Engineering of a Glycerol Utilization Pathway for Amino Acid Production by Corynebacterium glutamicum

The amino acid-producing organism Corynebacterium glutamicum cannot utilize glycerol, a stoichiometric by-product of biodiesel production. By heterologous expression of Escherichia coli glycerol utilization genes, C. glutamicum was engineered to grow on glycerol. While expression of the E. coli genes for glycerol kinase (glpK) and glycerol 3-phosphate dehydrogenase (glpD) was sufficient for growth on glycerol as the sole carbon and energy source, additional expression of the aquaglyceroporin gene glpF from E. coli increased growth rate and biomass formation. Glutamate production from glycerol was enabled by plasmid-borne expression of E. coli glpF, glpK, and glpD in C. glutamicum wild type. In addition, a lysine-producing C. glutamicum strain expressing E. coli glpF, glpK, and glpD was able to produce lysine from glycerol as the sole carbon substrate as well as from glycerol-glucose mixtures.     

Cell growth and P(3HB) accumulation from CO<Subscript>2</Subscript> of a carbon monoxide-tolerant hydrogen-oxidizing bacterium, <Emphasis Type="Italic">Ideonella</Emphasis> sp. O-1

Cell growth and accumulation of polyhydroxybutyric acid, P(3HB), from CO₂ in autotrophic condition of a newly isolated hydrogen-oxidizing bacterium, the strain O-1, was investigated. The bacterium, which was deposited in the Japan Collection of Microorganisms as JCM17105, autotrophically grows by assimilating H₂, O₂, and CO₂ as substrate. 16S rRNA gene sequence of the bacterium was the closest to Ideonella dechloratans (99%). Specific growth rate of the strain O-1 was faster than a hydrogen-oxidizing bacterium, Ralstonia eutropha, which is well-known P(3HB)-producing microorganism. The strain O-1 is tolerant to high O₂ concentration and it can grow above 30% (v/v) O₂, while the growth of R. eutropha and Alcaligenes latus was seriously inhibited. In culture medium containing 1 g/L (NH₄)₂SO₄, cell concentration of the strain O-1 and P(3HB) increased to 6.75 and 5.26 g/L, respectively. The content of P(3HB) in the cells was 77.9% (w/w). The strain O-1 was very tolerant to carbon monoxide (CO) and it grew even at 70% (v/v) CO, while the growth of R. eutropha and A. latus were seriously inhibited at 5% (v/v) CO. From these results, it is expected that the strain O-1 will be useful in the manufacture of P(3HB) because the industrial exhaust gas containing CO₂, H₂, and CO can be directly used as the substrate in the fermentation process.     

Metabolic engineering of Ralstonia eutropha for the biosynthesis of 2-hydroxyacid-containing polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are bio-based and biodegradable polyesters synthesized by numerous microorganisms. PHAs containing 2-hydroxyacids as monomer units have attracted much attention, but their production has not been efficient. Here, we metabolically engineered Ralstonia eutropha strains for the in vivo synthesis of PHAs containing 2-hydroxyacids as monomers. This was accomplished by replacing the R. eutropha phaC gene in the chromosome with either the R. eutropha phaC S506G A510K gene, which contains two point mutations, or the Pseudomonas sp. MBEL 6–19 phaC1437 gene. In addition, the R. eutropha phaAB genes in the chromosome were replaced with the Clostridium propionicum pct540 gene. All of the engineered R. eutropha strains produced PHAs containing 2-hydroxyacid monomers, including lactate and 2-hydroxybutyrate (2HB), along with 3-hydroxybutyrate (3HB) and/or 3-hydroxyvalerate (3HV), when they were cultured in nitrogen-free medium containing 5 g/L lactate or 4 g/L 2HB and 20 g/L glucose as carbon sources. Expression of the Escherichia coli ldhA gene in engineered R. eutropha strains allowed production of poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] from glucose as the sole carbon source. This is the first report on the production of 2-hydroxyacid-containing PHAs by metabolically engineered R. eutropha.     

