Sequencing microbial copolymers of 3-hydroxybutyric and 3-mercaptoalkanoic acids by NMR, electrospray ionization mass spectrometry, and size exclusion chromatography NMR

Copolymers of 3-hydroxybutyrate (3HB) and 3-mercaptopropionate (3MP) or 3-mercaptobutyrate (3MB) units and minor amounts of 3-hydroxypropionate (3HP), 3-hydroxyvalerate (3HV), or 3-mercaptovalerate (3MV) were investigated regarding their microstructure by NMR, electrospray ionization mass spectrometry, and size exclusion chromatography NMR. These copolymers were produced by Ralstonia eutropha strain H16 when cells were cultivated in a mineral salts medium with gluconate as a carbon source for growth and 3MP or 3MB as precursor substrates for incorporation of 3-mercaptoalkanoates. Mass spectrometry analysis of partially methanolyzed or pyrolyzed samples proved the presence of true copolymers or terpolymers. (13)C NMR spectroscopy of intact polymer samples, with values of average block length and degree of randomness deviating from a random sequence model, suggested microblock structures; however, composition analysis by (1)H NMR of fractions obtained by size exclusion chromatography showed significant variations with molecular weight, revealing the presence of blends of poly(3HB-co-3MP-co-3HP) or poly(3HB-co-3MB) with poly(3HB). The experimental NMR carbonyl dyad signal intensities were satisfactorily matched by a random sequence model when the presence of poly(3HB) was taken into account.     

Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16

Cysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. In Ralstonia eutropha H16, two Cdo homologues (CdoA and CdoB) have been identified previously. In vivo studies showed that Escherichia coli cells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropionate (3SP), whereas no 3SP could be detected in cells expressing CdoB. The objective of this study was to confirm these findings and to study both enzymes in detail by performing an in vitro characterization. The proteins were heterologously expressed and purified to apparent homogeneity by immobilized metal chelate affinity chromatography (IMAC). Subsequent analysis of the enzyme activities revealed striking differences with regard to their substrate ranges and their specificities for the transition metal cofactor, e.g., CdoA catalyzed the sulfoxidation of 3MP to a 3-fold-greater extent than the sulfoxidation of cysteine, whereas CdoB converted only cysteine. Moreover, the dependency of the activities of the Cdos from R. eutropha H16 on the metal cofactor in the active center could be demonstrated. The importance of CdoA for the metabolism of the sulfur compounds 3,3′-thiodipropionic acid (TDP) and 3,3′-dithiodipropionic acid (DTDP) by further converting their degradation product, 3MP, was confirmed. Since 3MP can also function as a precursor for polythioester (PTE) synthesis in R. eutropha H16, deletion of cdoA might enable increased synthesis of PTEs.     

Metabolic engineering of Escherichia coli for production of enantiomerically pure (R)-(--)-hydroxycarboxylic acids

A heterologous metabolism of polyhydroxyalkanoate (PHA) biosynthesis and degradation was established in Escherichia coli by introducing the Ralstonia eutropha PHA biosynthesis operon along with the R. eutropha intracellular PHA depolymerase gene. By with this metabolically engineered E. coli, enantiomerically pure (R)-3-hydroxybutyric acid (R3HB) could be efficiently produced from glucose. By employing a two-plasmid system, developed as the PHA biosynthesis operon on a medium-copy-number plasmid and the PHA depolymerase gene on a high-copy-number plasmid, R3HB could be produced with a yield of 49.5% (85.6% of the maximum theoretical yield) from glucose. By integration of the PHA biosynthesis genes into the chromosome of E. coli and by introducing a plasmid containing the PHA depolymerase gene, R3HB could be produced without plasmid instability in the absence of antibiotics. This strategy can be used for the production of various enantiomerically pure (R)-hydroxycarboxylic acids from renewable resources.     

