Engineering of Ralstonia eutropha H16 for Autotrophic and Heterotrophic Production of Methyl Ketones

Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H₂ and CO₂ under aerobic conditions. Under conditions of nutrient imbalance, R. eutropha produces copious amounts of poly[(R)-3-hydroxybutyrate] (PHB). Its ability to utilize CO₂ as a sole carbon source renders it an interesting new candidate host for the production of renewable liquid transportation fuels. We engineered R. eutropha for the production of fatty acid-derived, diesel-range methyl ketones. Modifications engineered in R. eutropha included overexpression of a cytoplasmic version of the TesA thioesterase, which led to a substantial (>150-fold) increase in fatty acid titer under certain conditions. In addition, deletion of two putative β-oxidation operons and heterologous expression of three genes (the acyl coenzyme A oxidase gene from Micrococcus luteus and fadB and fadM from Escherichia coli) led to the production of 50 to 65 mg/liter of diesel-range methyl ketones under heterotrophic growth conditions and 50 to 180 mg/liter under chemolithoautotrophic growth conditions (with CO₂ and H₂ as the sole carbon source and electron donor, respectively). Induction of the methyl ketone pathway diverted substantial carbon flux away from PHB biosynthesis and appeared to enhance carbon flux through the pathway for biosynthesis of fatty acids, which are the precursors of methyl ketones.     

Genomic view of energy metabolism in Ralstonia eutropha H16

Ralstonia eutropha is a strictly respiratory facultative lithoautotrophic beta-proteobacterium. In the absence of organic substrates, H2 and CO2 are used as sole sources of energy and carbon. In the absence of oxygen, the organism can respire by denitrification. The recent determination of the complete genome sequence of strain H16 provides the opportunity to reconcile the results of previous physiological and biochemical studies in light of the coding capacity. These analyses revealed genes for several isoenzymes, permit assignment of well-known physiological functions to previously unidentified genes, and suggest the presence of unknown components of energy metabolism. The respiratory chain is fueled by two NADH dehydrogenases, two uptake hydrogenases and at least three formate dehydrogenases. The presence of genes for five quinol oxidases and three cytochrome oxidases indicates that the aerobic respiration chain adapts to varying concentrations of dioxygen. Several additional components may act in balancing or dissipation of redox energy. Paralogous sets of nitrate reductase and nitric oxide reductase genes result in enzymatic redundancy for denitrification.     

A proteomic view of the facultatively chemolithoautotrophic lifestyle of Ralstonia eutropha H16

Ralstonia eutropha H16 is an H₂‐oxidizing, facultative chemolithoautotroph. Using 2‐DE in conjunction with peptide mass spectrometry we have cataloged the soluble proteins of this bacterium during growth on different substrates: (i) H₂ and CO₂, (ii) succinate and (iii) glycerol. The first and second conditions represent purely lithoautotrophic and purely organoheterotrophic nutrition, respectively. The third growth regime permits formation of the H₂‐oxidizing and CO₂‐fixing systems concomitant to utilization of an organic substrate, thus enabling mixotrophic growth. The latter type of nutrition is probably the relevant one with respect to the situation faced by the organism in its natural habitats, i.e. soil and mud. Aside from the hydrogenase and Calvin‐cycle enzymes, the protein inventories of the H₂‐CO₂‐ and succinate‐grown cells did not reveal major qualitative differences. The protein complement of the glycerol‐grown cells resembled that of the lithoautotrophic cells. Phosphoenolpyruvate (PEP) carboxykinase was present under all three growth conditions, whereas PEP carboxylase was not detectable, supporting earlier findings that PEP carboxykinase is alone responsible for the anaplerotic production of oxaloacetate from PEP. The elevated levels of oxidative stress proteins in the glycerol‐grown cells point to a significant challenge by ROS under these conditions. The results reported here are in agreement with earlier physiological and enzymological studies indicating that R. eutropha H16 has a heterotrophic core metabolism onto which the functions of lithoautotrophy have been grafted.     

Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16

The H(2)-oxidizing lithoautotrophic bacterium Ralstonia eutropha H16 is a metabolically versatile organism capable of subsisting, in the absence of organic growth substrates, on H(2) and CO(2) as its sole sources of energy and carbon. R. eutropha H16 first attracted biotechnological interest nearly 50 years ago with the realization that the organism's ability to produce and store large amounts of poly[R-(-)-3-hydroxybutyrate] and other polyesters could be harnessed to make biodegradable plastics. Here we report the complete genome sequence of the two chromosomes of R. eutropha H16. Together, chromosome 1 (4,052,032 base pairs (bp)) and chromosome 2 (2,912,490 bp) encode 6,116 putative genes. Analysis of the genome sequence offers the genetic basis for exploiting the biotechnological potential of this organism and provides insights into its remarkable metabolic versatility.     