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.     

Impact of Ralstonia eutropha's Poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on PHB Storage in Recombinant Escherichia coli

The model organism for polyhydroxybutyrate (PHB) biosynthesis, Ralstonia eutropha H16, possesses multiple isoenzymes of granules coating phasins as well as of PHB depolymerases, which degrade accumulated PHB under conditions of carbon limitation. In this study, recombinant Escherichia coli BL21(DE3) strains were used to study the impact of selected PHB depolymerases of R. eutropha H16 on the growth behavior and on the amount of accumulated PHB in the absence or presence of phasins. For this purpose, 20 recombinant E. coli BL21(DE3) strains were constructed, which harbored a plasmid carrying the phaCAB operon from R. eutropha H16 to ensure PHB synthesis and a second plasmid carrying different combinations of the genes encoding a phasin and a PHB depolymerase from R. eutropha H16. It is shown in this study that the growth behavior of the respective recombinant E. coli strains was barely affected by the overexpression of the phasin and PHB depolymerase genes. However, the impact on the PHB contents was significantly greater. The strains expressing the genes of the PHB depolymerases PhaZ1, PhaZ2, PhaZ3, and PhaZ7 showed 35% to 94% lower PHB contents after 30 h of cultivation than the control strain. The strain harboring phaZ7 reached by far the lowest content of accumulated PHB (only 2.0% [wt/wt] PHB of cell dry weight). Furthermore, coexpression of phasins in addition to the PHB depolymerases influenced the amount of PHB stored in cells of the respective strains. It was shown that the phasins PhaP1, PhaP2, and PhaP4 are not substitutable without an impact on the amount of stored PHB. In particular, the phasins PhaP2 and PhaP4 seemed to limit the degradation of PHB by the PHB depolymerases PhaZ2, PhaZ3, and PhaZ7, whereas almost no influence of the different phasins was observed if phaZ1 was coexpressed. This study represents an extensive analysis of the impact of PHB depolymerases and phasins on PHB accumulation and provides a deeper insight into the complex interplay of these enzymes.     

Physiological and Biochemical Characteristics and Capacity for Polyhydroxyalkanoates Synthesis in a Glucose-Utilizing Strain of Hydrogen-Oxidizing Bacteria, Ralstonia eutropha B8562

The physiological, biochemical, genetic, and cultural characteristics of the glucose-utilizing mutant strain Ralstonia eutropha B8562 were investigated in comparison with the parent strain R. eutropha B5786. The morphological, cultural, and biochemical characteristics of strain R. eutropha B8562 were similar to those of strain R. eutropha B5786. Genetic analysis revealed differences between the 16S rRNA gene sequences of these strains. The growth characteristics of the mutant using glucose as the sole carbon and energy source were comparable with those of the parent strain grown on fructose. Strain B8562 was characterized by high yields of polyhydroxyalkanoate (PHA) from different carbon sources (CO₂, fructose, and glucose). In batch culture with glucose under nitrogen limitation, PHA accumulation reached 90% of dry weight. In PHA, β-hydroxybutyrate was predominant (over 99 mol %); β-hydroxyvalerate (0.25–0.72 mol %) and β-hydroxyhexanoate (0.008–1.5 mol %) were present as minor components. The strain has prospects as a PHA producer on glucose-containing media.     

A Forward-Design Approach to Increase the Production of Poly-3-Hydroxybutyrate in Genetically Engineered Escherichia coli

Biopolymers, such as poly-3-hydroxybutyrate (P(3HB)) are produced as a carbon store in an array of organisms and exhibit characteristics which are similar to oil-derived plastics, yet have the added advantages of biodegradability and biocompatibility. Despite these advantages, P(3HB) production is currently more expensive than the production of oil-derived plastics, and therefore, more efficient P(3HB) production processes would be desirable. In this study, we describe the model-guided design and experimental validation of several engineered P(3HB) producing operons. In particular, we describe the characterization of a hybrid phaCAB operon that consists of a dual promoter (native and J23104) and RBS (native and B0034) design. P(3HB) production at 24 h was around six-fold higher in hybrid phaCAB engineered Escherichia coli in comparison to E. coli engineered with the native phaCAB operon from Ralstonia eutropha H16. Additionally, we describe the utilization of non-recyclable waste as a low-cost carbon source for the production of P(3HB).     

