Cometabolic biosynthesis of copolyesters consisting of 3-hydroxyvalerate and medium-chain-length 3-hydroxyalkanoates by Pseudomonas sp. DSY-82

A newly isolated strain, designated as Pseudomonas sp. DSY-82, synthesized medium-chain-length polyhydroxyalkanoate (MCL-PHA) copolyesters when grown on alkanoates from hexanoate to undecanoate as the sole carbon source. When used alone, butyrate and valerate supported the growth of the isolate but not PHA production. However, unusual polyesters containing 3-hydroxyvalerate, as well as various MCL 3-hydroxyalkanoate monomeric units, were synthesized when valerate was cofed with either nonanoate or 10-undecenoate, suggesting the formation of monomer units from both substrates. Concentrations of 3-hydroxyvalerate, 3-hydroxyoctanoate, and 3-hydroxydecanoate in the PHAs produced were significantly elevated by the addition of valerate, indicating that the incorporation of these monomer units to PHA occurred primarily through cometabolism. The total amount of these monomer units in the PHAs reached up to 30%. The PHAs produced in this study were most likely random copolyesters as determined by differential scanning calorimetric analysis. This is the first case of microbial synthesis of copolyesters consisting of 3-hydroxyvalerate and MCL 3-hydroxyalkanoate monomer units through cometabolism.     

Production of biodegradable copolyesters of 3-hydroxybutyrate and 4-hydroxybutyrate by Alcaligenes eutrophus

Summary New copolyesters of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) were produced by Alcaligenes eutrophus from various carbon sources of 4-hydroxybutyric acid, 4-chlorobutyric acid, 1,4-butanediol, and -butyrolactone. The composition of copolyesters varied from 0 to 37 mol% 4HB, depending on the carbon sources supplied. The biosynthetic pathway of copolyesters has been discussed. The copolyester film was biodegradable in soil and activated sludge. The rate of biodegradation was enhanced by the presence of 4HB units.     

Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126

A number of taxonomically-related bacteria have been identified which accumulate poly(hydroxyalkanoate) (PHA) copolymers containing primarily 3-hydroxyvalerate (3HV) monomer units from a range of unrelated single carbon sources. One of these, Rhodococcus sp. NCIMB 40126, was further investigated and shown to produce a copolymer containing 75 mol% 3HV and 25 mol% 3-hydroxybutyrate (3HB) from glucose as sole carbon source. Polyesters containing both 3HV and 3HB monomer units, together with 4-hydroxybutyrate (4HB), 5-hydroxyvalerate (5HV) or 3-hydroxyhexanoate (3HHx), were also produced by this organism from certain accumulation substrates. With valeric acid as substrate, almost pure (99 mol% 3HV) poly(3-hydroxyvalerate) was produced. N.m.r. analysis confirmed the composition of these polyesters. The thermal properties and molecular weight of the copolymer produced from glucose were comparable to those of PHB produced by Alcaligenes eutrophus.     

The biodegradation of poly-3-hydroxyalkanoates,PHAs, with long alkyl substitutents byPseudomonas maculicola

Utilizing a quantitative clear zone technique, the activity of an extracellular depolymerase system fromPseudomonas maculicola was investigated. Polymer degradation was influenced by the amount and availability of secondary carbon sources, with a simultaneous utilization of both sources. The initial carbon source in the liquid preculture also affected the eventual colony growth and polymer degradation. The enzyme solution was determined to readily degrade poly-3-hydroxyalkanoates (PHAs) with relatively long alkyl substituents at the 3 position: poly-3-hydroxyoctanoate (PHO), poly-3-hydroxynonanoate (PHN), and their copolymers (P[HO-co-HN]) and poly-3-hydroxyundecanoate (PHU). However, the system was unable to degrade either PHAs with shorter alkyl groups, including poly-3-hydroxybutyrate (PHB) and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P[HB-co-HV]) or PHAs with unusual substituents such as poly(3-hydroxy-5-phenylvaleric acid) (PHPV). It is proposed that degradation of these more bulky side chain polymers was prevented by the inability of the bacteria to assimilate their monomeric components, which inhibited the successful utilization of secondary carbon sources and thus inhibited colony growth.     