Novel pathway for catabolism of the organic sulfur compound 3,3'-dithiodipropionic acid via 3-mercaptopropionic acid and 3-Sulfinopropionic acid to propionyl-coenzyme A by the aerobic bacterium Tetrathiobacter mimigardefordensis strain DPN7

The hitherto unstudied microbial degradation of the organic disulfide 3,3'-dithiodipropionic acid (DTDP) was investigated with the recently described bacterium Tetrathiobacter mimigardefordensis strain DPN7(T) (DSM 17166(T); LMG 22922(T)), which is able to use DTDP as the sole carbon source for growth. 3-Mercaptopropionic acid (3MP) and 3-sulfinopropionic acid (3SP) were detected in the growth medium and occurred as intermediates during DTDP degradation. To identify genes coding for enzymes of DTDP catabolism, Tn5::mob-induced mutants of T. mimigardefordensis were generated. Screening of transposon mutant libraries yielded many mutants fully or partially impaired in utilizing DTDP as a carbon source. Mapping of the insertion loci in some mutants identified four disrupted open reading frames (ORFs) with putative metabolic functions. The ORFs were assigned function on the basis of homologies with lpdA (EC 1.8.1.4), cdo (EC 1.13.11.20), sucCD (EC 6.2.1.5), and acnB (EC 4.2.1.3). Tn5::mob insertions occurred additionally in the vicinity of heat shock protein-encoding genes. The predicted function of the LpdA homologue in T. mimigardefordensis is cleavage of the disulfide bond of DTDP to form two molecules of 3MP. Cdo catalyzes the conversion of the sulfhydryl group of 3MP, yielding the corresponding sulfinic acid, 3SP. SucCD exhibits thiokinase activity, ligating coenzyme A (CoA) with 3SP to form 3SP-CoA. Afterwards, an elimination of sulfite via a putative desulfinase is expected. acnB encodes a putative 2-methylisocitrate dehydratase. Therefore, a new pathway is proposed for the catabolism of DTDP via 3MP, 3SP, and 3SP-CoA toward propionyl-CoA, which is then further catabolized via the 2-methylcitric acid cycle in T. mimigardefordensis.     

Employing a recombinant strain of Advenella mimigardefordensis for biotechnical production of Homopolythioesters from 3,3'-dithiodipropionic acid

Advenella mimigardefordensis strain DPN7(T) was genetically modified to produce poly(3-mercaptopropionic acid) (PMP) homopolymer by exploiting the recently unraveled process of 3,3'-dithiodipropionic acid (DTDP) catabolism. Production was achieved by systematically engineering the metabolism of this strain as follows: (i) deletion of its inherent 3MP dioxygenase-encoding gene (mdo), (ii) introduction of the buk-ptb operon (genes encoding the butyrate kinase, Buk, and the phosphotransbutyrylase, Ptb, from Clostridium acetobutylicum), and (iii) overexpression of its own polyhydroxyalkanoate synthase (phaC(Am)). These measures yielded the potent PMP production strain A. mimigardefordensis strain SHX22. The deletion of mdo was required for adequate synthesis of PMP due to the resulting accumulation of 3MP during utilization of DTDP. Overexpression of the plasmid-borne buk-ptb operon caused a severe growth repression. This effect was overcome by inserting this operon into the genome. Polyhydroxyalkanoate (PHA) synthases from different origins were compared. The native PHA synthase of A. mimigardefordensis (phaC(Am)) was obviously the best choice to establish homopolythioester production in this strain. In addition, the cultivation conditions, including an appropriate provision of the carbon source, were further optimized to enhance PMP production. The engineered strain accumulated PMP up to approximately 25% (wt/wt) of the cell dry weight when cultivated in mineral salts medium containing glycerol as the carbon source in addition to DTDP as the sulfur-providing precursor. According to our knowledge, this is the first report of PMP homopolymer production by a metabolically engineered bacterium using DTDP, which is nontoxic, as the precursor substrate.     

