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Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] (PHBHHx) is a type of biopolyester of the polyhydroxyalkanoate group (PHA). Due to a wide range of properties resulting from the alteration of the (R)-3-hydroxyhexanoate (3HHx) composition, PHBHHx is getting a lot of attention as a substitute to conventional plastic materials for various applications. Cupriavidus necator H16 is the most promising PHA producer and has been genetically engineered to produce PHBHHx efficiently for many years. Nevertheless, the role of individual genes involved in PHBHHx biosynthesis is not well elaborated. C. necator H16 possesses six potential physiologically active β-ketothiolase genes identified by transcriptome analysis, i.e., phaA, bktB, bktC (h16_A0170), h16_A0462, h16_A1528, and h16_B0759. In this study, we focused on the functionality of these genes in vivo in relation to 3HHx monomer supply. Gene deletion experiments identified BktB and H16_A1528 as important β-ketothiolases for C6 metabolism in β-oxidation. Furthermore, in the bktB/h16_A1528 double-deletion strain, the proportion of 3HHx composition of PHBHHx produced from sugar was very low, whereas that from plant oil was significantly higher. In fact, the proportion reached 36.2 mol% with overexpression of (R)-specifc enoyl-CoA hydratase (PhaJ) and PHA synthase. Furthermore, we demonstrated high-density production (196 g/L) of PHBHHx with high 3HHx (32.5 mol%) by fed-batch fermentation with palm kernel oil. The PHBHHx was amorphous according to the differential scanning calorimetry analysis. KEY POINTS: • Role of six β-ketothiolases in PHBHHx biosynthesis was investigated in vivo. • Double-deletion of bktB/h16_A1528 results in high 3HHx composition with plant oil. • Amorphous PHBHHx with 32.5 mol% 3HHx was produced in high density by jar fermenter.

Abstract Fluorescence spectroscopy offers a cheap, simple, and fast approach to monitor poly(3-hydroxybutyrate) (PHB) formation, a biodegradable polymer belonging to the biodegradable polyester class polyhydroxyalkanoates. In the present study, a fluorescence and side scatter-based spectroscopic setup was developed to monitor in situ biomass, and PHB formation of biotechnological applied Cupriavidus necator strain. To establish PHB quantification of C. necator, the dyes 2,2-difluoro-4,6,8,10,12-pentamethyl-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaene (BODIPY493/503), ethyl 5-methoxy-1,2-bis(3-methylbut-2-enyl)-3-oxoindole-2-carboxylate (LipidGreen2), and 9-(diethylamino)benzo[a]phenoxazin-5-one (Nile red) were compared with each other. Fluorescence staining efficacy was obtained through 3D-excitation-emission matrix and design of experiments. The coefficients of determination were ≥ 0.98 for all three dyes and linear to the high-pressure liquid chromatography obtained PHB content, and the side scatter to the biomass concentration. The fluorescence correlation models were further improved by the incorporation of the biomass-related side scatter. Afterward, the resulting regression fluorescence models were successfully applied to nitrogen-deficit, phosphor-deficit, and NaCl-stressed C. necator cultures. The highest transferability of the regression models was shown by using LipidGreen2. The novel approach opens a tailor-made way for a fast and simultaneous detection of the crucial biotechnological parameters biomass and PHB content during fermentation. Key points • Intracellular quantification of PHB and biomass using fluorescence spectroscopy. • Optimizing fluorescence staining conditions and 3D-excitation-emission matrix. • PHB was best obtained by LipidGreen2, followed by BODIPDY493/503 and Nile red. Graphical abstract