Biosynthesis of poly(3-hydroxybutyrate-Co-3-hydroxyvalerate) by a mutant of Sphaerotilus natans

The copolyester of 3-hydroxybutyrate and 3- hydroxyvalerate was synthesized from the combined carbon sources of glucose and sodium propionate by a filamentaion-defective mutant of Sphaerotilus natans, which is a typical filamentous bacterium often found in activated sludge. The 3-hydroxyvalerate content in the produced polymer increased with increasing concentrations of propionate. Cell growth and polyester synthesis were observed even when 0.6% sodium propionate was added to the medium, when the 3-hydroxyvalerate content in the polymer produced was about 60 mol%. The monomer composition of the copolymer was also varied by aeration conditions, time of propionate feeding, and cultivation time. This strain flocculated in accordance with cell growth, allowing rapid and convenient separation of the biomass from the culture fluid.     

The accumulation of poly(3-hydroxyalkanoates) in Rhodobacter sphaeroides

In recent years industrial interest has been focussed on the evaluation of poly(3-hydroxyalkanoates) (PHA) as potentially biodegradable plastics for a wide range of technical applications. Studies have been carried out in order to optimize growth and culture conditions for the intracellular formation of PHA in the phototrophic, purple, non-sulfur bacterium Rhodobacter sphaeroides. Its potential to produce polyesters other than poly(3-hydroxybutyrate) (PHB) was investigated. On an industrial scale, the use of photosynthetic bacteria could harness sunlight as an energy source for the production of these materials. R. sphaeroides was grown anaerobically in the light on different carbon sources. Under nitrogenlimiting conditions a PHA content of up to 60 to 70% of the cellular dry weight was detected. In all of the cases studied, the storage polymer contained approximately 98 mol% of 3-hydroxybutyrate (HB) and 2 mol% 3-hydroxyvalerate (HV) monomer units. Decreasing light intensities did not stimulate PHA formation. Compared to Rhodospirillum rubrum (another member of the family of Rhodospirillaceae), R. sphaeroides showed a limited flexibility in its ability to form PHA with varying monomer unit compositions.     

Production of polyhydroxyalkanoates from methanol by a new methylotrophic bacterium Methylobacterium sp. GW2

A new bacterial strain, isolated from groundwater contaminated with explosives, was characterized as a pink-pigmented facultative methylotroph, affiliated to the genus Methylobacterium. The bacterial isolate designated as strain GW2 was found capable of producing the homopolymer poly-3-hydroxybutyrate (PHB) from various carbon sources such as methanol, ethanol, and succinate. Methanol acted as the best substrate for the production of PHB reaching 40 % w/w dry biomass. PHB accumulation was observed to be a growth-associated process, so that there was no need for two-step fermentation. Optimal growth occurred at 0.5 % (v/v) methanol concentration, and growth was strongly inhibited at concentration above 2 % (v/v). Methylobacterium sp. strain GW2 was also able to accumulate the copolyester poly-3-hydroxybutyrate-poly-3-hydroxyvalerate (PHB/HV) when valeric acid was supplied as an auxiliary carbon source to methanol. After 66 h, a copolymer content of 30 % (w/w) was achieved with a PHB to PHV ratio of 1:2. Biopolymers produced by strain GW2 had an average molecular weight ranging from 229,350 to 233,050 Da for homopolymer PHB and from 362,430 to 411,300 Da for the copolymer PHB/HV.     

