strains were found to have more than 80% of the theoretical value when converting carbohydrates into PHB and accumulated at least 50% of the cell dry weight as PHB. Ten strains incorporated 3-hydroxyvaleric acid units into the polymer from propionic acid of which 3 gave yields comparable to those of Alcaligenes eutrophus. Received: 19 May 1995/Received revision: 2 January 1996/Accepted: 22 January 1996     

Occurrence of 3 beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid as normal constituents in human blood

Three unconjugated C27 bile acids were found in plasma from healthy humans. They were isolated by liquid-solid extraction and anion-exchange chromatography and were identified by gas-liquid chromatography-mass spectrometry, microchemical reactions, and ultraviolet spectroscopy as 3 beta-hydroxy-5-cholestenoic, 3 beta,7 alpha-dihydroxy-5-cholestenoic, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acids. Their levels often exceeded those of the unconjugated C24 bile acids and the variations between individuals were smaller than for the C24 acids. The concentrations in plasma from 11 healthy subjects were 67.2 +/- 27.9 ng/ml (mean +/- SD) for 3 beta-hydroxy-5-cholestenoic acid, 38.9 +/- 25.6 ng/ml for 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 81.7 +/- 27.9 ng/ml for 7 alpha-hydroxy-3-oxo-4-cholestenoic acid. The levels of the individual acids were positively correlated to each other and not to the levels of the C24 acids. The cholestenoic acids were below the detection limit (20-50 ng/ml) in bile and C27 bile acids present in bile were not detected in plasma.     

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid.

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k₁), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k₁ value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k₁ value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k₁ values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.     

Biosynthesis and biotechnological production of degradable polyhydroxyalkanoic acid

The use of biodegradable polymers is one of the key solution to environmental problems and the development of biocompatible material. The impact of such a large commercial opportunity is one of the primary reasons for much interest in the field of microbial polyester, polyhydroxyalkanoic acid (PHA). Its valuable properties of biodegradability, biocompatibility and thermoplasticity have attracted considerable commercial interest, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] has been launched as the first market product. Recent advances in molecular genetics and microbial physiology of PHA biosynthesis have been uncovering the biosynthetic mechanics at molecular level, and extensive efforts for the developments of practical applications and cost-effective mass production of PHA will profell the commercialization of PHA towards the commodity market for biodegradable plastics. The biosynthesis of new members of PHA family with new monomer or unusual composition will also lead to the biotechnological production of tailor-made biopolymer for various applications.     

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k₁), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k₁ value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k₁ value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k₁ values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.     

Identification of boronic acids as antagonists of bacterial quorum sensing in Vibrio harveyi

Bacterial quorum sensing plays a very important role in the regulation of biofilm formation, virulence, conjugation, sporulation, and swarming mobility. Inhibitors of bacterial quorum sensing are important research tools and potential therapeutic agents. In this paper, we describe for the first time the discovery of several boronic acids as single digit micromolar inhibitors of bacterial quorum sensing in Vibrio harveyi.     

Molecular basis for biosynthesis and accumulation of polyhydroxyalkanoic acids in bacteria

Abstract The current knowledge on the structure and on the organization of polyhydroxyalkanoic acid (PHA)-biosynthetic genes from a wide range of different bacteria, which rely on different pathways for biosynthesis of this storage polyesters, is provided. Molecular data will be shown for genes of Alcaligenes eutrophus , purple non-sulfur bacteria, such as Rhodospirillum rubrum , purple sulfur bacteria, such as Chromatium vinosum , pseudomonads belonging to rRNA homology group I, such as Pseudomonas aeruginosa, Methylobacterium extorquens , and for the Gram-positive bacterium Rhodococcus ruber . Three different types of PHA synthases can be distinguished with respect to their substrate specificity and structure. Strategies for the cloning of PHA synthase structural genes will be outlined which are based on the knowledge of conserved regions of PHA synthase structural genes and of the PHA-biosynthetic routes in bacteria as well as on the heterologous expression of these genes and on the availability of mutants impaired in the accumulation of PHA. In addition, a terminology for the designation of PHAs and of proteins and genes relevant for the metabolism of PHA is suggested.     

