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

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

Poly-(3-hydroxybutyrate) production from whey by high-density cultivation of recombinant Escherichia coli

Recombinant Escherichia coli strain GCSC 6576, harboring a high-copy-number plasmid containing the Ralstonia eutropha genes for polyhydroxyalkanoate (PHA) synthesis and the E. coli ftsZ gene, was employed to produce poly-(3-hydroxybutyrate) (PHB) from whey. pH-stat fed-batch fermentation, using whey powder as the nutrient feed, produced cellular dry weight and PHB concentrations of 109 g l⁻¹ and 50 g l⁻¹ respectively in 47 h. When concentrated whey solution containing 210 g l⁻¹ lactose was used as the nutrient feed, cellular dry weight and PHB concentrations of 87 g l⁻¹ and 69 g l⁻¹ respectively could be obtained in 49 h by pH-stat fed-batch culture. The PHB content was as high as 80% of the cellular dry weight. These results suggest that cost-effective production of PHB is possible by fed-batch culture of recombinant E. coli using concentrated whey solution as a substrate. Received: 19 December 1997 / Received revision: 17 March 1998 / Accepted: 20 March 1998     

In vitro biosynthesis of poly(3-hydroxybutyric acid) by using purified poly(hydroxyalkanoic acid) synthase of Chromatium vinosum

Purified recombinant poly(hydroxyalkanoic acid) (PHA) synthase from Chromatium vinosum (PhaEC_Cv) was used to examine in vitro the specific synthase activity, turnover of R-(−)-3-hydroxybutyryl coenzyme A (3HB-CoA) and poly(3-hydroxybutyric acid) formation under various conditions. The 3HB-CoA consumption was terminated by a reaction-dependent inactivation of the PHA synthase. Salts (MgCl₂, CaCl₂, NaCl), proteins (bovine serum albumin, lysozyme, phasine) or detergent (Tween 20) increased the 3HB-CoA turnover to 2.5-fold. Specific PHA synthase activity was only partially affected by the added components. In general, a higher concentration of salt often inhibited the activity of PhaEC_Cv without affecting the yield according to 3HB-CoA turnover. NAD⁺ and NADP⁺ (2 mM) inhibited PhaEC_Cv completely, where-as NADH and NADPH did not. Macroscopic poly(3HB) granules were formed in vitro if PhaEC_Cv was incubated in the presence of sufficient amounts of 3HB-CoA and if MgCl₂ was present. The form and size of the granules synthesized in vitro were affected by the concentration of the PHA synthase protein as well as by bovine serum albumin and the GA24 protein, a poly(3HB)-granule-associated protein of Alcaligenes eutrophus. Scanning electron micrographs from the synthesized granules were obtained. The granules consisted of poly(3HB) that had a molar mass in the range (1–2) × 10⁶ g/mol. Received: 12 September 1997 / Received revision: 24 October 1997 / Accepted: 31 October 1997     

Candida bombicola : production of novel alkyl glycosides based on glucose/2-dodecanol

Candida bombicola produces glycolipids containing sophorose and a glycosidically/esterically bound ω- or (ω−1)-hydroxy C₁₆₍₁₈₎ acid. Here we describe novel glycolipids from this source. Glucose and 2-dodecanol were used for the cultivation of the yeast, one part of the racemic secondary alcohol being connected directly with a glucose or a sophorose unit. A relatively high content of yeast extract, up to 4 g l⁻¹, and subsequently higher biomass concentrations favoured the production of novel products. The provision of 150 g l⁻¹ glucose and 15 g l⁻¹ 2-dodecanol resulted in maximum production of 22 g l⁻¹ novel alkyl glycosides (more than 90% novel products). The molecular structures were analysed by gas chromatography, fast atom bombardment/mass spectrometry, ¹H- and ¹³C-nuclear magnetic resonance and optical rotation studies. Sophorose and glucose were detected as carbohydrate moieties, (S)-(+)-2-dodecanol (88%) was found to be the major lipid moiety. The new glycolipids are suitable biosurfactants, reducing the surface tension of water from 72 mN m⁻¹ to 32–38 mN m⁻¹. Received: 8 December 1997 / Received revision: 19 March 1998 / Accepted: 20 March 1998     