Characterization of microbial polythioesters: physical properties of novel copolymers synthesized by Ralstonia eutropha

Various samples of polythioesters with different contents of 3-mercaptopropionic acid (3MP) or 3-mercaptobutyric acid (3MB) as one comonomer and with 3-hydroxybutyric acid (3HB) as the second constituent were produced by cultivating cells of Ralstonia eutropha strain H16 in mineral salts medium containing 3MP or 3MB plus gluconate as carbon sources. Fermentations were done also at the 30-L scale. The various samples were cast as films from chloroform and the following were recorded: melting point, solid-state NMR, X-ray diffraction. The copolyester poly(3HB-co-3MP) displayed mutiple melting peaks corresponding to separate phases rich in 3MP and 3HB. The copolyester poly(3HB-co-3MB) displayed very low crystallinity and melting points higher than that of poly(3HB) when the 3HB content was 40% or less.     

Application of the BPEC pathway for large-scale biotechnological production of poly(3-mercaptopropionate) by recombinant Escherichia coli, including a novel in situ isolation method

Metabolically engineered Escherichia coli JM109 harboring plasmid pBPP1 and expressing the nonnatural BPEC pathway for synthesis of thermoplastic polyhydroxyalkanoates (PHA) and novel polythioesters (PTE) to provide suitable substrates of PHA synthase was investigated with respect to biotechnological production of poly(3-mercaptopropionate) [poly(3MP)]. Fed-batch fermentation processes were established at the 30- and 500-liter scales in stirred tank bioreactors to produce kilogram amounts of poly(3MP). Cultivation was done in a modified M9 mineral salts medium containing glucose or glycerol as the carbon and energy source and with 3-mercaptopropionic acid (3MP) as the precursor substrate for poly(3MP) biosynthesis provided from the late exponential growth phase. Approximately 23 g of cell dry matter (CDM) per liter and poly(3MP) cell contents of up to 45% (wt/wt) were the highest cell densities and polymer contents obtained, respectively. At best, 69.1% (wt/wt) of 3MP was converted into poly(3MP), indicating that 3MP was mostly used for poly(3MP) biosynthesis. Furthermore, a novel in situ process for rapid and convenient isolation of poly(3MP) from the cells in the bioreactor was developed. This was achieved by addition of sodium dodecyl sulfate to the cultivation broth immediately after the fermentation, heating to 90 degrees C for 20 min with intensive stirring, and subsequent washing steps. The purity of such in situ isolated poly(3MP) was more than 98%, as revealed by gas chromatographic and elemental sulfur analyses of the material isolated.     

Genetically modified strains of Ralstonia eutropha H16 with β-ketothiolase gene deletions for production of copolyesters with defined 3-hydroxyvaleric acid contents

β-Ketothiolases catalyze the first step of poly(3-hydroxybutyrate) [poly(3HB)] biosynthesis in bacteria by condensation of two acetyl coenzyme A (acetyl-CoA) molecules to acetoacetyl-CoA and also take part in the degradation of fatty acids. During growth on propionate or valerate, Ralstonia eutropha H16 produces the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)]. In R. eutropha, 15 β-ketothiolase homologues exist. The synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) could be significantly reduced in an 8-fold mutant (Lindenkamp et al., Appl. Environ. Microbiol. 76:5373-5382, 2010). In this study, a 9-fold mutant deficient in nine β-ketothiolase gene homologues (phaA, bktB, H16_A1713, H16_B1771, H16_A1528, H16_B0381, H16_B1369, H16_A0170, and pcaF) was generated. In order to examine the polyhydroxyalkanoate production capacity when short- or long-chain and even- or odd-chain-length fatty acids were provided as carbon sources, the growth and storage behavior of several mutants from the previous study and the newly generated 9-fold mutant were analyzed. Propionate, valerate, octanoate, undecanoic acid, or oleate was chosen as the sole carbon source. On octanoate, no significant differences in growth or storage behavior were observed between wild-type R. eutropha and the mutants. In contrast, during the growth on oleate of a multiple mutant lacking phaA, bktB, and H16_A0170, diminished poly(3HB) accumulation occurred. Surprisingly, the amount of accumulated poly(3HB) in the multiple mutants grown on gluconate differed; it was much lower than that on oleate. The β-ketothiolase activity toward acetoacetyl-CoA in H16ΔphaA and all the multiple mutants remained 10-fold lower than the activity of the wild type, regardless of which carbon source, oleate or gluconate, was employed. During growth on valerate as a sole carbon source, the 9-fold mutant accumulated almost a poly(3-hydroxyvalerate) [poly(3HV)] homopolyester with 99 mol% 3HV constituents.     

