Enhancing the synthesis of latex clearing protein by different cultivation strategies.

In this study, we improved the synthesis of the latex clearing protein from Gordonia polyisoprenivorans VH2 (Lcp1VH2), a key enzyme for the initial cleavage of the rubber backbone. Cultivations using a recombinant strain of Escherichia coli were optimized to overcome poor solubility of Lcp1VH2 and improve the production yields. Different cultivation temperatures and agitation rates were evaluated in the process to demonstrate their impact on the solubility of Lcp1VH2. A specific maximum production rate of 28.3 mg Lcp1VH2 g-1 cell dry weight h-1 was obtained at 25 °C and at agitation rates between 200-300 rpm. The activity of Lcp1VH2 was strongly influenced by variations in the cultivation temperature with a specific maximum activity of 0.81 U mg-1 in cultures incubated at 30 °C. Besides cultivation-based optimization, also the strategy of fusion protein expression with NusA was successfully applied. The in vivo solubility of the Lcp1VH2 fusion protein was calculated to be 73.1%, which means an enhancement of 5.7-fold in comparison to the solubility of the native Lcp1VH2. The fusion protein of Lcp1VH2 and NusA still exhibited oxygenase activity with polyisoprene latex as a substrate. In fact, NusA-His-Lcp1VH2 reached a 4-fold higher volumetric activity in comparison to Lcp1VH2. Oligo(cis-1,4-isoprene) molecules were produced as degradation products due to the cleavage of the polymer backbone by NusA-His-Lcp1VH2. The formation of oligo-isoprenoid molecules with molecular weights between 236 and 984 Da were confirmed by electrospray ionization-mass spectrometry analysis.

In vitro studies on the degradation of poly(cis-1,4-isoprene).

Cleavage of the backbone of poly(cis-1,4-isoprene) (IR) in solid rubber material was accomplished by the addition of partially purified latex clearing protein (Lcp1VH2 ) using a 200-mL enzyme reactor. Two strategies for the addition of Lcp1VH2 were studied revealing that the daily addition of 50 µg mL-1 of Lcp1VH2 for 5 days was clearly a more efficient regime in comparison to a one-time addition of 250 µg of Lcp1VH2 at the beginning. Soluble oligo(cis-1,4-isoprene) molecules occurred as degradation products and were identified by ESI-MS and GPC. Oxygenase activity of Lcp1VH2 with solid IR particles as substrate was shown for the first time by measuring the oxygen consumption in the reaction medium. A strong decrease of the dissolved oxygen concentration was detected at the end of the assay, which indicates an increase in the number of cleavage reactions. The oligo(cis-1,4-isoprene) molecules comprised 1 to 11 isoprene units and exhibited an average molecular weight (Mn ) of 885 g mol-1 . Isolation of the oligo(cis-1,4-isoprene) molecules was achieved by using silica gel column chromatography. The relative quantification of the isolated products was performed by HPLC-MS after derivatization with 2,4-dinitrophenilhydrazyne yielding a concentration of total degradation products of 1.62 g L-1 . Analysis of the polymer surface in samples incubated for 3 days with Lcp1VH2 via ATR-FTIR indicated the presence of carbonyl groups, which occurred upon the cleavage reaction. This study presents a cell-free bioprocess as an alternative rubber treatment that can be applied for the partial degradation of the polymer. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:890-899, 2018.

Cleavage of poly(cis-1,4-isoprene) rubber as solid substrate by cultures of Gordonia polyisoprenivorans.