Hydrogen production with high yield and high evolution rate by self-flocculated cells of Enterobacter aerogenes in a packed-bed reactor

Continuous hydrogen gas evolution by self-flocculated cells of Enterobacter aerogenes, a natural isolate HU-101 and its mutant AY-2, was performed in a packed-bed reactor under glucose-limiting conditions in a minimal medium. The flocs that formed during the continuous culture were retained even when the dilution rate was increased to 0.9 h⁻¹. The H₂ production rate increased linearly with increases in the dilution rate up to 0.67 h⁻¹, giving maximum H₂ production rates of 31 and 58 mmol l⁻¹ h⁻¹ in HU-101 and AY-2 respectively, at a dilution rate of more than 0.67 h⁻¹. The molar H₂ yield from glucose in AY-2 was maintained at about 1.1 at dilution rates between 0.08 h⁻¹ and 0.67 h⁻¹, but it decreased rapidly at dilution rates more than 0.8 h⁻¹. Received: 27 August 1997 / Received revision: 11 November 1997 / Accepted: 14 December 1997     

Fed-batch production of unsaturated medium-chain-length polyhydroxyalkanoates with controlled composition by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2440

Unsaturated medium-chain-length polyhydroxyalkanoates (MCL-PHA) were produced at a productivity of 0.63–1.09 g PHA l⁻¹ h⁻¹ with final PHA content ranging from 42.6 to 55.8% in single-stage, carbon-limited, fed-batch fermentations of Pseudomonas putida KT2440. A mixture of nonanoic acid (NA) and 10-undecenoic acid (UDA⁼) was fed exponentially to control growth rate. Varying the specific growth rate (0.14 h⁻¹ vs. 0.23 h⁻¹) at similar substrate feed ratios (NA:UDA⁼ = 5:1) had little effect on the final PHA content and relative composition. However, decreasing the NA:UDA⁼ ratio decreased the final amount of PHA produced from 56% with NA:UDA⁼ = 5.07:1 to only 42% at NA:UDA⁼ = 2.47:1. The molar fraction of all 3-hydroxyalkanoate monomers in the PHA product was relatively constant throughout each fermentation, indicating that the final product was homogeneous rather than a mixture of different copolymers. A linear relationship between unsaturation of the PHA produced and unsaturation of the carbon feed was found, which demonstrates the feasibility of producing unsaturated MCL-PHAs with controlled polymeric composition in a fed-batch process.     

Poly(hydroxyalkanoate) biosynthesis from triglyceride substrates

The biosynthesis of poly(hydroxyalkanoates) (PHA) by Pseudomonas resinovorans from triglyceride substrates was investigated. Each triglyceride, whether animal fat or vegetable oil, supported cellular growth to relatively high average cell yields (3.3 ± 0.2 g/l). PHA yields ranged from 1.1 g/l to 2.1 g/l, representing approximately 45% of the bacterial cell dry weight. The repeat-unit composition of the polymers was determined by gas chromatography (GC) and GC/mass spectrometry of the β-hydroxyalkanoate methyl esters from the hydrolyzed polymers. With the exception of PHA from soybean oil (PHA-soy), each polyester was composed of β-hydroxyacyl moieties with chain lengths ranging from C₄ to C₁₄, with C₈ and C₁₀ being the predominant species. PHA-soy contained an additional fraction (2%) of C₁₆ monomers. The alkyl side-chains of the PHA contained varying degrees of unsaturation. PHA from coconut oil was composed entirely of saturated side-chains, whereas PHA-soy contained 4.2 mol% olefinic groups in its side-chains. The increase in the degree of side-chain unsaturation caused decreased melting temperatures, enthalpies of fusion, and glass transition temperatures. The molar masses of the polymers were relatively constant and ranged from 6.5 × 10⁴ to 10.1 × 10⁴ g/mol. Received: 2 September 1997 / Received revision: 21 November 1997 / Accepted: 2 January 1998     

