Quantitative Mass-Spectrometric Sequencing of Chitosan Oligomers Revealing Cleavage Sites of Chitosan Hydrolases.

Partially acetylated chito-oligosaccharides (paCOS) have diverse bioactivities that turn them into promising compounds especially for medical and agricultural applications. These properties likely arise from different acetylation patterns, but determining the sequences of paCOS and producing paCOS with patterns of interest have proven difficult. We present a novel method for sequencing submicrogram amounts of paCOS using quantitative mass spectrometry, allowing one to rapidly analyze the substrate specificities of chitosan hydrolases that can be used to produce paCOS. The method involves four major steps: (i) acetylation of free amino groups in paCOS using a deuterated reagent; (ii) labeling the reducing end with an 18O-tag; (iii) quantifying paCOS using [13C2, 2H3]-labeled isotopologs as internal standards; (iv) sequencing paCOS by tandem MS. Eventually, this method will aid in developing enzymes with cleavage patterns optimized for producing paCOS with defined patterns of acetylation and specific bioactivities.

Evaluation of the antiproliferative activity of the leaves from Arctium lappa by a bioassay-guided fractionation.

Arctium lappa L. (Asteraceae) is used in folk medicine around the World, and shows several kinds of biological activity, particularly in vitro antitumor activity in different cell lines. This study evaluated the antiproliferative activity of the crude extract, semipurified fractions, and isolated compounds from the leaves of A. lappa, through bioassay-guided testing in Caco-2 cells. The crude extract was obtained with a 50% hydroethanolic extract and then partitioned with hexane, ethyl acetate, and n-butanol. The ethyl-acetate fraction (EAF) showed antiproliferative activity. This fraction was subjected to sequential column chromatography over silica gel to afford onopordopicrin (1), mixtures of 1 with dehydromelitensin-8-(4'-hydroxymethacrylate) (2), a mixture of 2 with dehydromelitensin (3), mixture of 1 with melitensin (4), dehydrovomifoliol (5), and loliolide (6). The compounds were identified by spectroscopic methods (NMR, MS) and comparison with literature data. This is the first description of compounds 2-5 from this species. The compounds tested in Caco-2 cells showed the following CC(50) (µg/mL) values: 1: 19.7 ± 3.4, 1 with 2: 24.6 ± 1.5, 2 with 3: 27 ± 11.7, 1 with 4: 42 ± 13.1, 6 30 ± 6.2; compound 5 showed no activity.

Oligonuclear gallium nitrogen cage compounds: molecular intermediates on the way from gallium hydrazides to gallium nitride.

Gallium hydrazides are potentially applicable as facile starting compounds for the generation of GaN by thermolysis. The decomposition pathways are, however, complicated and depend strongly on the substituents attached to the gallium atoms and the hydrazido groups. This paper describes some systematic investigations into the thermolysis of the gallium hydrazine adduct Bu(t)(3)Ga←NH(2)-NHMe (1a) and the dimeric gallium hydrazides [R(2)Ga(N(2)H(2)R')](2) (2b, R = Bu(t), R' = Bu(t); 2c, R = Pr(i), R' = Ph; 2d, R = Me, R' = Bu(t)) which have four- or five-membered heterocycles in their molecular cores. Heating of the adduct 1a to 170 °C gave the heterocyclic compound Bu(t)(2)Ga(µ-NH(2))[µ-N(Me)-N(=CH(2))]GaBu(t)(2) (3) by cleavage of N-N bonds and rearrangement. 3 was further converted at 400 °C into the tetrameric gallium cyanide (Bu(t)(2)GaCN)(4) (4). The thermolysis of the hydrazide (Bu(t)(2)Ga)(2)(NH-NHBu(t))(2) (2b) at temperatures between 270 and 420 °C resulted in cleavage of all N-N bonds and the formation of an octanuclear gallium imide, (Bu(t)GaNH)(8) (6). The trimeric dialkylgallium amide (Bu(t)(2)GaNH(2))(3) (5) was isolated as an intermediate. Thermolysis of the hydrazides (Pr(i)(2)Ga)(2)(NH-NHPh)(NH(2)-NPh) (2c) and (Me(2)Ga)(2)(NH-NHBu(t))(2) (2d) proceeded in contrast with retention of the N-N bonds and afforded a variety of novel gallium hydrazido cage compounds with four gallium atoms and up to four hydrazido groups in a single molecule: (Pr(i)Ga)(4)(NH-NPh)(3)NH (7), (MeGa)(4)(NH-NBu(t))(4) (8), (MeGa)(4)(NH-NBu(t))(3)NBu(t) (9), and (MeGa)(4)(NHNBu(t))(3)NH (10). Partial hydrolysis gave reproducibly the unique octanuclear mixed hydrazido oxo compound (MeGa)(8)(NHNBu(t))(4)O(4) (11).

