Engineering the genotype of Acinetobacter sp. strain ADP1 to enhance biosynthesis of cyanophycin

To study the importance of arginine provision and phosphate limitation for synthesis and accumulation of cyanophycin (CGP) in Acinetobacter sp. strain ADP1, genes encoding the putative arginine regulatory protein (argR) and the arginine succinyltransferase (astA) were inactivated, and the effects of these mutations on CGP synthesis were analyzed. The inactivation of these genes resulted in a 3.5- or 7-fold increase in CGP content, respectively, when the cells were grown on glutamate. Knockout mutations in both genes led to a better understanding of the effect of the addition of other substrates to arginine on CGP synthesis during growth of the cells of Acinetobacter sp. strain ADP1. Overexpression of ArgF (ornithine carbamoyltransferase), CarA-CarB (small and large subunits of carbamoylphosphate synthetase), and PepC (phosphoenolpyruvate carboxylase) triggered synthesis of CGP if amino acids were used as a carbon source whereas it was not triggered by gluconate or other sugars. Cells of Acinetobacter sp. strain ADP1, which is largely lacking genes for carbohydrate metabolism, showed a significant increase in CGP contents when grown on mineral medium supplemented with glutamate, aspartate, or arginine. The Acinetobacter sp. DeltaastA(pYargF) strain is unable to utilize arginine but synthesizes more arginine, resulting in CGP contents as high as 30% and 25% of cell dry matter when grown on protamylasse or Luria-Bertani medium, respectively. This recombinant strain overcame the bottleneck of the costly arginine provision where it produces about 75% of the CGP obtained from the parent cells grown on mineral medium containing pure arginine as the sole source of carbon. Phosphate starvation is the only known trigger for CGP synthesis in this bacterium, which possesses the PhoB/PhoR phosphate regulon system. Overexpression of phoB caused an 8.6-fold increase in CGP content in comparison to the parent strain at a nonlimiting phosphate concentration.     

Polyphosphate Kinase of Acinetobacter sp. Strain ADP1: Purification and Characterization of the Enzyme and Its Role during Changes in Extracellular Phosphate Levels

Polyphosphate (polyP) is a ubiquitous biopolymer whose function and metabolism are incompletely understood. The polyphosphate kinase (PPK) of Acinetobacter sp. strain ADP1, an organism that accumulates large amounts of polyP, was purified to homogeneity and characterized. This enzyme, which adds the terminal phosphate from ATP to a growing chain of polyP, is a 79-kDa monomer. PPK is sensitive to magnesium concentrations, and optimum activity occurs in the presence of 3 mM MgCl₂. The optimum pH was between pH 7 and 8, and significant reductions in activity occurred at lower pH values. The greatest activity occurred at 40°C. The half-saturation ATP concentration for PPK was 1 mM, and the maximum PPK activity was 28 nmol of polyP monomers per μg of protein per min. PPK was the primary, although not the sole, enzyme responsible for the production of polyP in Acinetobacter sp. strain ADP1. Under low-phosphate (P_i) conditions, despite strong induction of the ppk gene, there was a decline in net polyP synthesis activity and there were near-zero levels of polyP in Acinetobacter sp. strain ADP1. Once excess phosphate was added to the P_i-starved culture, both the polyP synthesis activity and the levels of polyP rose sharply. Increases in polyP-degrading activity, which appeared to be mainly due to a polyphosphatase and not to PPK working in reverse, were detected in cultures grown under low-P_i conditions. This activity declined when phosphate was added.     