Organic halogen removal from chlorinated humic ground water and lake water by nitrifying fluidized-bed biomass characterised by electron microscopy and molecular methods

The dechlorinating and genotoxicity-removing activities of nitrifying fluidized-bed reactor biomass towards chlorinated organic compounds in water were shown at level below 1 ppm. The removal rates of adsorbable organic halogens were 200 μg Cl (g VS day)⁻¹ for chlorinated humic ground water and 50 μyg Cl (g VS day)⁻¹ for chlorinated lake water when studied in batch mode. In a sequenced batch mode the removal rates μg Cl (g VS day)⁻¹] were 2000 from chlorohumus, 1400–1800 from chlorophenols in chlorinated ground water, and 430–720 from chlorohumus in chlorinated lake water. Genotoxicity was removed to a large extent (60%–80%) from the chlorinated waters upon incubation with nitrifying reactor biomass. 2,6-Di-, 2,4,6-tri and 2,3,4,6-tetrachlorophenols competed with chlorinated water organohalogens for dechlorination. The dechlorination of chlorophenols and chlorohumus required no ammonia and was not prevented by inhibitors of ammonia oxidation, nitrapyrin, parathion, sodium diethyldithiocarbamate, or allylthiourea. Electron microscopical inspection of the biomass showed the dominance of clusters of bacteria resembling known nitrifying species, Nitrosomonas, Nitrobacter, and Nitrosospira. This was supported by polymerase chain reaction amplification of the biomass DNA with four different primers, revealing the presence of 16S rDNA sequences assignable to the same species. The most intensive band obtained with the Nitroso4E primer was shown to be closely related to Nitrosomonas europaea by restriction analysis. Received: 27 March 1998 / Received revision: 30 July 1998 / Accepted: 31 July 1998     

Kinetic models for astaxanthin production by high cell density mixotrophic culture of the microalga Haematococcus pluvialis

High cell density cultivation of Haematococcus pluvialis for astaxanthin production was carried out in batch and fed-batch modes in 3.7-L bioreactors with stepwise increased light intensity control mode. A high cell density of 2.65 g L⁻¹ (batch culture) or 2.74 g L⁻¹ (fed-batch culture) was obtained, and total astaxanthin production in the fed-batch culture (64.36 mg L⁻¹) was about 20.5% higher than in the batch culture (53.43 mg L⁻¹). An unstructured kinetic model to describe the microalga culture system including cell growth, astaxanthin formation, as well as sodium acetate consumption was proposed. Good agreement was found between the model predictions and experimental data. The models demonstrated that the optimal light intensity for mixotrophic growth of H. pluvialis in batch or fed-batch cultures in a 3.7-L bioreactor was 90–360 μmol m⁻² s⁻¹, and that the stepwise increased light intensity mode could be replaced by a constant light intensity mode. Received 24 December 1998/ Accepted in revised form 23 April 1999     

A new route to l-threo-3-[4-(methylthio)phenylserine], a key intermediate for the synthesis of antibiotics: recombinant low-specificity d-threonine aldolase-catalyzed stereospecific resolution

A new enzymatic resolution process was established for the production of l-threo-3-[4-(methylthio)phenylserine] (MTPS), an intermediate for synthesis of antibiotics, florfenicol and thiamphenicol, using the recombinant low-specificity d-threonine aldolase from Arthrobacter sp. DK-38. Chemically synthesized dl-threo-MTPS was efficiently resolved with either the purified enzyme or the intact recombinant Escherichiacoli cells overproducing the enzyme. Under the optimized experimental conditions, 100 mM (22.8 g l⁻¹) l-threo-MTPS was obtained from 200 mM (45.5 g l⁻¹) dl-threo-MTPS, with a molar yield of 50% and a 99.6% enantiomeric excess. Received: 2 September 1998 / Received revision: 27 October 1998 / Accepted: 29 November 1998     