Potential biotechnological recycling processes for rubber products include the bacterial degradation of poly(cis-1,4-isoprene) (IR) in order to achieve its total biodegradation or its biotransformation into useful products. The actinomycete Gordonia polyisoprenivorans strain VH2 catalyzes the degradation of IR and enables its use as a sole carbon source via ß-oxidation. The initial cleavage reaction is catalyzed by the extracellular latex clearing protein (Lcp). This dioxygenase is the key enzyme for the formation of oligo(cis-1,4-isoprene) molecules with different lengths, i.e., numbers of isoprene units. For the first time, IR was used as a solid substrate in 2-l fermenters. Two different particle size fractions (63-500 and 500-1000 µm) and three stirring rates (300, 400 and 500 rpm) were evaluated in the process. An increase of the cell concentration was achieved by using smaller particles and by using lower stirring rates, reaching a final biomass concentration of 0.52 g l-1 at 300 rpm after 12 days of cultivation. In order to enhance the formation of oligo(cis-1,4-isoprene) molecules, a transposon insertion mutant (TH5) of G. polyisoprenivorans strain VH2 that has lost the ability to transport the partial degradation products into the cells was used, thereby allowing the accumulation of the degradation products in the culture supernatants. Propionate, glucose and glycerol were evaluated as additional carbon sources besides IR, and the highest yields were observed on propionate. In 2-l bioreactors with pH control, different feeding regimes were performed during cultivation by the addition of propionate every 24 or 48 h for 16 days. After liquid-liquid extraction and a derivatization with Girard's T reagent, the oligo(cis-1,4-isoprene) molecules were detected by ESI-MS. The mass distribution of the degradation products was affected by the selection of the extraction solvent, but no influence of longer cultivation periods was detected.

A simple, rapid and cost-effective process for production of latex clearing protein to produce oligopolyisoprene molecules.

Aiming at finding feasible alternatives for rubber waste disposal, the partial enzymatic degradation of poly(cis-1,4-isoprene)-containing materials represents a potential solution. The use of rubber-degrading enzymes and the biotransformation of rubber into new materials is limited by the high costs associated with the production and purification of the enzyme and the complexity of the process. This study presents a simple and low-cost procedure to obtain purified latex clearing protein (Lcp), an enzyme capable of cleaving the double bonds of poly(cis-1,4-isoprene) in presence of oxygen to produce different size of oligomers with terminal aldehyde and ketone groups, respectively. The gene coding for Lcp1VH2 from Gordonia polyisoprenivorans strain VH2 was overexpressed in Escherichia coli C41 (DE3), and by using an auto-induction medium high protein yields were obtained. The cultivation process was described and compared with an IPTG-inducible medium previously used. Purification of the enzyme was performed using salting out precipitation with ammonium sulfate. Different salt concentrations and pH were tested in order to find the optimal for purification, obtaining a concentration of 60mg Lcp per l. The enzymatic activity of the purified enzyme was measured by an oxygen consumption assay in the presence of polyisoprene latex. Volumetric activities of 0.16Uml-1 were obtained at optimal conditions of temperature and pH. The results showed an active and partial purified fraction of Lcp1VH2, able to cleave the backbone of poly(cis-1,4-isoprene) and to produce degradation products that were identified with staining methodologies (Schiff reagent for aldehyde groups and 2,4-DNPH for carbonyl groups) and characterized using nuclear magnetic resonance (NMR). Thirteen different storage conditions were tested for the purified enzyme analyzing the enzymatic activity after 1 and 3 months. Lcp1VH2, as an ammonium sulfate precipitate, was stable, easy to handle and sufficiently active for further analysis. The described methodology offers the possibility to upscale the process and to produce large amounts of this protein.

The NADH:flavin oxidoreductase Nox from Rhodococcus erythropolis MI2 is the key enzyme of 4,4'-dithiodibutyric acid degradation.

The reduction of the disulphide bond is the initial catabolic step of the microbial degradation of the organic disulphide 4,4'-dithiodibutyric acid (DTDB). Previously, an NADH:flavin oxidoreductase from Rhodococcus erythropolis MI2 designated as NoxMI2 , which belongs to the old yellow enzyme (OYE) family, was identified. In the present study, it was proven that NoxMI2 has the ability to cleave the sulphur-sulphur bond in DTDB. In silico analysis revealed high sequence similarities to proteins of the flavin mononucleotide (FMN) reductase family identified in many strains of R. erythropolis. Therefore, nox was heterologously expressed in the pET23a(+) expression system using Escherichia coli strain BL21(DE3) pLysS, which effectively produces soluble active NoxMI2 . NoxMI2 showed a maximum specific activity (Vmax ) of 3·36 µmol min-1  mg-1 corresponding to a kcat of 2·5 s-1 and an apparent substrate Km of 0·6 mmol l-1 , when different DTDB concentrations were applied. No metal cofactors were required. Moreover, NoxMI2 had very low activity with other sulphur-containing compounds like 3,3'-dithiodipropionic acid (8·0%), 3,3'-thiodipropionic acid (7·6%) and 5,5'-dithiobis(2-nitrobenzoic acid) (8·0%). The UV/VIS spectrum of NoxMI2 revealed the presence of the cofactor FMN. Based on results obtained, NoxMI2 adds a new physiological substrate and mode of action to OYE members. SIGNIFICANCE AND IMPACT OF THE STUDY: It was unequivocally demonstrated in this study that an NADH:flavin oxidoreductase from Rhodococcus erythropolis MI2 (NoxMI2 ) is able to cleave the xenobiotic disulphide 4,4'-dithiodibutyric acid (DTDB) into two molecules of 4-mercaptobutyric acid (4MB) with concomitant consumption of NADH. NoxMI2 showed a high substrate specificity as well as high heat stability. This study provides the first detailed characterization of the initial cleavage of DTDB, which is considered as a promising polythioester precursor.