盐单胞菌利用短链脂肪酸合成聚羟基脂肪酸酯

盐单胞菌(Halomonas)能够利用多种底物为碳源生长,由于其能在高盐条件下进行不灭菌的开放发酵,已被开发用作下一代生物技术的底盘细胞.包括乙酸、丙酸和丁酸在内的短链挥发性脂肪酸能够以生物质为原料制备,有望成为用于微生物发酵的新型碳源.利用10-50g/L浓度的丁酸为碳源对Halomonas sp.TD01和TD08...     

Fatty acid content in wild type and WZSL mutant of Euglena gracilis

The effects of growth conditions on fatty acid profilewere examined in the photosynthetic wild type and inthe spontaneous non-photosynthetic WZSL mutant of theunicellular flagellate Euglena gracilis. Inthe light, the amount of polyunsaturated fatty acids(PUFAs) is higher in the wild type than in the mutant,independent of the carbon source. Among importantPUFAs, linolenic acid (18:3 3) is present inhigh amount only in wild type cells grown in the lightwith any of the tested carbon sources. The content ofother PUFAs, such as arachidonic acid (20:46), EPA (20:5 3) and DHA (22:63), is not correlated with the presence oflight or chloroplasts.The main effect of the dark in both strains is tolower the content of PUFAs and mono-unsaturated fattyacids and to increase the content of saturated fattyacids with all the carbon sources.     

The cyclic 3'',5''-adenosine monophosphate receptor protein and regulation of cyclic 3'',5''-adenosine monophosphate synthesis in Escherichia coli.

Summary Rates of synthesis of cyclic 3,5-adenosine monophosphate (cAMP) were measured in cultures of Escherichia coli aerating without a carbon source. This technique provides a representative measure of adenylate cyclase activity in the absence of inhibition caused by transport of the carbon source. Adenylate cyclase activity was found to vary more than 20-fold depending on the carbon source that had been available during growth. Synthesis of cAMP in cells aerating in the absence of the carbon source was highest when cells had been grown with glucose or fructose which inhibit adenylate cyclase activity severely. Synthesis of cAMP was much lower when cells had been grown with glycerol or succinate which cause only minimal inhibition of the activity.The variation in cAMP synthesis due to different carbon sources requires a functional cAMP receptor protein (CRP). Crp^- mutants synthesize cAMP at comparable rates regardless of the carbon source that afforded growth. A novel mutant of E. coli having a CRP no longer dependent on cAMP has been isolated and characterized. Adenylate cyclase activity in this mutant no longer responds normally to variations in the carbon source.     

Cultivation of Escherichia coli with mixtures of 3-phenylpropionic acid and glucose: Dynamics of growth and substrate consumption

In technical as well as natural ecosystems, pollutants are often mineralised in the presence of easily degradable carbon sources. A laboratory model system consisting of Escherichia coli ML 30 growing with mixtures of 3-phenylpropionic acid (3ppa, pollutant) and glucose (easily degradable substrate) was investigated in batch and carbon-limited continuous culture. Untypically, a linear growth pattern was observed during batch cultivation with 3ppa as the only carbon/energy source. When exposed to mixtures of both substrates in batch culture, E. coli utilised the two compounds sequentially. However, 3ppa and glucose were consumed simultaneously in continous culture. Whereas a pulse of excess glucose to a batch culture growing with 3ppa led to the repression of 3ppa utilisation, an excess of glucose added into continuous culture did not inhibit the utilisation of 3ppa. During continuous cultivation the 3ppa-degrading enzyme system operated close to saturation.     