Physiological Conditions Conducive to High Cyanophycin Content in Biomass of Acinetobacter calcoaceticus Strain ADP1

The effects of the inorganic medium components, the initial pH, the incubation temperature, the oxygen supply, the carbon-to-nitrogen ratio, and chloramphenicol on the synthesis of cyanophycin (CGP) by Acinetobacter calcoaceticus strain ADP1 were studied in a mineral salts medium containing sodium glutamate and ammonium sulfate as carbon and nitrogen sources, respectively. Variation of all these factors resulted in maximum CGP contents of only about 3.5% (wt/wt) of the cell dry matter (CDM), and phosphate depletion triggered CGP accumulation most substantially. However, addition of arginine to the medium as the sole carbon source for growth promoted CGP accumulation most strikingly. This effect was systematically studied, and an optimized phosphate-limited medium containing 75 mM arginine and 10 mM ammonium sulfate yielded a CGP content of 41.4% (wt/wt) of the CDM at 30°C. The CGP content of the cells was further increased to 46.0% (wt/wt) of the CDM by adding 2.5 μg of chloramphenicol per ml of medium in the accumulation phase. These contents are by far the highest CGP contents of bacterial cells ever reported. CGP was easily isolated from the cells by using an acid extraction method, and this CGP contained about equimolar amounts of aspartic acid and arginine and no detectable lysine; the molecular masses ranged from 21 to 29 kDa, and the average molecular mass was about 25 kDa. Transmission electron micrographs of thin sections of cells revealed large CGP granules that frequently had an irregular shape with protuberances at the surface and often severely deformed the cells. A cphI::ΩKm mutant of strain ADP1 with a disrupted putative cyanophycinase gene accumulated significantly less CGP than the wild type accumulated, although the cells expressed cyanophycin synthetase at about the same high level. It is possible that the intact CphI protein is involved in the release of CGP primer molecules from initially synthesized CGP. The resulting lower concentration of primer molecules could explain the observed low rate of accumulation at similar specific activities.     

Increased Lysine Content Is the Main Characteristic of the Soluble Form of the Polyamide Cyanophycin Synthesized by Recombinant Escherichia coli

Cyanophycin, a polyamide of cyanobacterial or noncyanobacterial origin consisting of aspartate, arginine, and lysine, was synthesized in different recombinant strains of Escherichia coli expressing cphA from Synechocystis sp. strain PCC 6308 or PCC 6803, Anabaena sp. strain PCC 7120, or Acinetobacter calcoaceticus ADP1. The molar aspartate/arginine/lysine ratio of the water-soluble form isolated from a recombinant strain expressing CphA₆₃₀₈ was 1:0.5:0.5, with a lysine content higher than any ever described before. The water-insoluble form consisted instead of mainly aspartate and arginine residues and had a lower proportion of lysine, amounting to a maximum of only 5 mol%. It could be confirmed that the synthesis of soluble cyanobacterial granule polypeptide (CGP) is independent of the origin of cphA. Soluble CGP isolated from all recombinant strains contained a least 17 mol% lysine. The total CGP portion of cell dry matter synthesized by CphA₆₃₀₈ from recombinant E. coli was about 30% (wt/wt), including 23% (wt/wt) soluble CGP, by using terrific broth complex medium for cultivation at 30°C for 72 h. Enhanced production of soluble CGP instead of its insoluble form is interesting for further application and makes recombinant E. coli more attractive as a suitable source for the production of polyaspartic acid or dipeptides. In addition, a new low-cost, time-saving, effective, and common isolation procedure for mainly soluble CGP, suitable for large-scale application, was established in this study.     

Physiological conditions conducive to high cyanophycin content in biomass of Acinetobacter calcoaceticus strain ADP1