Isolation and characterization of new poly(3HB)-accumulating star-shaped cell-aggregates-forming thermophilic bacteria.

AIMS: This study aimed at isolating thermophilic bacteria that utilize cheap carbon substrates for the economically feasible production of poly(3-hydroxybutyrate), poly(3HB), at elevated temperatures. METHODS AND RESULTS: Thermophilic bacteria were enriched from an aerobic organic waste treatment plant in Germany, and from hot springs in Egypt. Using the viable colony staining method for hydrophobic cellular inclusions with Nile red in mineral salts medium (MSM) containing different carbon sources, six Gram-negative bacteria were isolated. Under the cultivation conditions used in this study, strains MW9, MW11, MW12, MW13 and MW14 formed stable star-shaped cell-aggregates (SSCAs) during growth; only strain MW10 consisted of free-living rod-shaped cells. The phylogenetic relationships of the strains as derived from 16S rRNA gene sequence comparisons revealed them as members of the Alphaproteobacteria. The 16S rRNA gene sequences of the isolates were very similar (>99% similarity) and exhibited similarities ranging from 93 to 99% with the most closely related species that were Chelatococcus daeguensis, Chelatococcus sambhunathii,Chelatococcus asaccharovorans, Bosea minatitlanensis, Bosea thiooxidans and Methylobacterium lusitanum. Strains MW9, MW10, MW13 and MW14 grew optimally in MSM with glucose, whereas strains MW11 and MW12 preferred glycerol as sole carbon source for growth and poly(3HB) accumulation. The highest cell density and highest poly(3HB) content attained were 4·8g l(-l) (cell dry weight) and 73% (w/w), respectively. Cells of all strains grew at temperatures between 37 and 55°C with the optimum growth at 50°C. CONCLUSIONS: New PHA-accumulating thermophilic bacterial strains were isolated and characterized to produce poly(3HB) from glucose or glycerol in MSM at 50°C. SSCAs formation was reported during growth. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report on the formation of SSCAs by PHA-accumulating bacteria and also by thermophilic bacteria. PHA-producing thermophiles can significantly reduce the costs of fermentative PHA production.

[Cloning and molecular organization of the polyhydroxyalkanoic acid synthase gene (phaC) of Ralstonia eutropha strain B5786].

Class I polyhydroxyalkanoic acid (PHA) synthase gene (phaC) of Ralstonia eutropha strain B5786 was cloned and characterized. R. eutropha B5786 features the ability to synthesize multicomponent PHAs with short- and medium-chain-length monomers from simple carbohydrate substrate. A correlation was made between the molecular structure of PHA synthase and substrate specificity and the ability of strain-producers to accumulate PHAs of this or that structure. A strong similarity of PHA synthase of R. eutropha strain B5786 with PHA synthase of R. eutropha strain H16, which, as opposed to strain B5786, enables to incorporate medium chain length PHAs if hexanoate is used as carbon source, exhibited 99%. A correlation between the structure of PHA synthase of B5786 strain with synthases of microorganisms which synthesize short and medium chain length PHAs similarly to B5786 strain, showed an identity level from 26 to 41% (homology with synthase of Rhodospirillum rubrum makes 41%, Ectothiorhodospira shaposhnikovii makes 26%, Aeromonas punctata makes 40%, Thiococcus pfennigii makes 28%, Rhodococcus ruber makes 38%, and with PhaCl and PhaC2 synthases of Pseudomonas sp. 61-3 makes 34 and 37%, respectively). This allows for speaking about the absence of a direct connection between the molecular organization of PHA synthases and their functional abilities, namely, the ability to synthesize PHAs of a particular composition.