Supplementation of Substrate Uptake Gene Enhances the Expression of rhIFN-β in High Cell Density Fed-Batch Cultures of Escherichia coli

Over-expression of recombinant proteins in Escherichia coli triggers a metabolic stress response which causes a sharp decline in both growth and product formation rates post induction. We identified a key down-regulated substrate utilization gene, glycerol kinase (glpK), whose up-regulation could help alleviate this stress response. In a proof of principal study conducted in shake flask cultures, the glpK gene under the “ara” promoter in a pPROLar.A122 vector was co-transformed along with the recombinant interferon-β (rhIFN-β) gene in a pET22b vector into E. coli BL-21(DE3) cells. Co-expression of glpK improved the expression levels of rhIFN-β in glycerol containing medium, while no such gain was observed in medium without glycerol. This study was extended to high cell density fed-batch cultures where exponential feeding of complex substrates was done to increase biomass and hence product titers. For this we first constructed a modified E. coli strain BL-21(glpK ⁺) where the glpK gene was inserted downstream of the ibpA promoter in the host chromosome. There was a significant improvement in growth as well as expression levels of rhIFN-β in this modified strain when the feed medium contained high glycerol. A final product concentration of 4.8 g/l of rhIFN-β was obtained with the modified strain which was 35 % higher than the control.     

Over-expression of 3-ketoacyl-ACP synthase III or malonyl-CoA-ACP transacylase gene induces monomer supply for polyhydroxybutyrate production in Escherichia coli HB101

A Pseudomonas sp. 61-3 malonyl-CoA-ACP transacylase gene (fabD _Ps) was cloned by Southern analysis using an equivalent gene of Escherichia coli (fabD _Ec) as a probe. Some recombinant E. coli HB101 strains harboring fabD _Ps, fabD _Ec, or E. coli 3-ketoacyl-ACP synthase III gene (fabH _Ec) with Aeromonas caviae polyhydroxyalkanoate synthase gene (phaC _Ac) were constructed and grown on one-stage cultivation in Luria-Bertani broth containing glucose as carbon source. These strains accumulated 5 to 11 wt% of poly (3-hydroxybutyrate) (PHB) within cells. Over-expression of fabH _Ec, fabD _Ec, or fabD _Ps has been suggested to lead the monomer-supply of (R)-3-hydroxybutyryl-CoA for PHB synthesis in E. coli cells.     

Improvement on the yield of polyhydroxyalkanotes production from cheese whey by a recombinant Escherichia coli strain using the proton suicide methodology

In this work Escherichia coli strain CML3-1 was engineered through the insertion of Cupriavidus necator P(3HB)-synthesis genes, fused to a lactose-inducible promoter, into the chromosome, via transposition-mediated mechanism. It was shown that polyhydroxyalkanotes (PHAs) production by this strain, using cheese whey, was low due to a significant organic acids (OA) synthesis. The proton suicide method was used as a strategy to obtain an E. coli mutant strain with a reduced OA-producing capacity, aiming at driving bacterial metabolism toward PHAs synthesis.Thirteen E. coli mutant strains were obtained and tested in shake flask assays, using either rich or defined media supplemented with lactose. P8-X8 was selected as the best candidate strain for bioreactor fed-batch tests using cheese whey as the sole carbon source. Although cell growth was considerably slower for this mutant strain, a lower yield of OA on substrate (0.04 Cmol_OA/Cmol_lac) and a higher P(3HB) production (18.88 g_P(3HB)/L) were achieved, comparing to the original recombinant strain (0.11 Cmol_OA/Cmol_lac and 7.8 g_P(3HB)/L, respectively). This methodology showed to be effective on the reduction of OA yield by consequently improving the P(3HB) yield on lactose (0.28 Cmol_P(3HB)/Cmol_lac vs 0.10 Cmol_P(3HB)/Cmol_lac of the original strain).     