An organogallium subfluoride and subhydroxide: three Ga-Ga bonds in single molecules.

Treatment of the digallium compound R2Ga-GaR2 1 [R = CH(SiMe3)2] with hydrogen fluoride or water in the presence of 2-aminonicotinic acid afforded the corresponding fluoride or hydroxide, R(X)Ga-Ga(X)R (X = F, OH), by substituent exchange and retainment of the Ga-Ga bonds. Both compounds form trimeric formula units in which three Ga-Ga bonds in a parallel orientation are bridged by six fluoro or hydroxo ligands.

A new male sex pheromone and novel cuticular cues for chemical communication in Drosophila.

BACKGROUND: In many insect species, cuticular hydrocarbons serve as pheromones that can mediate complex social behaviors. In Drosophila melanogaster, several hydrocarbons including the male sex pheromone 11-cis-vaccenyl acetate (cVA) and female-specific 7,11-dienes influence courtship behavior and can function as cues for short-term memory associated with the mating experience. Behavioral and physiological studies suggest that other unidentified chemical communication cues are likely to exist. To more fully characterize the hydrocarbon profile of the D. melanogaster cuticle, we applied direct ultraviolet laser desorption/ionization orthogonal time-of-flight mass spectrometry (UV-LDI-o-TOF MS) and analyzed the surface of intact fruit flies at a spatial resolution of approximately 200 mum. RESULTS: We report the chemical and spatial characterization of 28 species of cuticular hydrocarbons, including a new major class of oxygen-containing compounds. Via UV-LDI MS, pheromones previously shown to be expressed exclusively by one sex, e.g., cVA, 7,11-heptacosadiene, and 7,11-nonacosadiene, appear to be found on both male and female flies. In males, cVA colocalizes at the tip of the ejaculatory bulb with a second acetylated hydrocarbon named CH503. We describe the chemical structure of CH503 as 3-O-acetyl-1,3-dihydroxy-octacosa-11,19-diene and demonstrate a behavioral role for this compound as a long-lived inhibitor of male courtship. Like cVA, CH503 is transferred from males to females during mating. Unlike cVA, CH503 remains on the surface of females for at least 10 days. CONCLUSIONS: Oxygenated hydrocarbons comprise a major previously undescribed class of compounds on the Drosophila cuticular surface. A newly discovered long-chain acetate, CH503, serves as a mediator of courtship-related chemical communication.

Methyl jasmonate induced accumulation of kalopanaxsaponin I in Nigella sativa.

Hydroponically cultivated Nigella sativa L. plants treated with methyl jasmonate (MeJA) showed a twelve-fold increase in levels of the monodesmosidic triterpene saponins alpha-hederin and kalopanaxsaponin I (KsI) in the leaves. We will demonstrate that these two saponins accounted for approximately 10% of the dry plant matter, of which 93% was KsI and 7% alpha-hederin. To address the molecular basis of saponin induction by MeJA, we cloned and characterized the beta-amyrin synthase gene (NsbetaAS1) encoding one of the key enzymes in triterpene saponin biosynthesis. As expected, NsbetaAS1 transcription was induced by MeJA and led to the production of beta-amyrin when over-expressed in yeast.

Automated interface for hyphenation of planar chromatography with mass spectrometry.

A fully automated interface to couple high-performance thin-layer chromatography (HPTLC) with mass spectrometry (MS) is described. This universal hands-free interface connects intact normal-phase plates to any liquid chromatography/mass spectrometry (LC/MS) system without any adjustments or modifications to the mass spectrometer. The interface extracts the complete substance band with its depth profile and thus allows detections in the pg/band range. The high performance of the automated interface was evaluated through caffeine quantification in real samples, viz., energy drinks and pharmaceutical tablets, without internal standard. Following chromatographic separation on silica gel 60 F(254) HPTLC plates, caffeine bands were eluted from the plate by means of the automated interface to the electrospray ionization (ESI) source of a triple-quadrupole mass spectrometer. Since in full scan mode only the protonated molecule [M+H](+) was observed, caffeine quantification was performed using the selected-ion monitoring (SIM) mode at m/z 195. The validation showed highly reliable results for the linear range (R(2) = 0.9973), repeatability (RSD = 5.6%, n = 6) and intermediate precision (RSD = 1.5%, n = 3). Regarding accuracy the results obtained by HPTLC/MS were not statistically different (F-test, t-test) from those obtained by validated HPTLC/UV methods. Hence, this interface proved to be one of the most reliable and universal interfaces for HPTLC/MS.