Properties and biodegradability of ultra-high-molecular-weight poly[(R)-hydroxybutyrate] produced by a recombinant Escherichia coli.

Ultra-high-molecular-weight poly[(R)-3-hydroxybutyrate] (P(3HB)) (Mw = 3-11 x 10(6)) was produced from glucose by a recombinant Escherichia coli XL1-Blue (pSYL105) harboring Ralstonia eutropha H16 polyhydroxyalkanoate (PHA) biosynthesis genes. Morphology of ultra-high-molecular-weight P(3HB) granules in the recombinant cells was studied by transmission electron microscopy. The recombinant E. coli contained several P(3HB) granules within a cell. Freeze-fracture morphology of ultra-high-molecular-weight P(3HB) granules showed the needle-type as that of P(3HB) granules in R. eutropha. Both the P(3HB) granules in wet cells and wet native granules isolated from the recombinant cells proved to be amorphous on the X-ray diffraction patterns. Mechanical properties of ultra-high-molecular-weight P(3HB) films were markedly improved by stretching over 400%, resulting from high crystallinity and highly oriented crystal regions. Biodegradability of the films of ultra-high-molecular-weight P(3HB) was tested with an extracellular polyhydroxybutyrate depolymerase from Alcaligenes faecalis T1. The rate of enzymatic erosion of P(3HB) films was not dependent of the molecular weight but was dependent of the crystallinity. In addition, it is demonstrated that all ultra-high-molecular-weight P(3HB) films were completely degraded at 25 degrees C in a natural river freshwater within 3 weeks.     

Dihydrolipoamide Dehydrogenases of Advenella mimigardefordensis and Ralstonia eutropha Catalyze Cleavage of 3,3′-Dithiodipropionic Acid into 3-Mercaptopropionic Acid