Incorporation of 2-methyl-3-hydroxybutyric acid into polyhydroxyalkanoic acids by axenic cultures in defined media

Abstract Alcaligenes eutrophus and Burkholderia cepacia synthesized and accumulated a terpolyester consisting of 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and 2-methyl-3-hydroxybutyric acid (2Me3HB) if the cells were cultivated in a mineral salts medium containing tiglic acid as the sole carbon source or in combination with gluconic acid. The presence of 1–2 mol% of 2Me3HB in the polyester was confirmed by comparison with chemically synthesized methyl ester of 2Me3HB and by nuclear magnetic resonance spectrometry as well as by gas chromatography/mass spectrometry. This is the first report of the incorporation of 2Me3HB by axenic cultures cultivated under defined conditions.     

Identification of a 13-kDa protein associated with the polyhydroxyalkanoic acid granules from Acinetobacter spp.

Abstract Transferrin-binding proteins from Neisseria meningitidis vary among different isolates. We have identified and studied a hypervariable region adjacent to the carboxyl-end of the transferrin-binding domain of the Tbp2 molecule. The tbp2 genes from six strains of N. meningitidis were cloned and sequenced in this particular region. Sequence analysis of these regions along with five other sequences available from pathogenic Neisseria showed a common organisation of seven highly variable nucleotide stretches interspersed with six conserved nucleotide stretches. The variable regions correlated with the location of immunoreactive epitopes in polyclonal antisera raised to transferrin-binding proteins identified by peptide pin technology. Sequence analysis suggested a mosaic-like organisation of the tbp2 genes. Taken together, these data suggest that the antigenic variation in this part of the protein may result from a strong host immune pressure.     

Recent advances in the knowledge of the metabolism of bacterial polyhydroxyalkanoic acids and potential impacts on the production of biodegradable thermoplastics

Bacteria are able to synthesize a wide variety of different thermoplastic polyhydroxyalkanoic acids (PHA). The physiology of PHA metabolism has been studied in most detail in the aerobic hydrogen bacterium Alcaligenes eutrophus. Strains of this bacterium are already used for the production of biodegradable polyesters on a technological scale. The current knowledge on the pathways for PHA synthesis, on the genes essential for PHA biosynthesis and on genes affecting the accumulation of PHA in this bacterium will be reviewed.     

4-Methylumbelliferyl-alpha-glycosides of partially O-acetylated N-acetylneuraminic acids as substrates of bacterial and viral sialidases

4-O-Acetylated, 7-O-acetylated, and 9-O-acetylated 4-methylumbelliferyl-alpha-N-acetyl-neuraminic acids (Neu4,5Ac2-MU, Neu5,7Ac2-MU, Neu5,9Ac2-MU) were tested as substrates of sialidases of Vibrio cholerae and of Clostridium perfringens. Both sialidases were unable to hydrolyse Neu4,5Ac2-MU. This compound at 1 mM concentration did not inhibit significantly the cleavage of Neu5Ac-MU, the best substrate tested. The 4-O-acetylated sialic acid glycoside is hydrolysed slowly by the sialidase from fowl plague virus. The relative substrate specificity, reflected in V/Km of the Vibrio cholerae sialidase is Neu5Ac-MU much greater than Neu5,7Ac2-MU approximately Neu5,9Ac2-MU and of the clostridial enzyme it is Neu5Ac-MU greater than Neu5,9Ac2-MU greater than Neu5,7Ac2-MU. The affinities of both enzymes for the side-chain O-acetylated sialic acid derivatives are higher than for Neu5Ac-MU. The artificial, well-defined substrates, described here, provide the opportunity to quantify the influence of sialic acid O-acetylation on the hydrolysis of sialoglycoconjugates without the side effects introduced by other parts of more complex glycans.