Early Access; International audience; A methodology for plasmid expression level monitoring of eGFP expression suitable for dynamic processes was assessed during fermentation. This technique was based on the expression of a fluorescent biosensor (eGFP) encoded on a recombinant plasmid coupled to single-cell analysis. Fluorescence intensity at single-cell level was measured by flow cytometry. We demonstrated that promoter evaluation based on single-cell analysis versus classic global analysis brings valuable insights. Single-cell analysis pointed out the fact that intrinsic fluorescence increased with the strength of the promoter up to a threshold. Beyond that, cell permeability increases to excrete the fluorescent protein in the medium. The metabolic load due to the increase in the eGFP production in the case of strong constitutive promoters leads to slower growth kinetics compared with plasmid-free cells. With the strain Cupriavidus necator Re2133, growth rate losses were measured from 3% with the weak constitutive promoter P-lac to 56% with the strong constitutive promoter P-j5. Through this work, it seems crucial to find a compromise between the fluorescence intensity in single cells and the metabolic load; in our conditions, the best compromise found was the weak promoter P-lac. The plasmid expression level monitoring method was tested in the presence of a heterogeneous population induced by plasmid-curing methods. For all the identified subpopulations, the plasmid expression level heterogeneity was significantly detected at the level of fluorescence intensity in single cells. After cell sorting, growth rate and cultivability were assessed for each subpopulation. In conclusion, this eGFP biosensor makes it possible to follow the variations in the level of plasmid expression under conditions of population heterogeneity. Key Points center dot Development of a plasmid expression level monitoring method at the single-cell level by flow cytometry. center dot Promoter evaluation by single-cell analysis: cell heterogeneity and strain robustness. center dot Reporter system optimization for efficient subpopulation detection in pure cultures.

While crude glycerol is a cheap carbon source for industrial-scale cultivation of microorganisms, its application relies on fast growth and conversion. The biopolymer producing Cupriavidus necator H16 (synonym: Ralstonia eutropha H16) grows poorly on glycerol. The heterologous expression of glycerol facilitator glpF, glycerol kinase glpK, and glycerol dehydrogenase glpD from E. coli accelerated the growth considerably. The naturally occurring glycerol utilization is inhibited by low glycerol kinase activity. A limited heterotrophic growth promotes the dependency on autotrophic growth by carbon dioxide (CO

Polyphosphosphate kinases (PPKs) catalyse the reversible transfer of the γ-phosphate group of a nucleoside-triphosphate to a growing chain of polyphosphate. Most known PPKs are specific for ATP, but some can also use GTP as a phosphate donor. In this study, we describe the properties of a PPK2-type PPK of the β-proteobacterium Ralstonia eutropha. The purified enzyme (PPK2c) is highly unspecific and accepts purine nucleotides as well as the pyridine nucleotides including UTP as substrates. The presence of a polyP primer is not necessary for activity. The corresponding nucleoside diphosphates and microscopically detectable polyphosphate granules were identified as reaction products. PPK2c also catalyses the formation of ATP, GTP, CTP, dTTP and UTP from the corresponding nucleoside diphosphates, if polyP is present as a phosphate donor. Remarkably, the nucleoside-tetraphosphates AT(4)P, GT(4)P, CT(4)P, dTT(4)P and UT(4)P were also detected in substantial amounts. The low nucleotide specificity of PPK2c predestines this enzyme in combination with polyP to become a powerful tool for the regeneration of ATP and other nucleotides in biotechnological applications. As an example, PPK2c and polyP were used to replace ATP and to fuel the hexokinase-catalysed phosphorylation of glucose with only catalytic amounts of ADP., Projekt DEAL, Deutsche Forschungsgemeinschaft

Polyhydroxyalkanoates (PHA), polyesters accumulated by numerous prokaryotes in the form of intracellular granules, have been for decades considered being predominantly storage molecules. However, numerous recent discoveries revealed and emphasized their complex biological role for microbial cells. Most of all, it was repeatedly reported and confirmed that the presence of PHA granules in prokaryotic cells enhances stress resistance and robustness of microbes against various environmental stress factors such as high or low temperature, freezing, oxidative, and osmotic pressure. It seems that protective mechanisms of PHA granules are associated with their extraordinary architecture and biophysical properties as well as with the complex and deeply interconnected nature of PHA metabolism. Therefore, this review aims at describing novel and unexpected properties of PHA granules with respect to their contribution to stress tolerance of various prokaryotes including common mesophilic heterotrophic bacteria, but also extremophiles or photo-autotrophic cyanobacteria. KEY POINTS: • PHA granules present in bacterial cells reveal unique properties and functions. • PHA enhances stress robustness of bacterial cells.