Particle size effects in bioleaching of pyrite by acidophilic thermophile Sulfolobus metallicus (BC)

The effect of mineral particle size on the bioleaching of pyrite by the acidophilic thermophile Sulfolobus metallicus was investigated in a batch bioreactor. Decreasing the particle size from a mean diameter of 202 micron (size fraction: 150–180 micron) to a mean diameter of 42.5 micron (size fraction: 25–45 micron) enhanced the bioleaching rate from 0.05 kg m⁻³ h⁻¹ to 0.098 kg m⁻³ h⁻¹. The particle size distribution of the mineral in this range did not influence the morphology and growth kinetics of the cells. The values of specific growth rate (μ) and yield factor (Y) were 0.018–0.025 h⁻¹ and 0.67 × 10¹¹–1.45 × 10¹¹ cells (g iron)⁻¹, respectively. Decreasing the particle size of the mineral to a mean diameter of 6.40 micron (size fraction <25 micron) adversely influenced the activity of the cells. The presence of fine particles apparently damaged the structure of the cells, resulting in their inability to oxidise pyrite. Received: 11 December 1998 / Accepted: 9 April 1999     

Rhamnose lipids – biosynthesis, microbial production and application potential

Biosurfactants containing rhamnose and β-hydroxydecanoic acid and called rhamnolipids are reviewed with respect to microbial producers, their physiological role, biosynthesis and genetics, and especially their microbial overproduction, physicochemical properties and potential applications. With Pseudomonas species, more than 100 g l⁻¹ rhamnolipids were produced from 160 g l⁻¹ soybean oil at a volumetric productivity of 0.4 g l⁻¹h⁻¹. The individual rhamnolipids are able to lower the surface tension of water from 72 mN m⁻¹ to 25–30 mN m⁻¹ at concentrations of 10–200 mg l⁻¹. After initial testing, rhamnolipids seem to have potential applications in combating marine oil pollution, removing oil from sand and in combating zoosporic phytopathogens. Rhamnolipids are also a source of l-rhamnose, which is already used for the industrial production of high-quality flavor components. Received: 1 July 1998 / Received revision: 11 September 1998 / Accepted: 13 September 1998     

Batch and continuous cultivation of Anaerobiospirillum succiniciproducens for the production of succinic acid from whey

Batch and continuous cultivation of Anaerobiospirillum succiniciproducens were systematically studied for the production of succinic acid from whey. Addition of 2.5 g l⁻¹ yeast extract and 2.5 g l⁻¹ polypeptone per 10 g l⁻¹ whey was most effective for succinic acid production from both treated and nontreated whey. When 20 g l⁻¹ nontreated whey and 7 g l⁻¹ glucose were used as cosubstrates, the yield and productivity of succinic acid reached at the end of fermentation were 95% and 0.46 g (l h)⁻¹, respectively. These values were higher than those obtained using nontreated whey alone [93% and 0.24 g (l h)⁻¹ for 20 g l⁻¹ whey]. Continuous fermentation of A. succiniciproducens at an optimal dilution rate resulted in the production of succinic acid with high productivity [1.35 g (l h)⁻¹], high conversion yield (93%), and higher ratio of succinic acid to acetic acid (5.1:1) from nontreated whey. Received: 23 July 1999 / Received revision: 17 November 1999 / Accepted: 24 December 1999     

Physiological aspects of continuous lactic acid fermentations at high dilution rates

The effects of the substrate conditions on the volumetric productivity of Lactobacillus helveticus at different cell densities up to 60 g l⁻¹ in a continuous stirred-tank reactor with microfiltration to retain the biomass were investigated. At low dilution rates, D, the steady-state volumetric productivity, r _p, gradually increased to a maximum at D = 1.2–1.5 h⁻¹, because of reduced product inhibition. At higher D values, r _p unexpectedly decreased, although the substrate conditions further improved. The maxima of r _p at different cell densities coincided with a critical specific substrate utilization rate beyond which the cell metabolism seems to be controlled through a catabolic modulator factor, and r _p decreases. Received: 8 September 1997 / Received last revision: 31 December 1997 / Accepted: 2 January 1998     