Chromosome specificity of satellite DNAs: short- and long-range organization of a diverged dimeric subset of human alpha satellite from chromosome 3

The human alpha satellite DNA family, like many highly repeated satellite DNAs in eukaryotic genomes, is organized in distinct chromosome-specific subsets. As part of investigations into the molecular and evolutionary basis for the chromosome-specific nature of such subsets, we report the isolation and characterization of alpha satellite sequences specific for human chromosome 3. This subset is characterized by a predominant tandemly arranged 2.9 kb higher-order repeat unit which, in turn, consists of 17 tandem diverged monomer repeat units of 171 bp. Nucleotide sequence analysis reveals that the chromosome 3 higher-order repeat units are comprised, at least in part, of diverged dimeric ( 340 bp) sub-repeats and that this divergence accounts for the chromosome-specific behavior of this subset. Pulsed-field gel electrophoresis demonstrates that the chromosome 3 higher-order repeat units are localized in large domains, at least 1000 kb in length. Familial restriction fragment length polymorphisms associated with the satellite subset can be detected by pulsed field gel electrophoresis and may facilitate molecular analysis of interchromosomal variation.     

The relationship between sucrose hydrolysis,sorbitol formation and mineral ion concentration during bioethanol formation using Zymomonas mobilis 2716

Summary Investigations into the relationship between sucrose hydrolysis, sorbitol formation and mineral ion concentration during bioethanol formation by Zymomonas mobilis 2716 revealed two distinct phenomena responsible for carbon flow diversion, a sucrose effect and a salt effect. Neither of the two phenomena affects sucrose hydrolysis, but they divert carbon flow of the fructose monomer leading to its own accumulation, sorbitol or oligosaccharide formation. Sucrose concentrations in excess of 15% (w/v) led to sorbitol formation, the level of which may exceed 2% (w/v) depending upon glucose accumulation during sucrose hydrolysis. Increasing mineral ion concentrations led initially to carbon losses and finally to fructose accumulation instead of sorbitol formation. This carbon loss can be corrected by the addition of invertase, which in turn leads to an increase in sorbitol, fructose and ethanol. Potassium and chloride are the dominant ions responsible for suppression of sorbitol formation and fructose uptake, encouraging oligosaccharide formation. These fructooligosaccharides must be of a type which can be converted to fructose, sorbitol and ethanol through the action of invertase. The requirement of invertase addition to prevent fructooligosaccharide formation is indirect evidence that Z. mobilis 2716 does not produce invertase.Offprint requests to: H. W. Doelle     

Production of copolyesters of 3-hydroxybutyrate and 3-hydroxyvalerate by Alcaligenes eutrophus from butyric and pentanoic acids

Summary Copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) have been produced by Alcaligenes eutrophus in nitrogenfree culture solutions of butyric and pentanoic acids. When pentanoic acid was used as the sole carbon source, a copolyester with an unusually high 3HV fraction of 90 mol% was produced. Copolyesters with a wide range of compositions (0–90 mol% 3HV) were obtained by using butyric and pentanoic acids together as carbon sources. The biosynthetic pathways of poly(3-hydroxybutyrate) were investigated using [1-¹³C]acetate and [1-¹³C]butyrate. It is suggested that butyric and pentanoic acids are incorporated into the copolyester as 3HB and 3HV units respectively without decomposition of the carbon skeletons in the cell.     

Effect of different carbon and nitrogen sources on the growth and sporulation of Claviceps microcephal (Wallr) TUL.

The effect of different carbon and nitrogen compounds on growth and sporulation ofC. microcephala (Wallr.)Tul. causing ergot disease of Bajra has been studied. Nine different sources of carbon were used but cane sugar was found to be the best source for both, growth and sportulation of the fungus. Glucose, sucrose and maltose gave good growth but fair sporulation. Lactose and sorbitaol proved to be the poor sources. However, fungus failed to utilize starch, dextrin and mannitol.Nineteen nitrogen compounds were tried for the growth and sporulation of the fungus. Best growth and sporulation were supported by peptone and glycine. L-asparagine, DL-valine, Urea, magnesium nitrate and L-proline supported good growth and fair sporulation except DL-valine where it was excellent. Poor growth was obtained on L-isoleucin, ammonium sulphate, potassium nitrate,-alanine, ammonium chloride, DL-aspartic acid and DL-methionine. Fungus failed to utilize thio-urea.     