Polyhydroxyalkanoates (PHAs) are biopolymers synthesized by a wide range of bacteria, which serve as a promising candidate in replacing some conventional petrochemical-based plastics. PHA synthase (PhaC) is the key enzyme in the polymerization of PHA, and the crystal structures were successfully determined using the catalytic domain of PhaC from Cupriavidus necator (PhaCCn-CAT) and Chromobacterium sp. USM2 (PhaCCs-CAT). Here, we review the beneficial mutations discovered in PhaCs from a structural perspective. The structural comparison of the residues involved in beneficial mutation reveals that the residues are near to the catalytic triad, but not inside the catalytic pocket. For instance, Ala510 of PhaCCn is near catalytic His508 and may be involved in the open-close regulation, which presumably play an important role in substrate specificity and activity. In the class II PhaC1 from Pseudomonas sp. 61-3 (PhaC1Ps), Ser325 stabilizes the catalytic cysteine through hydrogen bonding. Another residue, Gln508 of PhaC1Ps is located in a conserved hydrophobic pocket which is next to the catalytic Asp and His. A class I, II-conserved Phe420 of PhaCCn is one of the residues involved in dimerization and its mutation to serine greatly reduced the lag phase. The current structural analysis shows that the Phe362 and Phe518 of PhaC from Aeromonas caviae (PhaCAc) are assisting the dimer formation and maintaining the integrity of the core beta-sheet, respectively. The structure-function relationship of PhaCs discussed in this review will serve as valuable reference for future protein engineering works to enhance the performance of PhaCs and to produce novel biopolymers.

Numerous prokaryotes accumulate polyhydroxyalkanoates (PHA) in the form of intracellular granules. The primary function of PHA is the storage of carbon and energy. Nevertheless, there are numerous reports that the presence of PHA granules in microbial cells enhances their stress resistance and fitness when exposed to various stress factors. In this work, we studied the protective mechanism of PHA granules against UV irradiation employing Cupriavidus necator as a model bacterial strain. The PHA-accumulating wild type strain showed substantially higher UV radiation resistance than the PHA non-accumulating mutant. Furthermore, the differences in UV-Vis radiation interactions with both cell types were studied using various spectroscopic approaches (turbidimetry, absorption spectroscopy, and nephelometry). Our results clearly demonstrate that intracellular PHA granules efficiently scatter UV radiation, which provides a substantial UV-protective effect for bacterial cells and, moreover, decreases the intracellular level of reactive oxygen species in UV-challenged cells. The protective properties of the PHA granules are enhanced by the fact that granules specifically bind to DNA, which in turn provides shield-like protection of DNA as the most UV-sensitive molecule. To conclude, the UV-protective action of PHA granules adds considerable value to their primary storage function, which can be beneficial in numerous environments.

Cupriavidus necator H16 is the most promising bacterium for industrial production of polyhydroxyalkanoates (PHAs) because of their remarkable ability to accumulate them in the cells. With genetic modifications, this bacterium can produce poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), which has better physical properties, as well as poly(3-hydroxybutyrate) (PHB) using plant oils and sugars as a carbon source. Considering production cost, sucrose is a very attractive raw material because it is inexpensive; however, this bacterium cannot assimilate sucrose. Here, we used the sucrose utilization (csc) genes of Escherichia coli W to generate C. necator strains that can assimilate sucrose. Especially, glucose-utilizing recombinant C. necator strains harboring the sucrose hydrolase gene (cscA) and sucrose permease gene (cscB) of E. coli W grew well on sucrose as a sole carbon source and accumulated PHB. In addition, strains introduced with a crotonyl-CoA reductase gene (ccr), ethylmalonyl-CoA decarboxylase gene (emd), and some other genetic modifications besides the csc genes and the glucose-utilizing mutations produced PHBHHx with a 3-hydroxyhexanoate (3HHx) content of maximum approximately 27 mol% from sucrose. Furthermore, when one of the PHBHHx-producing strains was cultured with sucrose solution in a fed-batch fermentation, PHBHHx with a 3HHx content of approximately 4 mol% was produced and reached 113 g/L for 65 h, which is approximately 1.5-fold higher than that produced using glucose solution.