Characterization of an extracellular lipase and its chaperone from Ralstonia eutropha H16

Lipase enzymes catalyze the reversible hydrolysis of triacylglycerol to fatty acids and glycerol at the lipid–water interface. The metabolically versatile Ralstonia eutropha strain H16 is capable of utilizing various molecules containing long carbon chains such as plant oil, organic acids, or Tween as its sole carbon source for growth. Global gene expression analysis revealed an upregulation of two putative lipase genes during growth on trioleate. Through analysis of growth and activity using strains with gene deletions and complementations, the extracellular lipase (encoded by the lipA gene, locus tag H16_A1322) and lipase-specific chaperone (encoded by the lipB gene, locus tag H16_A1323) produced by R. eutropha H16 was identified. Increase in gene dosage of lipA not only resulted in an increase of the extracellular lipase activity, but also reduced the lag phase during growth on palm oil. LipA is a non-specific lipase that can completely hydrolyze triacylglycerol into its corresponding free fatty acids and glycerol. Although LipA is active over a temperature range from 10 °C to 70 °C, it exhibited optimal activity at 50 °C. While R. eutropha H16 prefers a growth pH of 6.8, its extracellular lipase LipA is most active between pH 7 and 8. Cofactors are not required for lipase activity; however, EDTA and EGTA inhibited LipA activity by 83 %. Metal ions Mg²⁺, Ca²⁺, and Mn²⁺ were found to stimulate LipA activity and relieve chelator inhibition. Certain detergents are found to improve solubility of the lipid substrate or increase lipase-lipid aggregation, as a result SDS and Triton X-100 were able to increase lipase activity by 20 % to 500 %. R. eutropha extracellular LipA activity can be hyper-increased, making the overexpression strain a potential candidate for commercial lipase production or in fermentations using plant oils as the sole carbon source.     

Purification and characterization of the single-component nitric oxide reductase from Ralstonia eutropha H16

Nitric oxide (NO) reductase was purified from Ralstonia eutropha (formerly Alcaligenes eutrophus) using a two step chromatographic procedure. Unlike the common NO reductases, the enzyme consists of a single subunit of 75 kDa which contains both high-spin and low-spin heme b, but lacks heme c. One additional iron atom, probably a ferric non-heme iron, was identified per enzyme molecule. Whereas reduced cytochrome c was ineffective as electron donor, NO was reduced at a specific activity of 2.3 micromol/min per mg of protein in the presence of 2-methyl-1,4-naphthoquinol.     

Biotransformation of Eugenol to Ferulic Acid by a Recombinant Strain of Ralstonia eutropha H16

The gene loci ehyAB, calA, and calB, encoding eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase, respectively, which are involved in the first steps of eugenol catabolism in Pseudomonas sp. strain HR199, were amplified by PCR and combined to construct a catabolic gene cassette. This gene cassette was cloned in the newly designed broad-host-range vector pBBR1-JO2 (pBBR1-JO2ehyABcalAcalB) and transferred to Ralstonia eutropha H16. A recombinant strain of R. eutropha H16 harboring this plasmid expressed functionally active eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase. Cells of R. eutropha H16(pBBR1-JO2ehyABcalAcalB) from the late-exponential growth phase were used as biocatalysts for the biotransformation of eugenol to ferulic acid. A maximum conversion rate of 2.9 mmol of eugenol per h per liter of culture was achieved with a yield of 93.8 mol% of ferulic acid from eugenol within 20 h, without further optimization.     

Dynamics of Activity of the Key Enzymes of Polyhydroxyalkanoate Metabolism in Ralstonia eutropha B5786

The dynamics of accumulation of polyhydroxybutyrate (PHB) and the activities of key enzymes of PHB metabolism (-ketothiolase, acetoacetyl-CoA reductase, PHB synthase, D-hydroxybutyrate dehydrogenase, and PHB depolymerase) in the hydrogen bacterium Ralstonia eutropha B5786 were studied under various conditions of carbon nutrition and substrate availability. The highest activities of -ketothiolase, acetoacetyl-CoA reductase, and PHB synthase were recorded during acceleration of PHB synthesis. The activities of enzymes catalyzing PHB depolymerization (PHB depolymerase and D-hydroxybutyrate dehydrogenase) were low, being expressed only upon stimulated endogenous PHB degradation. The change of carbon source (CO₂ or fructose) did not affect the time course of the enzyme activity significantly.     