Halopiger aswanensis sp. nov., a polymer-producing and extremely halophilic archaeon isolated from hypersaline soil.

Strain 56(T) was isolated from a hypersaline soil in Aswan (Egypt). Cells were pleomorphic rods. The organism was neutrophilic, motile and required at least 1.7 M (10 % w/v) NaCl, but not MgCl(2), for growth; optimal growth occurred at > or =3.8 M (> or =22.5 %) NaCl. The strain was thermotolerant with an optimum temperature for growth of 40 degrees C, although growth was possible up to 55 degrees C. The G+C content of the DNA of the novel strain was 67.1 mol%. 16S rRNA gene sequence analysis revealed that strain 56(T) was a member of the phyletic group defined by the family Halobacteriaceae, showing the highest similarity to Halopiger xanaduensis SH-6(T) (99 %) and the next highest similarity of 94 % to other members of the family Halobacteriaceae. DNA-DNA hybridization revealed 27 % relatedness between strain 56(T) and Hpg. xanaduensis SH-6(T). Polar lipid analysis revealed the presence of the bis-sulfated glycolipid S(2)-DGD-1 as the sole glycolipid and the absence of the glycerol diether analogue phosphatidylglycerosulfate. Both C(20 x 20) and C(20 x 25) core lipids were present. Strain 56(T) accumulated large amounts of polyhydroxybutyrate and also secreted an exopolymer. Physiological and biochemical differences suggested that Hpg. xanaduanesis and strain 56(T) were sufficiently different to be separated into two distinct species. It is suggested that strain 56(T) represents a novel species of the genus Halopiger , for which the name Halopiger aswanensis sp. nov. is proposed. The type strain is strain 56(T) (=DSM 13151(T)=JCM 11628(T)).

Zobellella denitrificans strain MW1, a newly isolated bacterium suitable for poly(3-hydroxybutyrate) production from glycerol.

AIMS: To search for new bacteria for efficient production of polyhydroxyalkanoates (PHAs) from glycerol. METHODS AND RESULTS: Samples were taken from different environments in Germany and Egypt, and bacteria capable of growing in mineral salts medium with glycerol as sole carbon source were enriched. From a wastewater sediment sample in Egypt, a Gram-negative bacterium (strain MW1) was isolated that exhibited good growth and that accumulated considerable amounts of polyhydroxybutyrate (PHB) from glycerol and also from other carbon sources. The 16S rRNA gene sequence of this isolate exhibited 98.5% and 96.2% similarity to Zobellella denitrificans strain ZD1 and to Zobellella taiwanensis strain ZT1 respectively. The isolate was therefore affiliated as strain MW1 of Z. denitrificans. Strain MW1 grows optimally on glycerol at 41 degrees C and pH 7.3 and accumulated PHB up to 80.4% (w/w) of cell dry weight. PHB accumulation was growth-associated. Although it was not an absolute requirement, 20 g l(-1) sodium chloride enhanced both growth (5 g cell dry weight per litre) and PHB content (87%, w/w). Zobellella denitrificans strain MW1 is also capable to accumulate the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer if sodium propionate was used as cosubstrate in addition to glycerol. CONCLUSIONS: A new PHB-accumulating strain was isolated and identified. This strain is able to utilize glycerol for growth and PHB accumulation to high content especially in the presence of NaCl that will enable the utilization of waste glycerol from biodiesel industry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report on accumulation of PHA in a member of the new genus Zobellella. Furthermore, utilization of glycerol as the sole carbon source for fast growth and PHB biosynthesis, growth in the presence of NaCl and high PHB contents of the cells will make this newly isolated bacterium a potent candidate for industrial production of PHB from crude glycerol occurring as byproduct during biodiesel production.

Unravelling the C3/C4 carbon metabolism in Ralstonia eutropha H16.