The effects of the inorganic medium components, the initial pH, the incubation temperature, the oxygen supply, the carbon-to-nitrogen ratio, and chloramphenicol on the synthesis of cyanophycin (CGP) by Acinetobacter calcoaceticus strain ADP1 were studied in a mineral salts medium containing sodium glutamate and ammonium sulfate as carbon and nitrogen sources, respectively. Variation of all these factors resulted in maximum CGP contents of only about 3.5% (wt/wt) of the cell dry matter (CDM), and phosphate depletion triggered CGP accumulation most substantially. However, addition of arginine to the medium as the sole carbon source for growth promoted CGP accumulation most strikingly. This effect was systematically studied, and an optimized phosphate-limited medium containing 75 mM arginine and 10 mM ammonium sulfate yielded a CGP content of 41.4% (wt/wt) of the CDM at 30 degrees C. The CGP content of the cells was further increased to 46.0% (wt/wt) of the CDM by adding 2.5 microg of chloramphenicol per ml of medium in the accumulation phase. These contents are by far the highest CGP contents of bacterial cells ever reported. CGP was easily isolated from the cells by using an acid extraction method, and this CGP contained about equimolar amounts of aspartic acid and arginine and no detectable lysine; the molecular masses ranged from 21 to 29 kDa, and the average molecular mass was about 25 kDa. Transmission electron micrographs of thin sections of cells revealed large CGP granules that frequently had an irregular shape with protuberances at the surface and often severely deformed the cells. A cphI::OmegaKm mutant of strain ADP1 with a disrupted putative cyanophycinase gene accumulated significantly less CGP than the wild type accumulated, although the cells expressed cyanophycin synthetase at about the same high level. It is possible that the intact CphI protein is involved in the release of CGP primer molecules from initially synthesized CGP. The resulting lower concentration of primer molecules could explain the observed low rate of accumulation at similar specific activities.     

Exopolysaccharide Production and Peculiarities of C6-Metabolism in Acinetobacter sp. Grown on Carbohydrate Substrates

An Acinetobacter sp. strain grown on carbohydrate substrates (mono- and disaccharides, molasses, starch) was shown to synthesize exopolysaccharides (EPS). Glucose catabolism proved to proceed via the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways. Pyruvate entered the tricarboxylic acid cycle due to pyruvate dehydrogenase activity. Pyruvate carboxylation by pyruvate carboxylase was the anaplerotic reaction providing for the synthesis of intermediates for the constructive metabolism of Acinetobacter sp. grown on C₆-substrates. The C₆-metabolism in Acinetobacter sp. was limited by coenzyme A. Irrespective of the carbohydrate growth substrate (glucose, ethanol), the activities of the key enzymes of both C₂- and C₆-metabolism was high, except for the isocitrate lyase activity in glucose-grown bacteria. Isocitrate lyase activity was induced by C₂-compounds (ethanol or acetate). After their addition to glucose-containing medium, both substrates were utilized simultaneously, and an increase was observed in the EPS synthesis, as well as in the EPS yield relative to biomass. The mechanisms responsible for enhancing the EPS synthesis in Acinetobacter sp. grown on a mixture of C₂- and C₆-substrates are discussed.     

Cloning and Genetic Characterization of dca Genes Required for β-Oxidation of Straight-Chain Dicarboxylic Acids in Acinetobacter sp. Strain ADP1

A previous study of deletions in the protocatechuate (pca) region of the Acinetobacter sp. strain ADP1 chromosome revealed that genes required for utilization of the six-carbon dicarboxylic acid, adipic acid, are linked to the pca structural genes. To investigate the genes involved in adipate catabolism, a 33.8-kb SacI fragment, which corrects a deletion spanning this region, was cloned. In addition to containing known pca, qui, and pob genes (for protocatechuate, quinate, and 4-hydroxybenzoate dissimilation), clone pZR8000 contained 10 kb of DNA which was the subject of this investigation. A mutant strain of Escherichia coli DH5α, strain EDP1, was isolated that was able to utilize protocatechuate and 4-hydroxybenzoate as growth substrates when EDP1 cells contained pZR8000. Sequence analysis of the new region of DNA on pZR8000 revealed open reading frames predicted to be involved in β-oxidation. Knockouts of three genes implicated in β-oxidation steps were introduced into the chromosome of Acinetobacter sp. strain ADP1. Each of the mutants was unable to grow with adipate. Because the mutants were affected in their ability to utilize additional saturated, straight-chain dicarboxylic acids, the newly discovered 10 kb of DNA was termed the dca (dicarboxylic acid) region. Mutant strains included one with a deletion in dcaA (encoding an acyl coenzyme A [acyl-CoA] dehydrogenase homolog), one with a deletion in dcaE (encoding an enoyl-CoA hydratase homolog), and one with a deletion in dcaH (encoding a hydroxyacyl-CoA dehydrogenase homolog). Data on the dca region should help us probe the functional significance and interrelationships of clustered genetic elements in this section of the Acinetobacter chromosome.     