Identification of bitter off-taste compounds in the stored cold pressed linseed oil.

Whereas freshly pressed linseed oil provides a delicate nutty flavor, a lingering bitter off-taste is developing upon storage at room temperature. Using a sensory guided fractionation approach, the key bitter compound was identified in stored linseed oil, and its structure was determined as the methionine sulfoxide-containing, cyclic octapeptide cyclo(PLFIM OLVF) by means of FTIR, LC-MS, NMR spectroscopy, and amino acid analysis. Although this peptide is known in the literature as cyclolinopeptide E, the bitter taste activity of this compound has not previously been described. Sensory evaluation revealed a recognition threshold concentration of 12.3 micromol/L water.

Identification of poly(cis-1,4-Isoprene) degradation intermediates during growth of moderately thermophilic actinomycetes on rubber and cloning of a functional lcp homologue from Nocardia farcinica strain E1.

The enrichment and isolation of thermophilic bacteria capable of rubber [poly(cis-1,4-isoprene)] degradation revealed eight different strains exhibiting both currently known strategies used by rubber-degrading mesophilic bacteria. Taxonomic characterization of these isolates by 16S rRNA gene sequence analysis demonstrated closest relationships to Actinomadura nitritigenes, Nocardia farcinica, and Thermomonospora curvata. While strains related to N. farcinica exhibited adhesive growth as described for mycolic acid-containing actinomycetes belonging to the genus Gordonia, strains related to A. nitritigenes and T. curvata formed translucent halos on natural rubber latex agar as described for several mycelium-forming actinomycetes. For all strains, optimum growth rates were observed at 50 degrees C. The capability of rubber degradation was confirmed by mineralization experiments and by gel permeation chromatography (GPC). Intermediates resulting from early degradation steps were purified by preparative GPC, and their analysis by infrared spectroscopy revealed the occurrence of carbonyl carbon atoms. Staining with Schiff's reagent also revealed the presence of aldehyde groups in the intermediates. Bifunctional isoprenoid species terminated with a keto and aldehyde function were found by matrix-assisted laser desorption ionization-time-of-flight and electrospray ionization mass spectrometry analyses. Evidence was obtained that biodegradation of poly(cis-1,4-isoprene) is initiated by endocleavage, rather than by exocleavage. A gene (lcp) coding for a protein with high homology to Lcp (latex-clearing protein) from Streptomyces sp. strain K30 was identified in Nocardia farcinica E1. Streptomyces lividans TK23 expressing this Lcp homologue was able to cleave synthetic poly(cis-1,4-isoprene), confirming its involvement in initial polymer cleavage.

Neutral lipid biosynthesis in engineered Escherichia coli: jojoba oil-like wax esters and fatty acid butyl esters.

Wax esters are esters of long-chain fatty acids and long-chain fatty alcohols which are of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant (Simmondsia chinensis) is the main biological source of wax esters. Although it has a multitude of potential applications, the use of jojoba oil is restricted, due to its high price. In this study, we describe the establishment of heterologous wax ester biosynthesis in a recombinant Escherichia coli strain by coexpression of a fatty alcohol-producing bifunctional acyl-coenzyme A reductase from the jojoba plant and a bacterial wax ester synthase from Acinetobacter baylyi strain ADP1, catalyzing the esterification of fatty alcohols and coenzyme A thioesters of fatty acids. In the presence of oleate, jojoba oil-like wax esters such as palmityl oleate, palmityl palmitoleate, and oleyl oleate were produced, amounting to up to ca. 1% of the cellular dry weight. In addition to wax esters, fatty acid butyl esters were unexpectedly observed in the presence of oleate. The latter could be attributed to solvent residues of 1-butanol present in the medium component, Bacto tryptone. Neutral lipids produced in recombinant E. coli were accumulated as intracytoplasmic inclusions, demonstrating that the formation and structural integrity of bacterial lipid bodies do not require specific structural proteins. This is the first report on substantial biosynthesis and accumulation of neutral lipids in E. coli, which might open new perspectives for the biotechnological production of cheap jojoba oil equivalents from inexpensive resources employing recombinant microorganisms.