The catabolism of the disulfide 3,3′-dithiodipropionic acid (DTDP) is initiated by the reduction of its disulfide bond. Three independent Tn5::mob-induced mutants of Advenella mimigardefordensis strain DPN7^T were isolated that had lost the ability to utilize DTDP as the sole source of carbon and energy and that harbored the transposon insertions in three different sites of the same dihydrolipoamide dehydrogenase gene encoding the E3 subunit of the pyruvate dehydrogenase multi-enzyme complex of this bacterium (LpdA_Am). LpdA_Am was analyzed in silico and compared to homologous proteins, thereby revealing high similarities to the orthologue in Ralstonia eutropha H16 (PdhL_Re). Both bacteria are able to cleave DTDP into two molecules of 3-mercaptopropionic acid (3MP). A. mimigardefordensis DPN7^T converted 3MP to 3-sulfinopropionic acid, whereas R. eutropha H16 showed no growth with DTDP as the sole carbon source but was instead capable of synthesizing heteropolythioesters using the resulting cleavage product 3MP. Subsequently, the genes lpdA_Am and pdhL_Re were cloned, heterologously expressed in Escherichia coli applying the pET23a expression system, purified, and assayed by monitoring the oxidation of NADH. The physiological substrate lipoamide was reduced to dihydrolipoamide with specific activities of 1,833 mkat/kg of protein (LpdA_Am) or 1,667 mkat/kg of protein (PdhL_Re). Reduction of DTDP was also unequivocally detected with the purified enzymes, although the specific enzyme activities were much lower: 0.7 and 0.5 mkat/kg protein, respectively.In Advenella mimigardefordensis strain DPN7^T (15, 42), three independent mutants with an insertion of Tn5::mob in the lpdA gene coding for the E3 component of the pyruvate dehydrogenase multi-enzyme complex revealed an interesting phenotype: these mutants were fully impaired in utilizing 3,3′-dithiodipropionic acid (DTDP) as the sole carbon and energy source, whereas the growth on no other tested carbon sources was affected (41). Our main interest in the catabolism of DTDP is to unravel the pathway and to identify the involved enzymes. Furthermore, the application of this disulfide as precursor substrate for biotechnological production of polythioesters (PTE) (22) is of interest. Since poly(3-mercaptopropionate) (PMP) biosynthesis depends hitherto on supplying the harmful thiol 3-mercaptopropionic acid (3MP) (35), an improvement of the recombinant Escherichia coli system by heterologous expression of enzymes capable of cleaving the less toxic DTDP symmetrically into two molecules of 3MP, which are then polymerized, could be an important achievement toward large-scale biotechnological production of PMP.Two different enzyme systems catalyzing the conversion of disulfides into the corresponding thiols are already known and have been described in detail. (i) Enzymes belonging to the well-characterized family of pyridine-nucleotide disulfide oxidoreductases (25) contain a redox center formed by a disulfide bridge coupled to a flavin ring. They catalyze a simultaneous two-electron transfer via the enzymatic active disulfides associated with the pyridine nucleotides and flavin, toward the substrate (39, 40). (ii) An alternative disulfide reduction is catalyzed by enzymes using iron-sulfur clusters to cleave of disulfide substrates in two one-electron reduction steps (37). The disrupted gene in A. mimigardefordensis was designated lpdA_Am (EC 1.8.1.4), and it encodes a homodimeric flavoprotein, the dihydrolipoamide dehydrogenase LpdA_Am (i.e., the E3 component of the pyruvate dehydrogenase multi-enzyme complex of A. mimigardefordensis strain DPN7^T) belonging to the above-mentioned family of pyridine nucleotide-disulfide oxidoreductases. Enzymes of this class share high sequence and structural similarities and catalyze reduction of compounds which are linked by disulfide bonds (38). Alkylhydroperoxide reductases, coenzyme A disulfide reductases, glutathione reductases, mycothione reductases, thioredoxin reductases, and trypanothione reductases also, in addition to dihydrolipoamide dehydrogenases, belong to this family (3, 38). The physiological function of LpdA_Am is most probably the conversion of lipoamide to dihydrolipoamide, but the reduction of DTDP into two molecules of 3MP (Fig. ​(Fig.1)1) is also predicted, enabling the first step in DTDP catabolism in A. mimigardefordensis strain DPN7^T (41).Open in a separate windowFIG. 1.Reactions catalyzed by LpdA_Am and PdhL_Re. Presented are the enzymatic conversions of DTDP into two molecules of 3MP (A), lipoamide into dihydrolipoamide (B), and DTNB into two molecules of NTB (C). Abbreviations: DTDP, 3,3′-dithiodipropionic acid; 3MP, 3-mercaptopropionic acid; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); NTB, 2-nitro-5-thiobenzoic acid.Ralstonia eutropha H16 synthesizes copolymers of 3-hydroxybutyrate and 3MP, if 3MP (23) or DTDP (22) is supplied as a precursor in addition to a second utilizable carbon source. Although R. eutropha is not able to grow with DTDP as the sole carbon source, it must be capable of cleaving this organic disulfide symmetrically, because it synthesizes from it heteropolymers containing the resulting 3MP. Thus, R. eutropha must possess at least one gene encoding a DTDP-cleaving enzyme. Five genes coding for homologues of a dihydrolipoamide dehydrogenase (DHLDH), which in A. mimigardefordensis DPN7^T is obviously involved in DTDP degradation, are known to exist in the genome of R. eutropha H16 (27; M. Raberg, J. Bechmann, U. Brandt, J. Schlüter, B. Uischner, and A. Steinbüchel, unpublished data). Therefore, LpdA_Am and the five DHLDH paralogues of R. eutropha H16 were aligned and compared (Fig. ​(Fig.2).2). Subsequently, lpdA_Am and the gene encoding the DHLDH belonging to the pyruvate dehydrogenase complex of R. eutropha H16 (pdhL_Re) were cloned, heterologously expressed in Escherichia coli, purified, and assayed.Open in a separate windowFIG. 2.Phylogenetic relationships of the A. mimigardefordensis strain DPN7^T LpdA (boldface), R. eutropha H16 PdhL (boldface), and homologues. The neighbor-joining plot was derived from a CLUSTAL X alignment of amino acid sequences closely related to LpdA_Am. The amino acid sequence of the outer membrane protein P64K from Neisseria meningitidis was used as the outgroup. GenBank accession numbers are given in parentheses. Scale bar, 10% sequence divergence.     