AIMS: Detailed knowledge about the enzymes responsible for conversion of C(3) and C(4) compounds will be helpful to establish the bacterial strain Ralstonia eutropha as platform for the production of biotechnologically interesting compounds. Although various studies about these enzymes were accomplished in the past, some contradicting information about the enzyme pattern in this bacterium still exists. To resolve these discrepancies, the C(3) /C(4) metabolism was reinvestigated after the genome sequence of this bacterium became available. METHODS AND RESULTS: In silico analysis of genome sequence revealed putative genes coding for NAD(P)(+) -dependent malic enzymes (Mae), phoshoenolpyruvate carboxykinase (Pck), phosphoenolpyruvate carboxylase (Ppc), phosphoenolpyruvate synthase (Pps) and pyruvate carboxylase (Pyc). Reverse transcription PCR revealed constitutive expression of mae and pck genes, whereas no transcripts of pyc and ppc were found. Expression of active NADP(+) -dependent MaeB and Pck and absence of Pyc and Ppc was confirmed by spectrophotometric enzyme assays. CONCLUSIONS: The data reported in this study suggest that two enzymes, (i) MaeB and (ii) Pck, mediate between the C(3) and C(4) intermediates in R. eutropha H16. The enzymatic conversion of pyruvate into phosphoenolpyruvate (PEP) is catalysed by Pps, and an NADH(+) -dependent Mdh mediates the reversible conversion of malate and oxaloacetate. SIGNIFICANCE AND IMPACT OF THE STUDY: An increased knowledge of the enzymes mediating between C(3) and C(4) intermediates in R. eutropha will facilitate metabolic engineering.

Cyanophycin-degrading bacteria in digestive tracts of mammals, birds and fish and consequences for possible applications of cyanophycin and its dipeptides in nutrition and therapy.

AIMS: To determine the susceptibility of cyanophycin granule polypeptide (CGP) to degradation by several mammalian, avian and fish gut flora. METHODS AND RESULTS: Samples of gut flora were investigated for the occurrence of bacteria capable of CGP degradation. With all samples, a complete anaerobic degradation of CGP was achieved over incubation periods of only 12-48 h at 37 degrees C. CGP-degrading bacteria were detected in all samples, and they occurred in particular high titres in caecum flora from rabbit and sheep and in the digestive tract of carp fish. A total of 62 axenic cultures were isolated. All degraded CGP aerobically, 46 of them degraded CGP also anaerobically over incubation periods ranging from 24 h to 7 days. HPLC analysis revealed that all isolates degraded CGP to its constituting dipeptides. Eight strains were identified by 16S rRNA gene sequencing and were affiliated to the genera Bacillus, Brevibacillus, Pseudomonas, Streptomyces and Micromonospora. CONCLUSIONS: These data demonstrate for the first time the occurrence of a natural niche for CGP in the digestive tracts of animals. SIGNIFICANCE AND IMPACT OF THE STUDY: The biodegradability of CGP by gut flora provides a first confirmation for the potential applications of CGP and its dipeptides in nutrition and therapy as highly bio-available sources for arginine, lysine, aspartate and possibly also other amino acids.

[Biosynthesis of multicomponent polyhydroxyalkanoates by Wautersia eutropha].

The effect of carbon supply on polyhydroxyalkanoate (PHA) synthesis by bacteria Wautersia eutropha was studied. Synthesis of multicomponent PHA composed of short- and long-chain monomers (C4-C8) by two natural strains (H16 and B5786) under mixotrophic conditions (CO2 + alkane acids as cosubstrates) was demonstrated for the first time. The PHA composition was shown to be dependent on the cosubstrate type. In the presence of odd fatty acids, four- and five-component polymers were synthesized; hydroxybutyrate, hydroxyvalerate, and hydroxyheptanoate were the major monomers, while hydroxyhexanoate and hydroxyoctanoate were minor and irregular. In the presence of even fatty acids, PHA contained not only the corresponding molecules (hydroxyhexanoate and hydroxyoctanoate), but also hydroxyvalerate; synthesis of four-component PHA which contain mainly hydroxybutyrate and hydroxyhexanoate (up to 18 mol %) is therefore possible. A series of four- and five-component PHA was synthesized and their physicochemical characteristics were determined.

Functional analyses of genes involved in the metabolism of ferulic acid in Pseudomonas putida KT2440.