Production of poly(3-hydroxyalkanoates) from α, ω-alkanedioic acids and hydroxylated fatty acids by Alcaligenes sp.

Summary Poly(3-hydroxybutyrate) (P(3HB)) was produced from a series of , -alkanedioic acids of both even and odd carbon numbers by the Alcaligenes sp. AK201. In contrast, copolymers of 3HB and 3-hydroxyvalerate(P(3HB-co-3HV)) were produced from hydroxylated fatty acids of even carbon numbers such as 12-hydroxystearate and 2-hydroxyoctanoate. The biosynthetic pathways to poly(3-hydroxyalkanoates)(P(3HA)) are discussed.     

Accumulation of PHA and its copolyesters by Methylobacterium sp. KCTC 0048

Summary Methylobacterium sp. KCTC 0048 isolated from soil, could synthesize a variety of copolyesters when secondary carbon substrates were added to nitrogen-limited cultures containing methanol as a major carbon and energy source. The copolyester of 3-hydroxy-butyrate and 3-hydroxyvalerate, P(3HB-co-3HV) accumulated when valeric acid, pentanol or heptanoic acid was added to the nitrogen-limited medium containing methanol. The copolyester of 3-hydroxybutyrate and 4-hydroxybutyrate, P(3HB-co-4HB) was synthesized from 4-hydroxybutyrate, 1,4-butanediol, or -butyrolactone, and the copolyester of 3-hydroxybutyrate and 3-hydroxypropionate (P(3HB-co-3HP)), from 3-hydroxypropionate as the secondary carbon substrates, respectively.     

Hyperproduction of polyesters consisting of medium-chain-length hydroxyalkanoate monomers by strain Pseudomonas stutzeri 1317

Pseudomonas stutzeri strain 1317 was found to grow on various fatty acids, alcohols, diols, as well as glucose and gluconate for the synthesis of polyhydroxyalkanoates (PHA) with various monomer units. The PHA monomer structures were dependent on the type of fatty acids and alcohols, as well as the diols in the culture media. Only even number monomers, such as 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO) and 3-hydroxydecanoate (HD), were accumulated when even numbered fatty acids, alcohols, glucose and gluconate, as well as diol were used as carbon sources. Odd numbered fatty acids and odd numbered alcohols led to the formation of odd numbered monomers, such as 3-hydroxyvalerate (HV), 3-hydroxyheptanoate (HHp), 3-hydroxynonanoate (HN) and 3-hydroxyundecanoate (HU). The strain tolerated up to 1.5% of ethanol and made 8.3% of PHA when growth was conducted in 1.2% of ethanol. PHA formed up to 77% of cell dry weight when the strain was grown in tridecanoate. PHA synthesis was highly dependent on the nitrogen source. A depletion in nitrogen supply immediately resulted in PHA accumulation in cells grown in the glucose mineral medium.     

Non-conventional yeasts as producers of polyhydroxyalkanoates—genetic engineering of<Emphasis Type="Italic"> Arxula adeninivorans</Emphasis>

The non-conventional yeast Arxula adeninivorans was equipped with the genes phbA, phbB and phbC of the polyhydroxyalkanoate (PHA) biosynthetic pathway of Ralstonia eutropha, which encode -ketothiolase, NADPH-linked acetoacetyl-CoA reductase and PHA synthase, respectively. Arxula strains transformed solely with the PHA synthase gene (phbC) were able to produce PHA. However, the maximum content of the polymer detected in these strains was just 0.003% poly-3-hydroxybutyrate (PHB) and 0.112% poly-3-hydroxyvalerate (PHV). The expression of all three genes (phbA, phbB, phbC) resulted in small increases in the PHA content of the transgenic Arxula cells. However, under controlled cultivation conditions with minimal medium and ethanol as the carbon source, the recombinant yeast was able to accumulate up to 2.2% PHV and 0.019% PHB. Possible reasons for these differences are discussed.     

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. Bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.