Degradation of cyanophycin by Sedimentibacter hongkongensis strain KI and Citrobacter amalonaticus strain G Isolated from an anaerobic bacterial consortium.

Using a combination of various enrichment techniques, the strictly anaerobic, gram-positive, endospore-forming bacterium Sedimentibacter hongkongensis strain KI as revealed by 16S rRNA analysis and the gram-negative enterobacterium Citrobacter amalonaticus strain G as revealed by physiological tests were isolated from an anaerobic cyanophycin (CGP)-degrading bacterial consortium. S. hongkongensis strain KI is the first anaerobic bacterium with the ability to hydrolyze CGP to beta-Asp-Arg and beta-Asp-Lys dipeptides, as revealed by electrospray ionization-mass spectrometry and reversed-phase high-performance liquid chromatography analysis. However, these primary accumulated hydrolysis products were only partially used by S. hongkongensis strain KI, and significant growth on CGP did not occur. On the other hand, C. amalonaticus strain G did not degrade CGP but grew on the beta-linked iso-dipeptides formed in vitro by enzymatic CGP degradation or in vivo by metabolic activity of S. hongkongensis strain KI. Dipeptide utilization occurred at the highest rate if both strains were used in cocultivation experiments with CGP, indicating that cooperation between different bacteria occurs in anaerobic natural environments for complete CGP turnover. The amino acids obtained from the cleavage of dipeptides were fermented to ethanol, acetic acid, and succinic acid, as revealed by gas chromatographic analysis and by spectrophotometric enzyme assays.

Liquid chromatographic/mass spectrometric investigation on the reaction products in the peroxidase-catalyzed oxidation of o-phenylenediamine by hydrogen peroxide.

Mass spectrometric evidence was obtained to confirm that the main reaction product of the horseradish peroxidase (POD)-catalyzed oxidation of o-phenylenediamine (OPD) by hydrogen peroxide is 2,3-diaminophenazine. Although this reaction is one of the most widespread detection schemes in enzyme-linked immunosorbent assays (ELISAs), the literature data on the identity of the reaction product(s) have been strongly contradictory throughout the last few decades. Liquid chromatography with UV/Vis and mass spectrometric detection as well as exact mass measurements after LC fraction collection have led to the unambiguous identification of 2,3-diaminophenazine as main reaction product. 2,2'-Diaminoazobenzene, which is frequently described in other publications to be the major reaction product, was not detected at all.

Synthesis of novel lipids in Saccharomyces cerevisiae by heterologous expression of an unspecific bacterial acyltransferase.

The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) is the key enzyme in storage lipid accumulation in the gram-negative bacterium Acinetobacter calcoaceticus ADP1, mediating wax ester, and to a lesser extent, triacylglycerol (TAG) biosynthesis. Saccharomyces cerevisiae accumulates TAGs and steryl esters as storage lipids. Four genes encoding a DGAT (Dga1p), a phospholipid:diacylglycerol acyltransferase (Lro1p) and two acyl-coenzyme A:sterol acyltransferases (ASATs) (Are1p and Are2p) are involved in the final esterification steps in TAG and steryl ester biosynthesis in this yeast. In the quadruple mutant strain S. cerevisiae H1246, the disruption of DGA1, LRO1, ARE1, and ARE2 leads to an inability to synthesize storage lipids. Heterologous expression of WS/DGAT from A. calcoaceticus ADP1 in S. cerevisiae H1246 restored TAG but not steryl ester biosynthesis, although high levels of ASAT activity could be demonstrated for WS/DGAT expressed in Escherichia coli XL1-Blue in radiometric in vitro assays with cholesterol and ergosterol as substrates. In addition to TAG synthesis, heterologous expression of WS/DGAT in S. cerevisiae H1246 resulted also in the accumulation of fatty acid ethyl esters as well as fatty acid isoamyl esters. In vitro studies confirmed that WS/DGAT is capable of utilizing a broad range of alcohols as substrates comprising long-chain fatty alcohols like hexadecanol as well as short-chain alcohols like ethanol or isoamyl alcohol. This study demonstrated the highly unspecific acyltransferase activity of WS/DGAT from A. calcoaceticus ADP1, indicating the broad biocatalytic potential of this enzyme for biotechnological production of a large variety of lipids in vivo in prokaryotic as well as eukaryotic expression hosts.