Biotransformation of eugenol to ferulic acid by a recombinant strain of Ralstonia eutropha H16

The gene loci ehyAB, calA, and calB, encoding eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase, respectively, which are involved in the first steps of eugenol catabolism in Pseudomonas sp. strain HR199, were amplified by PCR and combined to construct a catabolic gene cassette. This gene cassette was cloned in the newly designed broad-host-range vector pBBR1-JO2 (pBBR1-JO2ehyABcalAcalB) and transferred to Ralstonia eutropha H16. A recombinant strain of R. eutropha H16 harboring this plasmid expressed functionally active eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase. Cells of R. eutropha H16(pBBR1-JO2ehyABcalAcalB) from the late-exponential growth phase were used as biocatalysts for the biotransformation of eugenol to ferulic acid. A maximum conversion rate of 2.9 mmol of eugenol per h per liter of culture was achieved with a yield of 93.8 mol% of ferulic acid from eugenol within 20 h, without further optimization.     

The genome organization of Ralstonia eutropha strain H16 and related species of the Burkholderiaceae

Ralstonia eutropha strain H16 is a facultatively chemolithoautotrophic, hydrogen-oxidizing bacterium belonging to the family Burkholderiaceae of the Betaproteobacteria. The genome of R. eutropha H16 consists of two chromosomes (Chr1, Chr2) and one megaplasmid (pHG1), and thus shows a multi-replicon architecture, which is characteristic for all members of the Burkholderiaceae sequenced so far. The genes for housekeeping cell functions are located on Chr1. In contrast, many characteristic traits of R. eutropha H16 such as the ability to switch between alternative lifestyles and to utilize a broad variety of growth substrates are primarily encoded on the smaller replicons Chr2 and pHG1. The latter replicons also differ from Chr1 by carrying a repA-associated origin of replication typically found on plasmids. Relationships between the individual replicons from various Burkholderiaceae genomes were studied by multiple sequence alignments and whole-replicon protein comparisons. While strong conservation of gene content and order among the largest replicons indicate a common ancestor, the resemblance between the smaller replicons is considerably lower, suggesting a species-specific origin of Chr2. The megaplasmids, however, in most cases do not show any taxonomically related similarities. Based on the results of the comparative studies, a hypothesis for the evolution of the multi-replicon genomes of the Burkholderiaceae is proposed.     

Studies on the aerobic utilization of synthesis gas (syngas) by wild type and recombinant strains of Ralstonia eutropha H16

The biotechnical platform strain Ralstonia eutropha H16 was genetically engineered to express a cox subcluster of the carboxydotrophic Oligotropha carboxidovoransOM5, including (i) the structural genes coxM, ‐S and ‐L, coding for an aerobic carbon monoxide dehydrogenase (CODH) and (ii) the genes coxD, ‐E, ‐F and ‐G, essential for the maturation of CODH. The cox_Oc genes expressed under control of the CO₂‐inducible promoter P_L enabled R. eutropha to oxidize CO to CO₂ for the use as carbon source, as demonstrated by ¹³CO experiments, but the recombinant strains remained dependent on H₂ as external energy supply. Therefore, a synthetic metabolism, which could be described as ‘carboxyhydrogenotrophic’, was established in R. eutropha. With this extension of the bacterium's substrate range, growth in CO‐, H₂‐ and CO₂‐containing artificial synthesis gas atmosphere was enhanced, and poly(3‐hydroxybutyrate) synthesis was increased by more than 20%.     

Production and purification of self-assembling peptides in Ralstonia eutropha

Self-assembling peptides have emerged as an attractive scaffold material for tissue engineering, yet the expense associated with solid phase chemical synthesis has limited their broad use. In addition, the fidelity of chemical synthesis constrains the length of polypeptides that can be produced homogeneously by this method. Template-derived biosynthesis by recombinant DNA technology may overcome both of these problems. However, recovery of polypeptides from recombinant protein expression systems typically involves multi-step purification schemes. In this study, we report an integrated approach to recombinantly produce and purify self-assembling peptides from the recently developed expression host Ralstonia eutropha. The purification is based on the specific affinity of carbohydrate binding modules (CBMs) to cellulose. In a first step, we identified CBMs that express well in R. eutropha by assembling a fusion library of green fluorescent protein (GFP) and CBMs and determining the fluorescence of cell-free extracts. Three GFP::CBM fusions were found to express at levels similar to GFP alone, of which two CBMs were able to mediate cellulose binding of the GFP::CBM fusion. These two CBMs were then fused to multiple repeats of the self-assembling peptide RAD16-I::E (N-RADARADARADARADAE-C). The fusion protein CBM::E::(RAD16-I::E)4 was expressed in R. eutropha and purified using the CBM's affinity for cellulose. Subsequent proteolytic cleavage with endoproteinase GluC liberated RAD16-I::E peptide monomers with similar properties to the chemically synthesized counterpart RAD16-I.     