Production of xylitol by Candida peltata

Candida peltata NRRL Y-6888 to ferment xylose to xylitol was evaluated under different fermentation conditions such as pH, temperature, aeration, substrate concentration and in the presence of glucose, arabinose, ethanol, methanol and organic acids. Maximum xylitol yield of 0.56 g g⁻¹ xylose was obtained when the yeast was cultivated at pH 6.0, 28°C and 200 rpm on 50 g L⁻¹ xylose. The yeast produced ethanol (0.41 g g⁻¹ in 40 h) from glucose (50 g L⁻¹) and arabitol (0.55 g g⁻¹ in 87 h) from arabinose (50 g L⁻¹). It preferentially utilized glucose > xylose > arabinose from mixed substrates. Glucose (10 g L⁻¹), ethanol (7.5 g L⁻¹) and acetate (5 g L⁻¹) inhibited xylitol production by 61, 84 and 68%, respectively. Arabinose (10 g L⁻¹) had no inhibitory effect on xylitol production. Received 24 December 1998/ Accepted in revised form 18 March 1999     

Chemo-enzymatic synthesis of polyhydroxyalkanoate (PHA) incorporating 2-hydroxybutyrate by wild-type class I PHA synthase from <Emphasis Type="Italic">Ralstonia eutropha</Emphasis>

A previously established improved two-phase reaction system has been applied to analyze the substrate specificities and polymerization activities of polyhydroxyalkanoate (PHA) synthases. We first analyzed the substrate specificity of propionate coenzyme A (CoA) transferase and found that 2-hydroxybutyrate (2HB) was converted into its CoA derivative. Then, the synthesis of PHA incorporating 2HB was achieved by a wild-type class I PHA synthase from Ralstonia eutropha. The PHA synthase stereoselectively polymerized (R)-2HB, and the maximal molar ratio of 2HB in the polymer was 9 mol%. The yields and the molecular weights of the products were decreased with the increase of the (R)-2HB concentration in the reaction mixture. The weight-average molecular weight of the polymer incorporating 9 mol% 2HB was 1.00 × 10⁵, and a unimodal peak with polydispersity of 3.1 was observed in the GPC chart. Thermal properties of the polymer incorporating 9 mol% 2HB were analyzed by DSC and TG-DTA. T _g, T _m, and T _d (10%) were observed at −1.1°C, 158.8°C, and 252.7°C, respectively. In general, major components of PHAs are 3-hydroxyalkanoates, and only engineered class II PHA synthases have been reported as enzymes having the ability to polymerize HA with the hydroxyl group at C2 position. Thus, this is the first report to demonstrate that wild-type class I PHA synthase was able to polymerize 2HB.     

Production of 1,3-propanediol by Clostridium butyricum in continuous culture with cell recycling

The continuous fermentation of 1,3-propanediol from glycerol by Clostridium butyricum was subjected to cell recycling by filtration using hollow-fibre modules made from polysulphone. The performance of the culture system was checked at a retention ratio (dilution rate/bleed rate) of 5, dilution rates between 0.2 h⁻¹ and 1.0 h⁻¹ and glycerol input concentrations of 32 g l⁻¹ and 56 g l⁻¹. The near-to-optimum propanediol concentration of 26.5 g l⁻¹ (for 56 g l⁻¹ glycerol) was maintained up to a dilution rate of 0.5 h⁻¹ and then decreased while the propanediol productivity was highest at 0.7 h⁻¹. The productivity could be increased by a factor of four in comparison to the continuous culture without cell recycling. By application of the model of Zeng and Deckwer [(1995) Biotechnol Prog 11: 71–79] for cultures under substrate excess, it was shown that the limitations resulted exclusively from product inhibition and detrimental influences from the cell recycling system, such as shear stress, were not involved. Received: 20 October 1997 / Received revision: 12 December 1997 / Accepted: 14 December 1997     