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.     

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.     

Microbial production of ethanol from acetate by engineered <Emphasis Type="Italic">Ralstonia eutropha</Emphasis>

This study was performed to produce ethanol from acetate using a genetically engineered Ralstonia eutropha. In order to genetically modify R. eutropha H16, phaCAB operon encoding metabolic pathway genes from acetyl-CoA to polyhydroxybutyrate (PHB) was deleted and adhE encoding an alcohol dehydrogenase from Escherichia coli was overexpressed for conversion of acetyl-CoA to ethanol. The resulting strain produced ethanol up to 170 mg/L when cultivated in minimal media supplemented with 5 g/L of acetate as a sole carbon source. Growth and ethanol production were optimized by adjusting nitrogen source (NH₄Cl) content and repetitive feeding of acetate into the bacterial culture, by which the ethanol production was reached to approximately 350 mg/L for 84 h.     

Genome-Based Analysis and Gene Dosage Studies Provide New Insight into 3-Hydroxy-4-Methylvalerate Biosynthesis in Ralstonia eutropha

Recombinant Ralstonia eutropha strain PHB⁻4 expressing the broad-substrate-specificity polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. strain 61-3 (PhaC1_Ps) synthesizes a PHA copolymer containing the branched side-chain unit 3-hydroxy-4-methylvalerate (3H4MV), which has a carbon backbone identical to that of leucine. Mutant strain 1F2 was derived from R. eutropha strain PHB⁻4 by chemical mutagenesis and shows higher levels of 3H4MV production than does the parent strain. In this study, to understand the mechanisms underlying the enhanced production of 3H4MV, whole-genome sequencing of strain 1F2 was performed, and the draft genome sequence was compared to that of parent strain PHB⁻4. This analysis uncovered four point mutations in the 1F2 genome. One point mutation was found in the ilvH gene at amino acid position 36 (A36T) of IlvH. ilvH encodes a subunit protein that regulates acetohydroxy acid synthase III (AHAS III). AHAS catalyzes the conversion of pyruvate to 2-acetolactate, which is the first reaction in the biosynthesis of branched amino acids such as leucine and valine. Thus, the A36T IlvH mutation may show AHAS tolerance to feedback inhibition by branched amino acids, thereby increasing carbon flux toward branched amino acid and 3H4MV biosynthesis. Furthermore, a gene dosage study and an isotope tracer study were conducted to investigate the 3H4MV biosynthesis pathway. Based on the observations in these studies, we propose a 3H4MV biosynthesis pathway in R. eutropha that involves a condensation reaction between isobutyryl coenzyme A (isobutyryl-CoA) and acetyl-CoA to form the 3H4MV carbon backbone.     

Integrative network analysis reveals active microRNAs and their functions in gastric cancer

Background

Ralstonia eutropha H16, found in both soil and water, is a Gram-negative lithoautotrophic bacterium that can utillize CO₂ and H₂ as its sources of carbon and energy in the absence of organic substrates. R. eutropha H16 can reach high cell densities either under lithoautotrophic or heterotrophic conditions, which makes it suitable for a number of biotechnological applications. It is the best known and most promising producer of polyhydroxyalkanoates (PHAs) from various carbon substrates and is an environmentally important bacterium that can degrade aromatic compounds. In order to make R. eutropha H16 a more efficient and robust biofactory, system-wide metabolic engineering to improve its metabolic performance is essential. Thus, it is necessary to analyze its metabolic characteristics systematically and optimize the entire metabolic network at systems level.