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.     

Establishment of an alternative phosphoketolase-dependent pathway for fructose catabolism in Ralstonia eutropha H16

The β-proteobacterium Ralstonia eutropha H16 utilizes fructose and gluconate as carbon sources for heterotrophic growth exclusively via the Entner–Doudoroff pathway with its key enzyme 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase. By deletion of the responsible gene eda, we constructed a KDPG aldolase-negative strain, which is disabled to supply pyruvate for energy metabolism from fructose or gluconate as sole carbon sources. To restore growth on fructose, an alternative pathway, similar to the fructose-6-phosphate shunt of heterofermentative bifidobacteria, was established. For this, the xfp gene from Bifidobacterium animalis, coding for a bifunctional xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp; Meile et al. in J Bacteriol 183:2929–2936, 2001), was expressed in R. eutropha H16 PHB⁻4 Δeda. This Xfp catalyzes the phosphorolytic cleavage of fructose 6-phosphate to erythrose 4-phosphate and acetylphosphate as well as of xylulose 5-phosphate to glyceralaldehyde 3-phosphate and acetylphosphate. The recombinant strain showed phosphoketolase (PKT) activity on either substrate, and was able to use fructose as sole carbon source for growth, because PKT is the only enzyme that is missing in R. eutropha H16 to establish the artificial fructose-6-phosphate shunt. The Xfp-expressing strain R. eutropha H16 PHB⁻4 Δeda (pBBR1MCS-3::xfp) should be applicable for a novel variant of a plasmid addiction system to stably maintain episomally encoded genetic information during fermentative production processes. Plasmid addiction systems are often used to ensure plasmid stability in many biotechnology relevant microorganisms and processes without the need to apply external selection pressure, like the addition of antibiotics. By episomal expression of xfp in a R. eutropha H16 mutant lacking KDPG aldolase activity and cultivation in mineral salt medium with fructose as sole carbon source, the growth of this bacterium was addicted to the constructed xfp harboring plasmid. This novel selection principle extends the applicability of R. eutropha H16 as production platform in biotechnological processes.     

Reduction of unusual iron-sulfur clusters in the H2-sensing regulatory Ni-Fe hydrogenase from Ralstonia eutropha H16

The regulatory Ni-Fe hydrogenase (RH) from Ralstonia eutropha functions as a hydrogen sensor. The RH consists of the large subunit HoxC housing the Ni-Fe active site and the small subunit HoxB containing Fe-S clusters. The heterolytic cleavage of H(2) at the Ni-Fe active site leads to the EPR-detectable Ni-C state of the protein. For the first time, the simultaneous but EPR-invisible reduction of Fe-S clusters during Ni-C state formation was demonstrated by changes in the UV-visible absorption spectrum as well as by shifts of the iron K-edge from x-ray absorption spectroscopy in the wild-type double dimeric RH(WT) [HoxBC](2) and in a monodimeric derivative designated RH(stop) lacking the C-terminal 55 amino acids of HoxB. According to the analysis of iron EXAFS spectra, the Fe-S clusters of HoxB pronouncedly differ from the three Fe-S clusters in the small subunits of crystallized standard Ni-Fe hydrogenases. Each HoxBC unit of RH(WT) seems to harbor two [2Fe-2S] clusters in addition to a 4Fe species, which may be a [4Fe-3S-3O] cluster. The additional 4Fe-cluster was absent in RH(stop). Reduction of Fe-S clusters in the hydrogen sensor RH may be a first step in the signal transduction chain, which involves complex formation between [HoxBC](2) and tetrameric HoxJ protein, leading to the expression of the energy converting Ni-Fe hydrogenases in R. eutropha.     