Protamylasse, a Residual Compound of Industrial Starch Production, Provides a Suitable Medium for Large-Scale Cyanophycin Production

Protamylasse is a residual compound occurring during the industrial production of starch from potatoes. It contains a variety of nutrients and all necessary minerals and could be used as a carbon, nitrogen, and energy source for the growth of bacteria and also for cyanophycin (CGP) biosynthesis. Media containing protamylasse as the sole compound diluted only in water were therefore examined for their suitability in CGP production. Among various bacterial strains investigated in this study, a recombinant strain of Escherichia coli DH1 harboring plasmid pMa/c5-914::cphA₆₈₀₃, which carries the cyanophycin synthetase structural gene (cphA) from Synechocystis sp. strain PCC6803, was found to be most suitable. Various cultivation conditions for high CGP contents were first optimized in shake flask cultures. The optimized conditions were then successfully applied to 30- and 500-liter fermentation scales in stirred tank reactors. A maximum CGP content of 28% (wt/wt) CGP per cell dry matter was obtained in 6% (vol/vol) protamylasse medium at an initial pH of 7.0 within a cultivation period of only 24 h. The CGP contents obtained with this recombinant strain employing protamylasse medium were higher than those obtained with the same strain cultivated in mineral salts medium or in expensive commercial complex media such as Luria-Bertani or Terrific broth. It was shown that most amino acids present in the protamylasse medium were almost completely utilized by the cells during cultivation. Exceptions were alanine, tryptophan, tyrosine, and most interestingly, arginine. Furthermore, CGP was easily isolated from protamylasse-grown cells by applying the acid extraction method. The CGP exhibited a molecular mass of about 26 to 30 kDa and was composed of 50% (mol/mol) aspartate, 46% (mol/mol) arginine, and 4% (mol/mol) lysine. The use of cheap residual protamylasse could contribute in establishing an economically and also ecologically feasible process for the biotechnological production of CGP.     

Acinetobacter sp. ADP1: an ideal model organism for genetic analysis and genome engineering

Acinetobacter sp. strain ADP1 is a naturally transformable gram-negative bacterium with simple culture requirements, a prototrophic metabolism and a compact genome of 3.7 Mb which has recently been sequenced. Wild-type ADP1 can be genetically manipulated by the direct addition of linear DNA constructs to log-phase cultures. This makes it an ideal organism for the automation of complex strain construction. Here, we demonstrate the flexibility and versatility of ADP1 as a genetic model through the construction of a broad variety of mutants. These include marked and unmarked insertions and deletions, complementary replacements, chromosomal expression tags and complex combinations thereof. In the process of these constructions, we demonstrate that ADP1 can effectively express a wide variety of foreign genes including antibiotic resistance cassettes, essential metabolic genes, negatively selectable catabolic genes and even intact operons from highly divergent bacteria. All of the described mutations were achieved by the same process of splicing PCR, direct transformation of growing cultures and plating on selective media. The simplicity of these tools make genetic analysis and engineering with Acinetobacter ADP1 accessible to laboratories with minimal microbial genetics expertise and very little equipment. They are also compatible with complete automation of genetic analysis and engineering protocols.     