Alkaline polygalacturonate lyase (PGL), one of the pectinolytic enzymes, has been widely used for the bioscouring of cotton fibers, biodegumming, and biopulp production. In our study, PGL from Bacillus subtilis was successfully immobilized on the surface of polyhydroxyalkanoate (PHA) nanogranules by fusing PGL to the N-terminal of PHA synthase from Ralstonia eutropha via a designed linker. The PGL-decorated PHA beads could be simply achieved by recombinant fermentation and consequent centrifugation. The fused PGL occupied 0.985% of the total weight of purified PHA granules, which was identified by mass spectrometer-based quantitative proteomics. The activity of immobilized PGL (184.67 U/mg PGL protein) was a little lower than that of the free PGL (215.93 U/mg PGL protein). The immobilization process did not affect the optimal pH and the optimal temperature of the PGL, but it did enhance the thermostability as well as the pH stability at certain conditions, which will extend the practicability of the immobilized PGL-PHA beads in the alkaline and generally harsh bioscouring process. Furthermore, the immobilized PGL still retained more than 60% of its initial activity after 8 cycles of reuse. Our study provided a novel and promising approach for cost-efficient in vivo PGL immobilization, contributing to wider commercialization of this environmental-friendly biocatalyst.

Statistical distribution of cell and poly[3-(R)-hydroxybutyrate] (PHB) granule size and number of granules per cell are investigated for PHB production in a five-stage cascade (5CSTR). Electron microscopic pictures of cells from individual cascade stages (R1–R5) were converted to binary pictures to visualize footprint areas for polyhydroxyalkanoate (PHA) and non-PHA biomass. Results for each stage were correlated to the corresponding experimentally determined kinetics (specific growth rate μ and specific productivity π). Log-normal distribution describes PHA granule size dissimilarity, whereas for R1 and R4, gamma distribution best reflects the situation. R1, devoted to balanced biomass synthesis, predominately contains cells with rather small granules, whereas with increasing residence time τ, maximum and average granule sizes by trend increase, approaching an upper limit determined by the cell’s geometry. Generally, an increase of intracellular PHA content and ratio of granule to cell area slow down along the cascade. Further, the number of granules per cell decreases with increasing τ. Data for μ and π obtained by binary picture analysis correlate well with the experimental results. The work describes long-term continuous PHA production under balanced, transient, and nutrient-deficient conditions, as well as their reflection on the granules size, granule number, and cell structure on the microscopic level. Electronic supplementary material The online version of this article (doi:10.1007/s00253-016-7844-6) contains supplementary material, which is available to authorized users.

With global CO

The present study addresses the synthesis and properties of polyhydroxyalkanoates (PHA) of different composition synthesized by Cupriavidus eutrophus B-10646 using glycerol as a carbon substrate. Poly(3-hydroxybutyrate) [P(3HB)] was effectively synthesized in fed-batch culture in a 30-L fermenter on glycerol of various purification degrees, with 99.5, 99.7, and 82.1% content of the main component. Purified glycerol (99.7%) was used for 150-L pilot scale fermentation. The total biomass and P(3HB) concentration reached 110 and 85.8 g/L, respectively, after 45 h of fed-batch fermentation. An average volumetric productivity of P(3HB) was 1.83 g/(L h). The degree of crystallinity and molecular weight of P(3HB) synthesized on glycerol were lower than and temperature characteristics were the same as those of P(3HB) synthesized on sugars.

Microbial production of solvents like acetone and butanol was a couple of the first industrial fermentation processes to gain global importance. These solvents are important feedstocks for the chemical and biofuel industry. Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H2 and CO2 under aerobic conditions. This bacterium is a natural producer of polyhydroxyalkanoate biopolymers. Recently, with the advances in the development of genetic engineering tools, the range of metabolites R. eutropha can produce has enlarged. Its ability to utilize various carbon sources renders it an interesting candidate host for synthesis of renewable biofuel and solvent production. This review focuses on progress in metabolic engineering of R. eutropha for the production of alcohols, terpenes, methyl ketones, and alka(e)nes using various resources. Biological synthesis of solvents still presents the challenge of high production costs and competition from chemical synthesis. Better understanding of R. eutropha biology will support efforts to engineer and develop superior microbial strains for solvent production. Continued research on multiple fronts is required to engineer R. eutropha for truly sustainable and economical solvent production.