Pseudomonas putida KT2440 is a physiologically extremely versatile non-pathogenic bacterium that is applied as a "biosafety strain" in biotechnological processes, as authorized by the USA National Institute of Health. Analysis of the P. putida KT2440 whole-genome sequence revealed the genetic organization of the genes fcs, ech, and vdh, which are essential for ferulic acid conversion to vanillic acid via vanillin. To confirm the physiological function of these structural genes as feruloyl-CoA synthetase (Fcs), enoyl-CoA hydratase/aldolase (Ech), and vanillin dehydrogenase (Vdh), respectively, they were cloned and expressed in Escherichia coli. Recombinant strains harboring fcs and ech were able to transform ferulic acid to vanillin. The enzyme activities of Fcs and Vdh were determined in protein extracts of these cells. The essential involvement of fcs, ech and vdh in the catabolism of ferulic acid in P. putida KT2440 was proven by separately inactivating each gene by insertion of Omega-elements. The corresponding mutant strains KT2440 fcsOmegaKm, KT2440 echOmegaKm, and KT2440 vdhOmegaKm were not able to grow on ferulic acid. The potential application of P. putida KT2440 and the mutant strains in biotechnological vanillin production process is discussed.

Sphingopyxis chilensis sp. nov., a chlorophenol-degrading bacterium that accumulates polyhydroxyalkanoate, and transfer of Sphingomonas alaskensis to Sphingopyxis alaskensis comb. nov.

The taxonomic position of a chlorophenol-degrading bacterium, strain S37T, was investigated. The 16S rDNA sequence indicated that this strain belongs to the genus Sphingopyxis, exhibiting high sequence similarity to the 16S rDNA sequences of Sphingomonas alaskensis LMG 18877T (98.8%), Sphingopyxis macrogoltabida LMG 17324T (98.2%), Sphingopyxis terrae IFO 15098T (95%) and Sphingomonas adhaesiva GIFU 11458T (92%). These strains (except Sphingopyxis terrae IFO 15098T, which was not investigated) and the novel isolate accumulated polyhydroxyalkanoates consisting of 3-hydroxybutyric acid and 3-hydroxyvaleric acid from glucose as carbon source. The G + C content of the DNA of strain S37T was 65.5 mol%. The major cellular fatty acids of this strain were octadecenoic acid (18 : 1omega7c), heptadecenoic acid (17 : 1omega6c) and hexadecanoic acid (16 : 0). The results of DNA-DNA hybridization experiments and its physiological characteristics clearly distinguished the novel isolate from all known Sphingopyxis species and indicated that the strain represents a novel Sphingopyxis species. Therefore, the species Sphingopyxis chilensis sp. nov. is proposed, with strain S37T (=LMG 20986T =DSM 14889T) as the type strain. The transfer of Sphingomonas alaskensis to the genus Sphingopyxis as Sphingopyxis alaskensis comb. nov. is also proposed.

Triacylglycerols in prokaryotic microorganisms.

Triacylglycerols (TAG) are fatty acid triesters of glycerol; there are diverse types of TAG with different properties depending on their fatty acid composition. The occurrence of TAG as reserve compounds is widespread among eukaryotic organisms such as yeast, fungi, plants and animals, whereas occurrence of TAG in bacteria has only rarely been described. However, accumulation of TAG seems to be widespread among bacteria belonging to the actinomycetes group, such as species of Mycobacterium, Streptomyces, Rhodococcus and Nocardia. Fatty acids in acylglycerols in cells of Rhodococcus opacus PD630 accounted for up to 87% of the cellular dry weight. TAG biosynthesis, justifying an oleaginous status, seems to be restricted mainly to this group of bacteria, but occurs to a minor extent also in a few other bacteria. The compositions and structures of bacterial TAG vary considerably depending on the microorganism and on the carbon source, and unusual acyl moieties, such as phenyldecanoic acid and 4,8,12 trimethyl tridecanoic acid, are also included. The principal function of bacterial TAG seems to be as a reserve compound. Other functions that have been discussed include regulation of cellular membrane fluidity by keeping unusual fatty acids away from membrane phospholipids, or acting as a sink for reducing equivalents. In recent years, basic aspects of the physiology and biochemistry of bacterial TAG accumulation, and the molecular biology of the lipid inclusion bodies have been reported. TAG are used for nutritional, therapeutic and pharmaceutical purposes and serve as a source of oleochemicals.

In vivo evolution of the Aeromonas punctata polyhydroxyalkanoate (PHA) synthase: isolation and characterization of modified PHA synthases with enhanced activity.