Hyperproduction of poly(4-hydroxybutyrate) from glucose by recombinant Escherichia coli

ABSTRACT: BACKGROUND: Poly(4-hydroxybutyrate) [poly(4HB)] is a strong thermoplastic biomaterial with remarkable mechanical properties, biocompatibility and biodegradability. However, it is generally synthesized when 4-hydroxybutyrate (4HB) structurally related substrates such as gamma-butyrolactone, 4-hydroxybutyrate or 1,4-butanediol (1,4-BD) are provided as precursor which are much more expensive than glucose. At present, high production cost is a big obstacle for large scale production of poly(4HB). RESULTS: Recombinant Escherichia coli strain was constructed to achieve hyperproduction of poly(4-hydroxybutyrate) [poly(4HB)] using glucose as a sole carbon source. An engineering pathway was established in E. coli containing genes encoding succinate degradation of Clostridium kluyveri and PHB synthase of Ralstonia eutropha. Native succinate semialdehyde dehydrogenase genes sad and gabD in E. coli were both inactivated to enhance the carbon flux to poly(4HB) biosynthesis. Four PHA binding proteins (PhaP or phasins) including PhaP1, PhaP2, PhaP3 and PhaP4 from R. eutropha were heterologously expressed in the recombinant E. coli, respectively, leading to different levels of improvement in poly(4HB) production. Among them PhaP1 exhibited the highest capability for enhanced polymer synthesis. The recombinant E. coli produced 5.5 g L-1 cell dry weight containing 35.4% poly(4HB) using glucose as a sole carbon source in a 48 h shake flask growth. In a 6-L fermentor study, 11.5 g L-1 cell dry weight containing 68.2% poly(4HB) was obtained after 52 h of cultivation. This was the highest poly(4HB) yield using glucose as a sole carbon source reported so far. Poly(4HB) was structurally confirmed by gas chromatographic (GC) as well as 1H and 13C NMR studies. CONCLUSIONS: Significant level of poly(4HB) biosynthesis from glucose can be achieved in sad and gabD genes deficient strain of E. coli JM109 harboring an engineering pathway encoding succinate degradation genes and PHB synthase gene, together with expression of four PHA binding proteins PhaP or phasins, respectively. Over 68% poly(4HB) was produced in a fed-batch fermentation process, demonstrating the feasibility for enhanced poly(4HB) production using the recombinant strain for future cost effective commercial development.     

Proteomic examination of Ralstonia eutropha in cellular responses to formic acid

In this study, Ralstonia eutropha was used to elucidate protein changes in response to formic acid. Sixty-three differentially expressed proteins in relation to formic acid in R. eutropha were found with 1-D PAGE and nano-LC-MS/MS. Among the proteins with decreased expression, four were involved in the shikimate pathway and three proteins in the pyrimidine biosynthesis pathway. With the increased expression of proteins, a dramatic change occurred in the induction of ion transporters in relation to maintenance of the acid-base balance. A detoxification process of formic acid in the bacteria might be related to a membrane enzyme, formate hydrogenylase. Three proteins in polyhydroxyalkanoate synthesis were enhanced and five proteins in glutathione biosynthesis increased in response to formic acid. Three enzymes in mevalonate biosynthesis and heat shock proteins were also elevated in the cells. Therefore, formic acid might have an inhibitory effect on aromatic amino acid production and pyrimidine biosynthesis in R. eutropha. R. eutropha cells seemed to attempt to overcome the effects of formic acid by increasing ion transporters and proteins that metabolized formic acid.     