Methylotrophic biosynthesis using methanol as a feedstock is a promising and attractive method to solve the over-dependence of the bioindustry on sugar feedstocks derived from grains that are used for food. In this study, we introduced and engineered the mevalonate pathway into Methylobacterium extorquens AM1 to achieve high mevalonate production from methanol, which could be a platform for terpenoid synthesis. We first constructed a natural operon (MVE) harboring the mvaS and mvaE genes from Enterococcus faecalis as well as an artificial operon (MVH) harboring the hmgcs1 gene from Blattella germanica and the tchmgr gene from Trypanosoma cruzi that encoded enzymes with the highest reported activities. We achieved mevalonate titers of 56 and 66 mg/L, respectively, in flask cultivation. Introduction of the phaA gene from Ralstonia eutropha into the operon MVH increased the mevalonate titer to 180 mg/L, 3.2-fold higher than that of the natural operon MVE. Further modification of the expression level of the phaA gene by regulating the strength of the ribosomal binding site resulted in an additional 20 % increase in mevalonate production to 215 mg/L. A fed-batch fermentation of the best-engineered strain yielded a mevalonate titer of 2.22 g/L, which was equivalent to an overall yield and productivity of 28.4 mg mevalonate/g methanol and 7.16 mg/L/h, respectively. The production of mevalonate from methanol, which is the initial, but critical step linking methanol with valuable terpenoids via methylotrophic biosynthesis, represents a proof of concept for pathway engineering in M. extorquens AM1.

Microbial polyhydroxyalkanoates (PHA) are a family of biopolyesters with properties similar to petroleum plastics such as polyethylene (PE) or polypropylene (PP). Polyhydroxybutyrate (PHB) is the most common PHA known so far. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a technology recently developed to control gene expression levels in eukaryotic and prokaryotic genomes, was employed to regulate PHB synthase activity influencing PHB synthesis. Recombinant Escherichia coli harboring an operon of three PHB synthesis genes phaCAB cloned from Ralstonia eutropha, was transformed with various single guided RNA (sgRNA with its guide sequence of 20–23 bases) able to bind to various locations of the PHB synthase PhaC, respectively. Depending on the binding location and the number of sgRNA on phaC, CRISPRi was able to control the phaC transcription and thus PhaC activity. It was found that PHB content, molecular weight, and polydispersity were approximately in direct and reverse proportion to the PhaC activity, respectively. The higher the PhaC activity, the more the intracellular PHB accumulation, yet the less the PHB molecular weights and the wider the polydispersity. This study allowed the PHB contents to be controlled in the ranges of 1.47–75.21% cell dry weights, molecular weights from 2 to 6 millions Dalton and polydispersity of 1.2 to 1.43 in 48 h shake flask studies. This result will be very important for future development of ultrahigh molecular weight PHA useful to meet high strength application requirements.

Ralstonia eutropha H16 is a well-studied bacterium with respect to biosynthesis of polyhydroxyalkanoates (PHAs), which has attracted attentions as biodegradable bio-based plastics. However, this strain shows quite poor growth on glycerol of which bulk supply has been increasing as a major by-product of biodiesel industries. This study examined enhancement of glycerol assimilation ability of R. eutropha H16 by introduction of the genes of aquaglyceroporin (glpF) and glycerol kinase (glpK) from Escherichia coli. Although introduction of glpFK Ec into the strain H16 using a multi-copy vector was not successful, a recombinant strain possessing glpFK Ec within the chromosome showed much faster growth on glycerol than H16. Further analyses clarified that weak expression of glpK Ec alone allowed to establish efficient glycerol assimilation pathway, indicating that the poor growth of H16 on glycerol was caused by insufficient kination activity to glycerol, as well as this strain had a potential ability for uptake of extracellular glycerol. The engineered strains expressing glpFK Ec or glpK Ec produced large amounts of poly[(R)-3-hydroxybutyrate] [P(3HB)] from glycerol with much higher productivity than H16. Unlike other glycerol-utilizable wild strains of R. eutropha, the H16-derived engineered strains accumulated P(3HB) with no significant decrease in molecular weights on glycerol, and the polydispersity index of the glycerol-based P(3HB) synthesized by the strains expressing glpFK Ec was lower than those by the parent strains. The present study demonstrated possibility of R. eutropha H16-based platform for production of useful compounds from inexpensive glycerol.