Naturally Transformable Acinetobacter sp. Strain ADP1 Belongs to the Newly Described Species Acinetobacter baylyi

Genotypic and phenotypic analyses were carried out to clarify the taxonomic position of the naturally transformable Acinetobacter sp. strain ADP1. Transfer tDNA-PCR fingerprinting, 16S rRNA gene sequence analysis, and selective restriction fragment amplification (amplified fragment length polymorphism analysis) indicate that strain ADP1 and a second transformable strain, designated 93A2, are members of the newly described species Acinetobacter baylyi. Transformation assays demonstrate that the A. baylyi type strain B2^T and two other originally identified members of the species (C5 and A7) also have the ability to undergo natural transformation at high frequencies, confirming that these five strains belong to a separate species of the genus Acinetobacter, characterized by the high transformability of its strains that have been cultured thus far.     

Thio Wax Ester Biosynthesis Utilizing the Unspecific Bifunctional Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase of Acinetobacter sp. Strain ADP1

The bifunctional wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) from Acinetobacter sp. strain ADP1 (formerly Acinetobacter calcoaceticus ADP1) mediating the biosyntheses of wax esters and triacylglycerols was used for the in vivo and in vitro biosynthesis of thio wax esters and dithio wax esters. For in vitro biosynthesis, 5′His₆WS/DGAT comprising an N-terminal His₆ tag was purified from the soluble protein fraction of Escherichia coli Rosetta(DE3)pLysS (pET23a::5′His₆atf). By employing SP-Sepharose high-pressure and Ni-nitrilotriacetic acid fast-protein liquid chromatographies, a 19-fold enrichment with a final specific activity of 165.2 nmol mg of protein⁻¹ min⁻¹ was achieved by using 1-hexadecanol and palmitoyl-CoA as substrates. Incubation of purified 5′His₆WS/DGAT with 1-hexadecanethiol and palmitoyl-CoA as substrates resulted in the formation of palmitic acid hexadecyl thio ester (10.4% relative specific activity of a 1-hexadecanol control). Utilization of 1,8-octanedithiol and palmitoyl-CoA as substrates led to the formation of 1-S-monopalmitoyloctanedithiol and minor amounts of 1,8-S-dipalmitoyloctanedithiol (59.3% relative specific activity of a 1-hexadecanol control). The latter dithio wax ester was efficiently produced when 1-S-monopalmitoyloctanedithiol and palmitoyl-CoA were used as substrates (13.4% specific activity relative to that of a 1-hexadecanol control). For the in vivo biosynthesis of thio wax esters, the knockout mutant Acinetobacter sp. strain ADP1acr1ΩKm, which is unable to produce fatty alcohols, was used. Cultivation of Acinetobacter sp. strain ADP1acr1ΩKm in the presence of gluconate, 1-hexadecanethiol, and oleic acid in nitrogen-limited mineral salts medium resulted in the accumulation of unusual thio wax esters that accounted for around 1.19% (wt/wt) of the cellular dry weight and consisted mainly of oleic acid hexadecyl thioester as revealed by gas chromatography-mass spectrometry.     

Accumulation of Cyanophycin Granules as a Result of Phosphate Limitation in Agmenellum quadruplicatum

Phosphate-limited growth of the blue-green alga Agmenellum quadruplicatum resulted in the accumulation of cyanophycin granule polypeptide (CGP), which is a 1:1 co-polymer of aspartic acid and arginine. The progressive accumulation of CGP began after depletion of phosphate from the medium. CGP increased in concentration much faster than the increase in cell number. Electron microscopy indicated that both the number of cyanophycin granules per cell section and the diameter of each granule increased as phosphate starvation progressed. A marked decrease in the electron density of the inter-thylakoidal areas took place concurrently with the accumulation of CGP. At the same time a progessive decrease in the pigment concentration of cells and in the rate of nitrate uptake was observed. Thirty-two hours after phosphate depletion from the medium up to 28% of total cellular nitrogen was found in CGP.     