Results

We present the lithoautotrophic genome-scale metabolic model of R. eutropha H16 based on the annotated genome with biochemical and physiological information. The stoichiometic model, RehMBEL1391, is composed of 1391 reactions including 229 transport reactions and 1171 metabolites. Constraints-based flux analyses were performed to refine and validate the genome-scale metabolic model under environmental and genetic perturbations. First, the lithoautotrophic growth characteristics of R. eutropha H16 were investigated under varying feeding ratios of gas mixture. Second, the genome-scale metabolic model was used to design the strategies for the production of poly[R-(-)-3hydroxybutyrate] (PHB) under different pH values and carbon/nitrogen source uptake ratios. It was also used to analyze the metabolic characteristics of R. eutropha when the phosphofructokinase gene was expressed. Finally, in silico gene knockout simulations were performed to identify targets for metabolic engineering essential for the production of 2-methylcitric acid in R. eutropha H16.

Conclusion

The genome-scale metabolic model, RehMBEL1391, successfully represented metabolic characteristics of R. eutropha H16 at systems level. The reconstructed genome-scale metabolic model can be employed as an useful tool for understanding its metabolic capabilities, predicting its physiological consequences in response to various environmental and genetic changes, and developing strategies for systems metabolic engineering to improve its metabolic performance.     

Metabolic Engineering of Escherichia coli for Enhanced Production of (R)- and (S)-3-Hydroxybutyrate