Spent coffee grounds (SCG), an important waste product of the coffee industry, contain approximately 15 wt% of coffee oil. The aim of this work was to investigate the utilization of oil extracted from SCG as a substrate for the production of poly(3-hydroxybutyrate) (PHB) by Cupriavidus necator H16. When compared to other waste/inexpensive oils, the utilization of coffee oil resulted in the highest biomass as well as PHB yields. Since the correlation of PHB yields and the acid value of oil indicated a positive effect of the presence of free fatty acids in oil on PHB production (correlation coefficient R 2 = 0.9058), superior properties of coffee oil can be probably attributed to the high content of free fatty acids which can be simply utilized by the bacteria culture. Employing the fed-batch mode of cultivation, the PHB yields, the PHB content in biomass, the volumetric productivity, and the Y P/S yield coefficient reached 49.4 g/l, 89.1 wt%, 1.33 g/(l h), and 0.82 g per g of oil, respectively. SCG are annually produced worldwide in extensive amounts and are disposed as solid waste. Hence, the utilization of coffee oil extracted from SCG is likely to improve significantly the economic aspects of PHB production. Moreover, since oil extraction decreased the calorific value of SCG by only about 9 % (from 19.61 to 17.86 MJ/kg), residual SCG after oil extraction can be used as fuel to at least partially cover heat and energy demands of fermentation, which should even improve the economic feasibility of the process.

Lipid and fatty acid metabolism has been well studied in model microbial organisms like Escherichia coli and Bacillus subtilis. The major precursor of fatty acid biosynthesis is also the major product of fatty acid degradation (β-oxidation), acetyl-CoA, which is a key metabolite for all organisms. Controlling carbon flux to fatty acid biosynthesis and from β-oxidation allows for the biosynthesis of natural products of biotechnological importance. Ralstonia eutropha can utilize acetyl-CoA from fatty acid metabolism to produce intracellular polyhydroxyalkanoate (PHA). R. eutropha can also be engineered to utilize fatty acid metabolism intermediates to produce different PHA precursors. Metabolism of lipids and fatty acids can be rerouted to convert carbon into other value-added compounds like biofuels. This review discusses the lipid and fatty acid metabolic pathways in R. eutropha and how they can be used to construct reagents for the biosynthesis of products of industrial importance. Specifically, how the use of lipids or fatty acids as the sole carbon source in R. eutropha cultures adds value to these biotechnological products will be discussed here.

2-Hydroxyalkanoates (2HAs) have become the new monomeric constituents of bacterial polyhydroxyalkanoates (PHAs). PHAs containing 2HA monomers, lactate (LA), glycolate (GL), and 2-hydroxybutyrate (2HB) can be synthesized by engineered microbes in which the broad substrate specificities of PHA synthase and propionyl-CoA transferase are critical factors for the incorporation of the monomers into the polymer chain. LA-based polymers, such as P[LA-co-3-hydroxybutyrate (3HB)], have the properties of pliability and stretchiness which are distinctly different from those of the rigid poly(lactic acid) (PLA) and P(3HB) homopolymers. This versatile platform is also applicable to the biosynthesis of GL- and 2HB-based polymers. In the case of the synthesis of 2HB-based polymers, the enantiospecificity of PHA synthase enabled the production of isotactic (R)-2HB-based polymers, including P[(R)-2HB], from racemic precursors of 2HB. P(2HB) is a pliable material, in contrast to PLA. Furthermore, to obtain a new 2HA-polymerizing PHA synthase, the class I PHA synthase from Ralstonia eutropha was engineered so as to achieve the first incorporation of LA units. The analysis of the polymer synthesized using this new LA-polymerizing PHA synthase unexpectedly focused a spotlight on the studies on block copolymer biosynthesis.