Microbial production of poly-D-3-hydroxybutyrate from CO2

This short review covers the biotechnological aspects of the production of poly-D-3-hydroxybutyric acid, P(3HB), from H2, O2 and CO2 by autotrophic culture of the hydrogen-oxidizing bacterium, Ralstonia eutropha. Considering the efficiency of utilization of a gas mixture as substrate, a practical fermentation process using R. eutropha for the mass production of P(3HB) from CO2 should be designed on the basis of a recycled-gas, closed-circuit culture system. Also, maintaining the O2 concentration in a gas phase lower than 6.9% (v/v) is essential to prevent the gas mixture from exploding. Our study, using an explosion-proof fermentation bench plant and a two-stage culture system with a newly designed air-lift fermenter, demonstrated that very high P(3HB) yield and productivity could be obtained while the O2 concentration was maintained below 6.9%. However, a study on the continuous production of P(3HB) from CO2 by chemostat culture of R. eutropha revealed that the productivity and content of P(3HB) in the cells was considerably lower than by fed-batch culture. It is deduced that the use of the hydrogen-oxidizing bacterium, Alcaligenes latus, which accumulates P(3HB) even in the exponential growth phase, will be useful for the effective production of P(3HB) from CO2.     

Mobilization of poly(3-hydroxybutyrate) in Ralstonia eutropha

Ralstonia eutropha H16 degraded (mobilized) previously accumulated poly(3-hydroxybutyrate) (PHB) in the absence of an exogenous carbon source and used the degradation products for growth and survival. Isolated native PHB granules of mobilized R. eutropha cells released 3-hydroxybutyrate (3HB) at a threefold higher rate than did control granules of nonmobilized bacteria. No 3HB was released by native PHB granules of recombinant Escherichia coli expressing the PHB biosynthetic genes. Native PHB granules isolated from chromosomal knockout mutants of an intracellular PHB (i-PHB) depolymerase gene of R. eutropha H16 and HF210 showed a reduced but not completely eliminated activity of 3HB release and indicated the presence of i-PHB depolymerase isoenzymes.     

Formation of short chain length/medium chain length polyhydroxyalkanoate copolymers by fatty acid beta-oxidation inhibited Ralstonia eutropha

Ralstonia eutropha has been considered as a bacterium, incorporating hydroxyalkanoates of less than six carbons only into polyhydroxyalkanoates (PHAs). Cells of the wild type cultivated with sodium octanoate as the carbon source in the presence of the fatty acid beta-oxidation inhibitor sodium acrylate synthesized PHAs composed of the medium chain length hydroxyalkanoates (3HA(MCL)) 3-hydroxyhexanoate (3HHx) and 3-hydroxyoctanoate (3HO) as well as of 3-hydroxybutyrate and 3-hydroxyproprionate as revealed by gas chromatography, (1)H NMR spectroscopy, and mass spectroscopy. The characterization of the polymer as a tetrapolymer was confirmed by differential solvent extraction and measurement of melting and glass transition temperature depression in the purified polymer compared to PHB. These data suggested that the R. eutropha PHA synthase is capable of incorporating longer chain substrates than suggested by previous in vitro studies. Furthermore, expression of the class II PHA synthase gene phaC1 from P. aeruginosa in R. eutropha resulted in the accumulation of PHAs consisting of 3HA(MCL) contributing about 3-5% to cellular dry weight. These PHAs were composed of nearly equal molar fractions of 3HO and 3-hydroxydecanoate (3HD) with traces of 3HHx. These data indicated that 3HA(MCL)-CoA thioesters were diverted from the fatty acid beta-oxidation pathway towards PHA biosynthesis in recombinant R. eutropha.     