Synthetic metabolic pathways have been constructed for the production of enantiopure (R)- and (S)-3-hydroxybutyrate (3HB) from glucose in recombinant Escherichia coli strains. To promote maximal activity, we profiled three thiolase homologs (BktB, Thl, and PhaA) and two coenzyme A (CoA) removal mechanisms (Ptb-Buk and TesB). Two enantioselective 3HB-CoA dehydrogenases, PhaB, producing the (R)-enantiomer, and Hbd, producing the (S)-enantiomer, were utilized to control the 3HB chirality across two E. coli backgrounds, BL21Star(DE3) and MG1655(DE3), representing E. coli B- and K-12-derived strains, respectively. MG1655(DE3) was found to be superior for the production of each 3HB stereoisomer, although the recombinant enzymes exhibited lower in vitro specific activities than BL21Star(DE3). Hbd in vitro activity was significantly higher than PhaB activity in both strains. The engineered strains achieved titers of enantiopure (R)-3HB and (S)-3HB as high as 2.92 g liter⁻¹ and 2.08 g liter⁻¹, respectively, in shake flask cultures within 2 days. The NADPH/NADP⁺ ratio was found to be two- to three-fold higher than the NADH/NAD⁺ ratio under the culture conditions examined, presumably affecting in vivo activities of PhaB and Hbd and resulting in greater production of (R)-3HB than (S)-3HB. To the best of our knowledge, this study reports the highest (S)-3HB titer achieved in shake flask E. coli cultures to date.The synthesis of chiral molecules is of significant interest in the pharmaceutical industry because frequently one stereoisomer of a drug has efficacy while the other has either substantially reduced or no activity or may even have adverse effects (20, 23). Additionally, chiral molecules serve as building blocks for many pharmaceuticals and high-value compounds. Thus, the ability to prepare chiral molecules with high optical purity is important. Stereoselective chemical processes generally employ expensive chiral catalysts, require harsh physical conditions and solvents, and suffer from extensive by-product formation. In contrast, enzyme-catalyzed reactions are highly stereoselective and can be performed in aqueous solutions under mild conditions (21). As a result, the use of biological processes for chiral molecule production has been extensively investigated (4, 28, 32, 36). One example of such a process is the biosynthesis of 3-hydroxybutyric acid (3HB), a versatile chiral molecule containing one hydroxyl group and one carboxyl group, used as a building block for the synthesis of optically active fine chemicals, such as vitamins, antibiotics, pheromones, and flavor compounds (5, 6, 18, 27).The biosynthesis of 3HB has typically been achieved by two different mechanisms: depolymerization (in vitro or in vivo) of microbially synthesized poly-(R)-3-hydroxybutyric acid (PHB) (8, 13) or direct synthesis of 3HB without a PHB intermediate (9, 12, 15). However, due to the stereospecific constraints of PHB synthesis, in which polymers are composed exclusively of (R)-3HB monomer units, the synthesis of (S)-3HB from PHB is effectively impossible. In contrast, direct synthesis of both enantiopure (R)-3HB and (S)-3HB is possible. Pathways facilitating (R)-3HB synthesis have been constructed in Escherichia coli by simultaneous expression of phaA (encoding acetoacetyl coenzyme A [CoA] thiolase) and phaB [encoding (R)-3HB-CoA dehydrogenase] from Ralstonia eutropha H16, and ptb (encoding phosphotransbutyrylase) and buk (encoding butyrate kinase) from Clostridium acetobutylicum ATCC 824 (9). In addition to the use of ptb and buk to catalyze the conversion of (R)-3HB-CoA to (R)-3HB, tesB (encoding thioesterase II from E. coli) has also been used for the direct hydrolysis of (R)-3HB-CoA to yield (R)-3HB (15). The production of (S)-3HB in E. coli has recently been reported using a biosynthetic pathway consisting of phaA from R. eutropha H16, hbd [encoding (S)-3HB-CoA dehydrogenase] from C. acetobutylicum ATCC 824, and bch (encoding 3-hydroxyisobutyryl-CoA hydrolase) from Bacillus cereus ATCC 14579 (12).In E. coli, the synthesis of both enantiomers of 3HB begins with the condensation of two molecules of acetyl-CoA, catalyzed by a thiolase, to give acetoacetyl-CoA (Fig. ​(Fig.1).1). The acetoacetyl-CoA is then reduced either to (R)-3HB-CoA via ketone reduction mediated by an NADPH-dependent (R)-3HB-CoA dehydrogenase (PhaB) or to (S)-3HB-CoA via an NADH-dependent (S)-3-HB-CoA dehydrogenase (Hbd). (R)-3HB-CoA and (S)-3HB-CoA can each be further modified via a suitable CoA removal reaction to form (R)-3HB and (S)-3HB, respectively. In an effort to increase chiral 3HB production, it is essential to identify a thiolase capable of efficiently catalyzing the first reaction in the 3HB biosynthetic pathways, to draw acetyl-CoA from competing endogenous pathways. Thus, we examined three different thiolases (BktB and PhaA from R. eutropha H16 and Thl from C. acetobutylicum ATCC 824) to determine which is most proficient for 3HB synthesis. (R)-3HB-CoA and (S)-3HB-CoA synthesized via the reduction reaction catalyzed by PhaB and Hbd, respectively, must be converted to their respective free acid forms before transport or diffusion out of the cell. We have compared two sets of CoA-removing enzyme mechanisms, including the phosphotransbutyrylase (Ptb) and butyrate kinase (Buk) system encoded by the ptb-buk operon from C. acetobutylicum ATCC 824 and acyl-CoA thioesterase II (TesB) from E. coli MG1655. Moreover, it has long been argued whether B strains or K-12 strains of E. coli would serve as better hosts for the biosynthesis of small molecules. Microarrays and Northern blot analyses have suggested that several metabolic pathways, including the tricarboxylic acid (TCA) cycle, glyoxylate shunt, glycolysis, and fatty acid degradation are different between these two strains (22, 25, 34, 35), implying that they may differ significantly in their abilities to supply significant levels of acetyl-CoA as the precursor for 3HB synthesis. Thus, we have also compared 3HB synthesis across two representative E. coli strains: BL21Star(DE3) (B strain) and MG1655(DE3) (K-12 strain). 3HB chirality was examined and verified by high-performance liquid chromatography (HPLC) analysis using a chiral stationary phase to provide separation.Open in a separate windowFIG. 1.Schematic representation of (S)-3HB or (R)-3HB synthesis from glucose in engineered E. coli. BktB, acetoacetyl-CoA thiolase from R. eutropha H16; Thl, acetoacetyl-CoA thiolase from C. acetobutylicum ATCC 824; PhaA, acetoacetyl-CoA thiolase from R. eutropha H16; Hbd, (S)-3HB-CoA dehydrogenase from C. acetobutylicum ATCC 824; PhaB, (R)-3HB-CoA dehydrogenase from R. eutropha H16; Ptb, phosphotransbutyrylase from C. acetobutylicum ATCC 824; Buk, butyrate kinase from C. acetobutylicum ATCC 824; TesB, acyl-CoA thioesterase II from E. coli MG1655.Altogether, we have explored the production of each stereoisomer of 3HB across different strains of E. coli, different thiolases, and different CoA removal systems to engineer E. coli strains for enhanced chiral 3HB production.     