AtoSC two-component system (TCS) upregulates the high-molecular weight poly-(R)-3-hydroxybutyrate (PHB) biosynthesis in recombinant phaCAB + Escherichia coli strains, with the Cupriavidus necator phaCAB operon. We report here that AtoSC upregulates also the copolymer P(3HB-co-3HV) biosynthesis in phaCAB + E. coli. Acetoacetate-induced AtoSC maximized P(3HB-co-3HV) to 1.27 g/l with a 3HV fraction of 25.5 % wt. and biopolymer content of 75 % w/w in a time-dependent process. The atoSC locus deletion in the ∆atoSC strains resulted in 4.5-fold P(3HB-co-3HV) reduction, while the 3HV fraction of the copolymer was restricted to only 6.4 % wt. The ∆atoSC phenotype was restored by extrachromosomal introduction of AtoSC. Deletion of the atoDAEB operon triggered a significant decrease in P(3HB-co-3HV) synthesis and 3HV content in ∆atoDAEB strains. However, the acetoacetate-induced AtoSC in those strains increased P(3HB-co-3HV) to 0.8 g/l with 21 % 3HV, while AtoC or AtoS expression increased P(3HB-co-3HV) synthesis 3.6- or 2.4-fold, respectively, upon acetoacetate. Complementation of the ∆atoDAEB phenotype was achieved by the extrachromosomal introduction of the atoSCDAEB regulon. Individual inhibition of β-oxidation and mainly fatty acid biosynthesis pathways by acrylic acid or cerulenin, respectively, reduced P(3HB-co-3HV) biosynthesis. Under those conditions, introduction of atoSC or atoSCDAEB regulon was capable of upregulating biopolymer accumulation. Concurrent inhibition of both the fatty acid metabolic pathways eliminated P(3HB-co-3HV) production. P(3HB-co-3HV) upregulation in phaCAB + E. coli by AtoSC signaling through atoDAEB operon and its participation in the fatty acids metabolism interplay provide additional perceptions of AtoSC critical involvement in E. coli regulatory processes towards biotechnologically improved polyhydroxyalkanoates biosynthesis.

Polyhydroxybutyrate (PHB), the best known polyhydroxyalkanoates (PHA) has been believed to change intracellular metabolic flow and oxidation/reduction state, as well as enhance stress resistance of the host. In this study, a PHB biosynthesis pathway, which contains phaCAB operon genes from Ralstonia eutropha, was introduced into an L-tryptophan producing Escherichia coli strain GPT1002. The expression of the PHB biosynthesis genes resulted in PHB accumulation inside the cells and improved the L-tryptophan production. Quantitative real-time PCR analysis showed that the transcription of tryptophan operon genes in GPT2000 increased by 1.9 to 4.3 times compared with the control, indicating that PHB biosynthesis in engineered E. coli changed the physiological state of the host. Xylose was added into the medium as co-substrate to enhance the precursor supply for PHB biosynthesis. The addition of xylose improved both extracellular L-tryptophan production and intracellular PHB accumulation. Moreover, we obtained 14.4 g l(-1) L-tryptophan production and 9.7 % PHB (w/w) accumulation in GPT2000 via fed-batch cultivation.

Biotrickling filters (BTFs) are often subjected to transient-state operation due to different variations in the operation of industrial-scale sources of pollution. In this research, performance of a laboratory-scale BTF packed with pall ring and pumice (1:1 v/v) and inoculated with Ralstonia eutropha was evaluated for the treatment of toluene vapor under various transient conditions. The experiments were performed at empty bed residence times (EBRTs) of 45 and 90 s and toluene inlet concentration in the range of 0.5–4 g m−3. The transient-state experiments consisted of a sudden increase in inlet gas concentration, sudden change of trickling liquid rate, intermittent loading for 10 h day−1, aeration without toluene loading during shutdown periods, and long-term starvation. The maximum elimination capacity (ECmax) was 280 g m−3 h−1 under continuous loading. The removal efficiency (RE) reached 90 % in intermittent loading experiments at toluene inlet concentration of 3 g m−3 in less than 1 h after loading initiation. RE dropped to 50 % due to 4.5-fold increase in the inlet loading rate (ILR) during shock load experiment. The system became completely active after 24 h, when the BTF was subjected to a long-term starvation period for 7 days. The results showed that aeration at non-toluene loading periods could improve the BTF performance under intermittent loading condition.

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., United States. Dept. of Energy, United States. Advanced Research Projects Agency-Energy

Class I polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha (PhaCRe) was engineered so as to acquire an unusual lactate (LA)-polymerizing activity. To achieve this, the site-directed saturation mutagenesis of PhaCRe was conducted at position 510, which corresponds to position 481 in the initially discovered class II LA-polymerizing PHA synthase (PhaC1PsSTQK), a mutation in which (Gln481Lys) was shown to be essential to its LA-polymerizing activity (Taguchi et al., Proc Natl Acad Sci USA 105(45):17323-17327, 2008). The LA-polymerizing activity of the PhaCReA510X mutants was evaluated based on the incorporation of LA units into the P[3-hydroxybutyrate(3HB)] backbone in vivo using recombinant Escherichia coli LS5218. Among 19 PhaCRe(A510X) mutants, 15 synthesized P (LA-co-3HB), indicating that the 510 residue plays a critical role in LA polymerization. The polymer synthesized by PhaCReA510S was fractionated using gel permeation chromatography in order to remove the low molecular weight fractions. The (13)C and (1)H NMR analyses of the high molecular weight fraction revealed that the polymer was a P(7 mol% LA-co-3HB) copolymer with a weight-averaged molecular weight of 3.2 × 10(5) Da. Interestingly, the polymer contained an unexpectedly high ratio of an LA-LA -LA triad sequence, suggesting that the polymer synthesized by PhaCRe mutant may not be a random copolymer, but presumably had a block sequence.