Studies on the biodegradability of polythioester copolymers and homopolymers by polyhydroxyalkanoate (PHA)-degrading bacteria and PHA depolymerases.

The biodegradability of microbial polythioesters (PTEs), a novel class of biopolymers which were discovered recently and can be produced by polyhydroxyalkanoate (PHA)-accumulating bacteria, was studied. Using poly(3-hydroxybutyrate- co-3-mercaptopropionate) [poly(3HB- co-3MP)] as sole carbon source for screening, 22 new bacterial strains were isolated and characterized. Interestingly, none of the PHA-degrading bacteria was able to utilize the homopolymer poly(3MP) as a carbon source for growth or to form clear zones on poly(3MP)-containing agar plates. The extracellular PHA depolymerases from two strains ( Schlegelella thermodepolymerans, Pseudomonas indica K2) were purified to electrophoretic homogeneity and biochemically characterized. The PHA depolymerase of S. thermodepolymerans exhibited a temperate optimum of about 75 degrees C to 80 degrees C and was stable at 70 degrees C for more than 24 h. Regarding the substrate specificities of the PHA depolymerase of S. thermodepolymerans, enzyme activities decreased significantly with increasing 3MP content of the copolymer substrates. Interestingly, no activity could be detected with homoPTEs consisting only of 3MP or of 3-mercaptobutyrate. Similar results were obtained with the PHA depolymerases PhaZ2, PhaZ5 and PhaZ7 of Paucimonas lemoignei which were also investigated. The PHA depolymerase of Ps. indica K2 did not cleave any of the investigated polymers containing 3MP. Gas chromatography, infrared and (1)H-NMR spectrometry and matrix-assisted laser desorption/ionization time-of-flight analysis revealed that 3MPs containing oligomers were enriched in the water-insoluble fraction remaining after partial digestion of poly(3HB- co-3MP) by purified poly(3HB) depolymerase of S. thermodepolymerans. In contrast, 3HB was enriched in the water-soluble fraction, which also contained 3HB- co-3MP dimer obtained by partial digestion of this copolymer by the enzyme. This study clearly indicates that PHA depolymerases are specific for oxoester linkages of PHAs and that the thioester bonds of PTEs cannot be cleaved by this type of enzyme.

(Butadiene)metallocene/B(C6F5)3 pathway to catalyst systems for stereoselective methyl methacrylate polymerization: evidence for an anion dependent metallocene catalyzed polymerization process.

The ansa-zirconocene dichlorides [Me(2)Si(C(5)H(4))(3-R-C(5)H(3))]ZrCl(2) 7a-e (R = H, CH(3), cyclohexyl, -CHMe(2), -CMe(3)) were reacted with butadiene-magnesium to yield the respective (eta(4)-butadiene)metallocenes 17a-e. The chiral examples give a mixture of two s-cis and two s-trans diastereomers. The strong Lewis acid B(C(6)F(5))(3) adds selectively to a terminal butadiene carbon atom to yield the (butadiene)metallocene/B(C(6)F(5))(3) betaine complexes 18a-e. Initially, the formation of the Z-18 isomers is preferred. These consecutively rearrange to the thermodynamically favored isomers E-18. The dipolar systems 18 are active single component metallocene catalysts for the stereospecific polymerization of methyl methacrylate. With increasing steric bulk of the attached single alkyl substituent an increasingly isotactic poly(methyl methacrylate) is obtained. A similar trend is observed in the methyl methacrylate polymerization at the [Me(2)Si(C(5)H(4))(3-R-C(5)H(3))]ZrCH(3)(+) catalysts (9a-e) that were conventionally prepared by methyl abstraction from the corresponding ansa-zirconocene dimethyl complexes by treatment with B(C(6)F(5))(3). A comparison of the poly(methyl methacrylates) obtained at these two series of catalysts has revealed substantial differences in stereoselectivity that probably originate from an influence of the respective counteranions. An initial reactive intermediate of methyl methacrylate addition to the dipolar single component metallocene catalyst E-18a was experimentally observed and characterized by NMR spectroscopy at 253 K. The subsequently formed series of [PMMA-C(4)H(6)(-)B(C(6)F(5))(3)](-) anion oligomers (at the catalyst 18c) was monitored (after quenching) and characterized by electrospray mass spectrometry.