Accumulation of Amino Acids in Rhizobium sp. Strain WR1001 in Response to Sodium Chloride Salinity

Rhizobium sp. strain WR1001, isolated from the Sonoran Desert by Eskew and Ting, was found to be able to grow in defined medium containing NaCl up to 500 mM, a concentration approaching that of sea water. Therefore, it is a valuable strain for studying the biochemical basis of salt tolerance. Intracellular free glutamate was found to increase rapidly in response to osmotic stress by NaCl. It accounted for 88% of the amino acid pool when the bacterium was grown in 500 mM NaCl. The role of glutamate dehydrogenase in glutamate biosynthesis was examined in several Rhizobium strains. Both NADH- and NADPH-dependent glutamate dehydrogenase activities in various Rhizobium strains were observed. The range of activity differed considerably depending on the particular strain. KCl (500 mM) did not stimulate glutamate dehydrogenase activity, as reported in a number of bacterial strains by Measures. The low activity of glutamate dehydrogenase in Rhizobium sp. strain WR1001 apparently cannot fulfill a biosynthetic function of glutamate formation in response to medium NaCl concentrations.     

Synergism of VAM and Rhizobium on Production and Metabolism of IAA in Roots and Root Nodules of Vigna Mungo

A novel Acinetobacter strain, Ud-4, possessing a strong capacity to degrade edible, lubricating, and heavy oil was isolated from seawater in a fishing port located in Toyama, Japan. It was identified by morphological and physiological analyses and 16S rDNA sequencing. This strain could utilize five types of edible oils (canola oil, olive oil, sesame oil, soybean oil, and lard), lubricating oil, and C-heavy oil as the sole carbon source for growth in M9 medium. The strain grew well and heavily degraded edible oils in Luria–Bertani medium during a 7-day culture at 25°C; it also degraded all kinds of oils in artificial seawater medium for marine bacteria. Furthermore, this strain was capable of degrading almost all C10–C25 n-alkanes in C-heavy oil during a 4-week culture. Oligonucleotide primers specific to two catabolic genes involved in the degradation of n-alkanes (Acinetobacter sp. alkM) and triglyceride (Acinetobacter sp. lipA) allowed amplification of these genes in strain Ud-4. To our knowledge, this is the first report on the isolation of a bacterium that can efficiently degrade both edible and mineral oils.     

Identification of Novel Genes Involved in Long-Chain n-Alkane Degradation by Acinetobacter sp. Strain DSM 17874

Acinetobacter sp. strain DSM 17874 is capable of utilizing n-alkanes with chain lengths ranging from that of decane (C₁₀H₂₂) to that of tetracontane (C₄₀H₈₂) as a sole carbon source. Two genes encoding AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, have been shown to be involved in the degradation of n-alkanes with chain lengths of from 10 to 20 C atoms in this strain. Here, we describe a novel high-throughput screening method and the screening of a transposon mutant library to identify genes involved in the degradation of n-alkanes with C chain lengths longer than 20, which are solid at 30°C, the optimal growth temperature for Acinetobacter sp. strain DSM 17874. A library consisting of approximately 6,800 Acinetobacter sp. strain DSM 17874 transposon mutants was constructed and screened for mutants unable to grow on dotriacontane (C₃₂H₆₆) while simultaneously showing wild-type growth characteristics on shorter-chain n-alkanes. For 23 such mutants isolated, the genes inactivated by transposon insertion were identified. Targeted inactivation and complementation studies of one of these genes, designated almA and encoding a putative flavin-binding monooxygenase, confirmed its involvement in the strain's metabolism of long-chain n-alkanes. To our knowledge, almA represents the first cloned gene shown to be involved in the bacterial degradation of long-chain n-alkanes of 32 C's and longer. Genes encoding AlmA homologues were also identified in other long-chain n-alkane-degrading Acinetobacter strains.     