In vivo random mutagenesis of the polyhydroxyalkanoate (PHA) synthase gene from Aeromonas punctata was performed employing the mutator strain Escherichia coli XL1-Red. About 200,000 mutants were screened on Nile red-containing medium and five mutants with enhanced fluorescence were selected. Four of these mutants exhibited enhanced in vivo and in vitro PHA synthase activity. Mutant M1, which carried the single mutation F518I, showed a five-fold increase in specific PHA synthase activity, whereas the corresponding mediated PHA accumulation increased by 20%, as compared with the wild-type PHA synthase. Mutant M2, which carried the single mutation V214G, showed a two-fold increase in specific PHA synthase activity and PHA accumulation only increased by 7%. Overall, the in vitro activities of the overproducing mutants ranged from 1.1- to 5-fold more than the wild-type activity, whereas the amounts of accumulated PHA ranged over 107-126% of that of the wild type. Moreover, all mutants mediated synthesis of PHAs with an increased weight average molar mass, but the molar fractions of 3-hydroxybutyrate and 3-hydroxyhexanoate remained almost constant. In vivo random mutagenesis proved to be a versatile tool to isolate mutants exerting improved properties with respect to PHA biosynthesis.

Transformation of the Pseudonocardiaceae amycolatopsis sp. strain HR167 is highly dependent on the physiological state of the cells.

The Pseudonocardiaceae Amycolatopsis sp. strain HR167 is used in a biotransformation process to produce vanillin from ferulic acid. To make this strain accessible for genetic engineering, a direct mycelium transformation system developed for Amycolatopsis mediterranei [Madon and Hotter (1991) J Bacteriol 173: 6325-6331] was applied and optimized for Amycolatopsis sp. strain HR167. The physiological state of the cells had a major influence on the transformation rate. The highest transformation rate of about 7x10(5) transformants per microgram of DNA was obtained with mycelium harvested 6.5-7.5 h after the culture has reached the stationary growth phase. When cells were harvested outside of this time slot, the transformation rate drastically decreased. The density of the mycelium suspensions used in the transformation mixture and the methylation state of the plasmid DNA used for the transformation were also crucial parameters. With plasmid DNA isolated from Escherichia coli ET12567, transformation rates were 3,500-fold higher than those obtained with DNA isolated from E. coli XL1-Blue.

Cloning, characterization and comparison of the Pseudomonas mendocina polyhydroxyalkanoate synthases Phac1 and PhaC2.

This study describes a comparison of the polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 of Pseudomonas mendocina. The P mendocina pha gene locus, encoding two PHA synthase genes [phaC1Pm and phaC2pm flanking a PHA depolymerase gene (phaZ)], was cloned, and the nucleotide sequences of phaC1Pm (1,677 bp), phaZ (1,034 bp), and phaC2pm (1,680 bp) were determined. The amino acid sequences deduced from phaC1Pm and phaC2pm showed highest similarities to the corresponding PHA synthases from other pseudomonads sensu stricto. The two PHA synthase genes conferred PHA synthesis to the PHA-negative mutants P. putida GPp104 and Ralstonia eutropha PHB-4. In P. putida GPp 104, phaC1Pm and phaC2Pm mediated PHA synthesis of medium-chain-length hydroxyalkanoates (C6-C12) as often reported for other pseudomonads. In contrast, in R. eutropha PHB-4, either PHA synthase gene also led to the incorporation of 3-hydroxybutyrate (3HB) into PHA. Recombinant strains of R. eutropha PHB-4 harboring either P. mendocina phaC gene even accumulated a homopolyester of 3HB during cultivation with gluconate, with poly(3HB) amounting to more than 80% of the cell dry matter if phaC2 was expressed. Interestingly, recombinant cells harboring the phaC1 synthase gene accumulated higher amounts of PHA when cultivated with fatty acids as sole carbon source, whereas recombinant cells harboring PhaC2 synthase accumulated higher amounts when gluconate was used as carbon source in storage experiments in either host. Furthermore, isogenic phaC1 and phaC2 knock-out mutants of P. mendocina provided evidence that PhaC1 is the major enzyme for PHA synthesis in P. mendocina, whereas PhaC2 contributes to the accumulation of PHA in this bacterium to only a minor extent, and then only when cultivated on gluconate.