From Waste to Plastic: Synthesis of Poly(3-Hydroxypropionate) in Shimwellia blattae

In recent years, glycerol has become an attractive carbon source for microbial processes, as it accumulates massively as a by-product of biodiesel production, also resulting in a decline of its price. A potential use of glycerol in biotechnology is the synthesis of poly(3-hydroxypropionate) [poly(3HP)], a biopolymer with promising properties which is not synthesized by any known wild-type organism. In this study, the genes for 1,3-propanediol dehydrogenase (dhaT) and aldehyde dehydrogenase (aldD) of Pseudomonas putida KT2442, propionate-coenzyme A (propionate-CoA) transferase (pct) of Clostridium propionicum X2, and polyhydroxyalkanoate (PHA) synthase (phaC1) of Ralstonia eutropha H16 were cloned and expressed in the 1,3-propanediol producer Shimwellia blattae. In a two-step cultivation process, recombinant S. blattae cells accumulated up to 9.8% ± 0.4% (wt/wt [cell dry weight]) poly(3HP) with glycerol as the sole carbon source. Furthermore, the engineered strain tolerated the application of crude glycerol derived from biodiesel production, yielding a cell density of 4.05 g cell dry weight/liter in a 2-liter fed-batch fermentation process.     

Effect of puuC overexpression and nitrate addition on glycerol metabolism and anaerobic 3-hydroxypropionic acid production in recombinant Klebsiella pneumoniae ΔglpKΔdhaT

3-Hydroxypropionic acid (3-HP), an industrially important platform chemical, is used as a precursor during the production of many commercially important chemicals. Recently, recombinant strains of K. pneumoniae overexpressing an NAD⁺-dependent γ-glutamyl-γ-aminobutyraldehyde dehydrogenase (PuuC) enzyme of K. pneumoniae DSM 2026 were shown to produce 3-HP from glycerol without the addition coenzyme B₁₂, which is expensive. However, 3-HP production in K. pneumoniae is accompanied with NADH generation, and this always results in large accumulation of 1,3-propanediol (1,3-PDO) and lactic acid. In this study, we investigated the potential use of nitrate as an electron acceptor both to regenerate NAD⁺ and to prevent the formation of byproducts during anaerobic production of 3-HP from glycerol. Nitrate addition could improve NAD⁺ regeneration, but decreased glycerol flux towards 3-HP production. To divert more glycerol towards 3-HP, a novel recombinant strain K. pneumoniae ΔglpKΔdhaT (puuC) was developed by disrupting the glpK gene, which encodes glycerol kinase, and the dhaT gene, which encodes 1,3-propanediol oxidoreductase. This strain showed improved cellular NAD⁺ concentrations and a high carbon flux towards 3-HP production. Through anaerobic cultivation in the presence of nitrate, this recombinant strain produced more than 40±3 mM 3-HP with more than 50% yield on glycerol in shake flasks and 250±10 mM 3-HP with approximately 30% yield on glycerol in a fed-batch bioreactor.     

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.