Production of poly-\-hydroxybutyrate in Acinetobacter spp. isolated from activated sludge

Two strains of Acinetobacter sp. isolated from activated sludge actively removing phosphate were examined for their abilities to produce poly-\-hydroxybutyrate (PHB). When yield-limited by phosphate, strain RA3117 contained material that stained with Sudan Black, but contained only 0.9% PHB on a dry weight basis. This strain contained no sudanophilic material or PHB when limited by ammonia or sulphate. When strain RA3757 was limited by phosphate, ammonia or sulphate it produced 2.0, 7.8 and 11.5% PHB, respectively, on a dry weight basis. \-Ketothiolase and acetoacetyl-coenzyme A (CoA) reductase were only observed in RA3757 cell-free extracts. \-Ketothiolase was produced both in cells with and without PHB whereas acetoacetyl-CoA reductase was found only in cells accumulating PHB. When RA3757 was grown in ammonia-limiting medium with acetate, butyrate, caproate or ethanol as carbon source, similar levels of PHB were produced. When cells were grown on valerate, RA3757 produced 5.6 poly-\-hydroxyvalerate and 0.9% PHB on a dry weight basis. Correspondence to: J. W. May     

Sequencing and analysis of plasmids pAV1 and pAV2 ofAcinetobacter venetianus VE-C3 involved in diesel fuel degradation

Acinetobacter venetianus strain VE-C3 was isolated in the Venice lagoon (Italy) as a strain able to degrade diesel fuel oil. This strain possesses genes of the alkane monoxygenase complex responsible forn-alkane degradation and carries two plasmids, pAV1 (10820 bp) and pAV2 (15135 bp), which were supposed from the analysis of Alk mutant strains to harbour genetic determinants for hydrocarbon degradation. In this work we determined the nucleotide sequence of both plasmids and showed the presence of a putative aldehyde dehydrogenase gene, essential for hydrocarbon degradation, on plasmid pAV2, and of an ORF similar toalkL gene present on pAV1 plasmid. These data, combined with genetic reports indicating that strains lacking one of the two plasmids or carrying transposon insertion on pAV1, are defective inn-alkane degradation, suggest a complex genomic organisation of genes involved in alkane degradation inA. venetianus VE-C3. In this bacterium these genes are carried by both the chromosome and the plasmids, while inAcinetobacter sp. strain ADP1 and M1 all the genes for alkane monoxygenase complex are located only on the chromosome.     

Optimization of cyanophycin production in recombinant strains of Pseudomonas putida and Ralstonia eutropha employing elementary mode analysis and statistical experimental design

Elementary mode analysis was applied to simulate conditions for cyanophycin (CGP) biosynthesis and to optimize its production in bacteria. The conclusions from these simulations were confirmed by experiments with recombinant strains of the wild types and polyhydroxyalkanoate (PHA)-negative mutants of Ralstonia eutropha and Pseudomonas putida expressing CGP synthetase genes (cphA) of Synechocystis sp. strain PCC6308 or Anabaena sp. strain PCC7120. In particular, the effects of suitable precursor substrates and of oxygen supply as well as of the capability to accumulate PHA in addition to CGP biosynthesis were investigated. Since CGP consists of the amino acids aspartate and arginine, the tricarboxylic acid cycle (TCC), which provides intermediates for biosynthesis of these amino acids, seems to be important. Excretion of intermediates of the TCC upon cultivation at restricted oxygen supply and conversion of fumarate mainly to malate and to only little succinate in the absence of oxygen indicated that TCC intermediates for arginine and aspartate biosynthesis were provided by the oxidative or reductive parts of the TCC, respectively. The following important conclusions were made from the experiments and the simulations: (i) external arginine additionally supplied to the medium, (ii) oxygen limitation, and (iii) absence of PHA accumulation exerted positive effects on CGP accumulation. These conclusions were utilized to obtain CGP contents in the cells of as high as 17.9% (w x w(-1)) during cultivation of the investigated bacteria at the 30-L scale using mineral salts medium. Such high CGP contents were previously not obtained with these bacteria at a 30-L scale, even if complex media were used.