Transformation of Saccharopolyspora erythraea by electroporation of germinating spores: construction of propionyl Co-A carboxylase mutants

The introduction of plasmid DNA into germinating spores of an industrially improved strain of Saccharopolyspora erythraea was accomplished by electroporation. Various parameters affecting the efficiency of electroporation were examined. The most critical factor was the extent of spore germination. Electrocompetence was limited to a 4-h period following the initial emergence of the germ tube. Electroporation efficiencies as high as 2 × 10⁵ CFU μg⁻¹ of plasmid DNA were obtained using electrocompetent germlings. The optimal field strength was 12–14 kV cm⁻¹ with a pulse duration of 15–20 ms. Electrocompetent germlings were stored at −80°C without a significant decrease in transformation efficiency. The utility of this protocol was demonstrated by isolating a propionyl-CoA carboxylase mutant through targeted gene disruption and replacement. Received 3 April 1998/ Accepted in revised form 28 September 1998     

Biotransformation of citronellol by the basidiomycete Cystoderma carcharias in an aerated-membrane bioreactor

The basidiomycete Cystoderma carcharias transformed citronellol into 3,7-dimethyl-1,6,7-octanetriol as the main product. 3,7-Dimethyl-6,7-epoxy-1-octanol was identified as important intermediary product of the biotransformation, and the allylic diols 2,6-dimethyl-2-octene-1,8-diol, 3,7-dimethyl-5-octene-1,7-diol and 3,7-dimethyl-7-octene-1,6-diol were found to be minor products. Microbial formation of rose oxide, a flavour-impact component, was observed for the first time. The formation of the main products was inhibited by 70% after addition of 0.1 mmol l⁻¹ cytochrome monooxygenase inhibitors. Formation of 3,7-dimethyl-1,6,7-octanetriol was effective in a bioreactor with aeration over a coil of a hydrophobic microporous polypropene capillary membrane. Production rates of up to 150 mg l⁻¹ day⁻¹ were reached and led to a product concentration of 866 mg l⁻¹ (conversion rate: 52%). The total loss of the added volatile substrate via the exhaust air was 4.5% when this aeration method was used. Received: 30 July 1998 / Received revision: 2 November 1998 / Accepted: 7 November 1998     

Continuous production of l(+)-lactic acid by Lactobacillus casei in two-stage systems

A two-stage two-stream chemostat system and a two-stage two-stream immobilized upflow packed-bed reactor system were used for the study of lactic acid production by Lactobacillus casei subsp casei. A mixing ratio of D ₁₂/D ₂= 0.5 (D = dilution rate) resulted in optimum production, making it possible to generate continuously a broth with high lactic acid concentration (48 g l⁻¹) and with a lowered overall content of initial yeast extract (5  g l⁻¹), half the concentration supplied in the one-step process. In the two-stage chemostat system, with the first stage at pH 5.5 and 37 °C and a second stage at pH 6.0, a temperature change from 40 °C to 45 °C in the second stage resulted in a 100% substrate consumption at an overall dilution rate of 0.05 h⁻¹. To increase the cell mass in the system, an adhesive strain of L. casei was used to inoculate two packed-bed reactors, which operated with two mixed feedstock streams at the optimal conditions found above. Lactic acid fermentation started after a lag period of cell growth over foam glass particles. No significant amount of free cells, compared with those adhering to the glass foam, was observed during continuous lactic acid production. The extreme values, 57.5 g l⁻¹ for lactic acid concentration and 9.72 g l⁻¹ h⁻¹ for the volumetric productivity, in upflow packed-bed reactors were higher than those obtained for free cells (48 g l⁻¹  and 2.42 g l⁻¹ h⁻¹) respectively and the highest overall l(+)-lactic acid purity (96.8%) was obtained in the two-chemostat system as compared with the immobilized-cell reactors (93%). Received: 4 December 1997 / Received revision: 23 February 1998 / Accepted: 14 March 1998