Isolation and characterization of gram-positive cyanophycin-degrading bacteria-kinetic studies on cyanophycin depolymerase activity in aerobic bacteria.

This study is the first report on the extracellular degradation of cyanophycin (CGP) by Gram-positive bacteria. Three different Gram-positive bacteria were isolated from forest soil that were able to utilize CGP as the sole carbon source for growth. The isolates were assigned to species of the genera Bacillus and Micromonospora. From one of the isolates, which was taxonomically affiliated as Bacillus megaterium strain BAC19, the extracellular CGP depolymerase (extracellular CGPase; CphEBm) was purified to electrophoretic homogeneity by fast protein liquid chromatography and affinity binding to an arginine-agarose column. The purified enzyme was specific for hydrolytic cleavage of CGP, and inhibitor studies indicated that CphEBm is a serine-type peptidase. As CGP degradation products, (beta-Asp-Arg)2 tetrapeptides in addition to beta-Asp-Arg dipeptides occurred, which were identified by electrospray ionization mass spectrometry analysis. Furthermore, a novel quantitative enzyme assay was developed for kinetic studies on CGP depolymerases. For CphEBm, as well as for the extracellular CGPase of Pseudomonas anguilliseptica strain BI (CphEPa), KM values of 2.2 and 1.0 microM, respectively, for CGP were determined.

A simple device for the extraction of TLC spots: direct coupling with an electrospray mass spectrometer.

A new device is described which allows the recovery of compounds from thin layer chromatograms in short times, within a small volume, and without contamination. This apparatus can be coupled online to an electrospray mass spectrometer, but can also be used with other detectors or for micropreparations.

Selective determination of hydrogen peroxide by adduct formation with a dinuclear iron(III) complex and flow injection analysis/tandem mass spectrometry.

A highly selective method for the determination of hydrogen peroxide is presented. In a flow injection analysis (FIA) instrument, the analyte is brought into contact with a dinuclear heptadentate iron(III) complex. The formation of the peroxide adduct is quantified using electrospray tandem mass spectrometry (ESI-MS/MS). Selected reaction monitoring (SRM) based on the transition from the triply charged peroxide adduct with m/z = 251.2 to the triply charged fragment ion of m/z = 240.5 is performed. The limit of detection for hydrogen peroxide is 10(-7) mol dm(-3), limit of quantification is 3 x 10(-7) mol dm(-3), and a linear range of 2.5 decades starting at the limit of quantification is observed.

Identification of novel sulfur-containing bacterial polyesters: biosynthesis of poly(3-hydroxy-S-propyl-omega-thioalkanoates) containing thioether linkages in the side chains.

This study describes the biosynthesis of novel sulfur-containing polyhydroxyalkanoates (PHAs), which consist exclusively of hydroxypropylthioalkanoic acid containing thioether groups in the side chains. In addition, the utilization of alkylthioalkanoic acids (=thia fatty acids) by various bacteria was investigated. Based on feedings with propylthiooctanoic acid (PTO) or propylthiohexanoic acid, the metabolically engineered PHA-negative mutant PHB(-)4 of Ralstonia eutropha, which harbours plasmid pBBR1::phaC1 expressing the PHA synthase of Pseudomonas mendocina, synthesized two novel poly(3-hydroxy-S-propyl-omega-thioalkanoic) acids [poly(3HPTA)s]. A terpolyester consisting of 3-hydroxypropylthiobutyric acid (3HPTB), 3-hydroxypropylthiohexanoic acid (3HPTHx) and 3-hydroxypropyl- thiooctanoic acid (3HPTO) was synthesized from PTO, whereas a co-polyester of 3HPTB and 3HPTHx was synthesized from propylthiohexanoic acid. Fed-batch fermentation of R. eutropha PHB(-)4(pBBR1::phaC1) on PTO was done on a 26-litre scale, providing a cell density of 7.3 g l(-1), from which 45 g of the novel poly(3HPTB-co-3HPTHx-co-3HPTO) were isolated. The chemical structures of the poly(3HPTA)s were identified by gas chromatography/mass spectrometry, elemental sulfur analysis, partial pyrolysis and detailed mass spectrometric analysis, exhibiting 3HPTB, 3HPTHx and 3HPTO as constituents. These novel, hitherto undescribed, constituents of PHAs were randomly distributed in the co-polyesters.