Roles of Multiple Acetoacetyl Coenzyme A Reductases in Polyhydroxybutyrate Biosynthesis in Ralstonia eutropha H16

The bacterium Ralstonia eutropha H16 synthesizes polyhydroxybutyrate (PHB) from acetyl coenzyme A (acetyl-CoA) through reactions catalyzed by a β-ketothiolase (PhaA), an acetoacetyl-CoA reductase (PhaB), and a polyhydroxyalkanoate synthase (PhaC). An operon of three genes encoding these enzymatic steps was discovered in R. eutropha and has been well studied. Sequencing and analysis of the R. eutropha genome revealed putative isologs for each of the PHB biosynthetic genes, many of which had never been characterized. In addition to the previously identified phaB1 gene, the genome contains the isologs phaB2 and phaB3 as well as 15 other potential acetoacetyl-CoA reductases. We have investigated the roles of the three phaB isologs by deleting them from the genome individually and in combination. It was discovered that the gene products of both phaB1 and phaB3 contribute to PHB biosynthesis in fructose minimal medium but that in plant oil minimal medium and rich medium, phaB3 seems to be unexpressed. This raises interesting questions concerning the regulation of phaB3 expression. Deletion of the gene phaB2 did not result in an observable phenotype under the conditions tested, although this gene does encode an active reductase. Addition of the individual reductase genes to the genome of the ΔphaB1 ΔphaB2 ΔphaB3 strain restored PHB production, and in the course of our complementation experiments, we serendipitously created a PHB-hyperproducing mutant. Measurement of the PhaB and PhaA activities of the mutant strains indicated that the thiolase reaction is the limiting step in PHB biosynthesis in R. eutropha H16 during nitrogen-limited growth on fructose.Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by a wide range of bacteria as carbon and energy reserves. PHAs are typically stored when organisms are in an environment in which carbon is plentiful but the lack of another nutrient limits normal cell growth. It has been found that in environments with fluctuating carbon levels, PHA producers have crucial advantages over rival species (14). In addition to their importance in the microbial world, these polymers have been studied for their potential uses in biodegradable consumer goods (12) and medical products (22) and as chemical precursors (4). Although many PHA monomers have been discovered, the most common are 3-hydroxyalkanoates (32). Common PHAs are typically characterized by their constituent monomers as short-chain-length polymers (SCL-PHA; C₄ and C₅ monomers) or medium-chain-length polymers (MCL-PHA; C₆ and longer monomers).The model organism used to study PHA biosynthesis is the Gram-negative bacterium Ralstonia eutropha. This organism accumulates a high percentage of its cell dry weight (CDW) as SCL-PHA under nutrient limitation. When grown on sugars or plant oils, R. eutropha makes poly(3-hydroxybutyrate) (PHB) almost exclusively, although the addition of precursors such as propionate to the growth medium can lead to incorporation of 3-hydroxyvalerate into the polymer chain as well (2). An operon of biosynthetic genes from R. eutropha encoding enzymes sufficient for synthesis of PHB from acetyl coenzyme A (acetyl-CoA), which consisted of phaC-phaA-phaB, was discovered in the late 1980s (25, 26, 36). In this pathway, two molecules of acetyl-CoA are condensed by a β-ketothiolase (PhaA) and the resulting acetoacetyl-CoA is reduced by a reductase (PhaB) to form (R)-3-hydroxybutyryl-CoA (HB-CoA), which is the substrate for the PHA synthase (PhaC). Sequencing and analysis of the R. eutropha genome revealed the existence of putative isologs for each of the PHA synthetic genes (29). While the existence of alternate β-ketothiolases was already known (39), most of the potential isologs identified had never been characterized.Our group wanted to better understand how acetoacetyl-CoA reduction occurs in R. eutropha. In addition to the earlier-identified phaB gene, now referred to as phaB1 (GeneID, 4249784), the genes phaB2 (GeneID, 4249785) and phaB3 (GeneID, 4250155) were discovered on R. eutropha chromosome 1. Fifteen other potential isologs were also found to encode amino acid sequences that could potentially indicate acetoacetyl-CoA reductase activity (29). The roles of the newly discovered genes in PHB biosynthesis were unclear, especially given the results of an earlier biochemical study that suggested there was a single NADPH-dependent acetoacetyl-CoA reductase in R. eutropha (10). In order to determine the roles of the reductase genes in R. eutropha, we deleted phaB1, phaB2, and phaB3 from the genome both individually and in combination. In addition to characterizing these newly discovered genes, we also hoped to eliminate or diminish formation of HB-CoA by stopping the reduction reaction. Efforts to purify the PHA synthase from R. eutropha have been complicated by the high levels of PHB made by this organism (7). Studying formation and growth of PHB granules is difficult because PHB accumulates at a high rate, causing individual granules to coalesce and become indistinct (44). We therefore believed that an R. eutropha strain with decreased HB-CoA synthesis would be a useful experimental tool and could also serve as a platform for engineering new PHA synthesis pathways into R. eutropha.