Application of the BPEC Pathway for Large-Scale Biotechnological Production of Poly(3-Mercaptopropionate) by Recombinant Escherichia coli, Including a Novel In Situ Isolation Method

Metabolically engineered Escherichia coli JM109 harboring plasmid pBPP1 and expressing the nonnatural BPEC pathway for synthesis of thermoplastic polyhydroxyalkanoates (PHA) and novel polythioesters (PTE) to provide suitable substrates of PHA synthase was investigated with respect to biotechnological production of poly(3-mercaptopropionate) [poly(3MP)]. Fed-batch fermentation processes were established at the 30- and 500-liter scales in stirred tank bioreactors to produce kilogram amounts of poly(3MP). Cultivation was done in a modified M9 mineral salts medium containing glucose or glycerol as the carbon and energy source and with 3-mercaptopropionic acid (3MP) as the precursor substrate for poly(3MP) biosynthesis provided from the late exponential growth phase. Approximately 23 g of cell dry matter (CDM) per liter and poly(3MP) cell contents of up to 45% (wt/wt) were the highest cell densities and polymer contents obtained, respectively. At best, 69.1% (wt/wt) of 3MP was converted into poly(3MP), indicating that 3MP was mostly used for poly(3MP) biosynthesis. Furthermore, a novel in situ process for rapid and convenient isolation of poly(3MP) from the cells in the bioreactor was developed. This was achieved by addition of sodium dodecyl sulfate to the cultivation broth immediately after the fermentation, heating to 90°C for 20 min with intensive stirring, and subsequent washing steps. The purity of such in situ isolated poly(3MP) was more than 98%, as revealed by gas chromatographic and elemental sulfur analyses of the material isolated.     

以葡萄糖为唯一碳源合成聚-4-羟基丁酸的重组大肠杆菌的构建

为实现重组大肠杆菌以葡萄糖为唯一碳源合成均聚的P( 4HB) ,PCR扩增大肠杆菌编码谷氨酸:琥珀酰半缩醛转氨基酶基因(gabT) ,谷氨酸脱羧酶基因(gadA)以及富养罗尔斯通氏菌(Ralstoniaeutropha)H16的4_羟基丁酸脱氢酶基因(gadB) ,并组装到携带富养罗尔斯通氏菌(Ralstoniaeutropha)H16的PHA聚合酶基因(phaC)和克氏梭菌(Clostridiumkluyveri)中编码4_羟基丁酸:CoA转移酶基因(orfZ)的重组质粒pKESS5 3上,形成一个大的操纵元。携带重组质粒的大肠杆菌获得从三羧酸循环的中间物———α_酮戊二酸到P( 4HB)的代谢途径。结果表明,重组大肠杆菌可以以葡萄糖为唯一碳源合成均聚的P( 4HB) ,当向以葡萄糖为唯一碳源的无机培养基添加蛋白胨、酵母提取物、酪蛋白水解物时,P( 4HB)的含量可以高达菌体干重的30 %。     

Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha

Wild-type Ralstonia eutropha H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Production of these branched-chain alcohols was initiated during nitrogen or phosphorus limitation in the engineered R. eutropha. One mutant strain not only produced over 180?mg/L branched-chain alcohols in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three potential carbon sinks (ilvE, bkdAB, and aceE), the production titer improved to 270?mg/L isobutanol and 40?mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the R. eutropha mutant grew and produced more than 14?g/L branched-chain alcohols over the duration of 50?days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcohols and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis.     

The synthesis of hydroxybutyrate and hydroxyvalerate copolymers by the bacterium Ralstonia eutropha

The paper deals with the study of the synthesis of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) copolymers by the bacterium Ralstonia eutropha B-5786 grown under different carbon nutrition conditions (growth on carbon dioxide, fructose, and CO2-valerate and fructose-valerate mixtures). The parameters to be analyzed included the yield of biomass, the yield, synthesis rate, and composition of copolymers, the activity of the key enzymes of polyhydroxyalkanoate (PHA) synthesis (beta-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase), the maximum tolerable concentration of valerate to the bacterium, and the conditions that govern the incorporation of hydroxyvalerate to copolymers. This allowed the relationship between cultivation conditions and the proportion of monomers in the copolymers to be deduced. We were able to synthesize a range of 3HB/3HV copolymers and found that the thermal characteristics and the degree of crystallinity of these copolymers depend on the molar fraction of 3HV.