A recent study of the UV-generated glucose-utilizing mutant Ralstonia eutropha G⁺1 comprising transcriptomic and proteomic analyses revealed clear evidence that glucose is transported by the N-acetylglucosamine-specific phosphotransferase system (PTS(Nag)), which is overexpressed in this mutant due to a derepression of the encoding nag operon by an identified insertion mutation in nagR (Raberg et al., Appl Environ Microbiol 77:2058-2070, 2011). The inability of the defined deletion mutant R. eutropha G⁺1∆nagFEC to utilize glucose confirms this finding. Furthermore, a missense mutation in nagE (membrane component comprising the cell membrane spanning EIIC(Nag) and the cytosolic domain EIIB(Nag)) was identified, which yields a substitution of an alanine by threonine at aa 153 of NagE and may affect glucose specificity of the mutated PTS(Nag) in R. eutropha G⁺1. The investigation of various generated deletion and substitution mutants of R. eutropha H16 and G⁺1 in this study was able to elucidate these phenomena. It could be shown that the porin NagC, encoded by nagC being part of the nag operon, is not necessary, while NagE is required and is probably responsible for glucose transport through the cell membrane. The intracellular phosphorylation of glucose is obviously mediated by the glucokinase GLK and not by NagF (cytosolic component comprising the three soluble domains EIIA(Nag), HPr(Nag), and EI(Nag)). Our data clearly indicate that the derepression of the nag operon is essential for glucose uptake. The point mutation in NagE is not an essential prerequisite for glucose transport although it increased glucose transport as observed in this study.

The recombinant strain of Ralstonia eutropha H16-PHB(-)4-∆eda (pBBR1MCS-2::cphA (6308)/eda (H16)) presenting a 2-keto-3-desoxy-phosphogluconate (KDPG) aldolase (eda) gene-dependent catabolic addiction system for plasmid maintenance when using gluconate or fructose as sole carbon source was used in this study. The effects of the initial pH, the nitrogen-to-carbon ratio, the inorganic components of medium, the oxygen supply, and the different carbon and nitrogen sources on the cell dry matter (CDM) and the cyanophycin granule polypeptide (CGP) content of the cells were studied in a mineral salts medium (MSM) without any additional amino acids or CGP precursor substrates. The experiments were designed to systematically find out the optimal conditions for growth of cells to high densities and for high CGP contents of the cells. Maximum contents of water-insoluble CGP and water-soluble CGP, contributing to 47.5% and 5.8% (w/w) of CDM, respectively, were obtained at the 30-L scale cultivation when cells were cultivated in MSM medium containing sufficient supplements of fructose, NH(3), K(2)SO(4), MgSO(4)[Symbol: see text]7H(2)O, Fe(Ш)NH(4)-citrate, CaCl(2)[Symbol: see text]2H(2)O, and trace elements (SL6). The molecular masses of water-insoluble and water-soluble CGP ranged from 25 to 31 kDa and from 15 to 21 kDa, respectively. High cell densities of up to 82.8 g CDM/L containing up to 37.8% (w/w) water-insoluble CGP at the 30-L scale cultivation were also obtained. This is by far the best combination of high cell density and high cellular CGP contents ever reported, and it showed that efficient production of CGP at the industrial scale in white biotechnology could be achieved.

We have previously reported in vivo biosynthesis of polylactic acid (PLA) and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] employing metabolically engineered Escherichia coli strains by the introduction of evolved Clostridium propionicum propionyl-CoA transferase (Pct(Cp)) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1(Ps6-19)). Using this in vivo PLA biosynthesis system, we presently report the biosynthesis of PHAs containing 2-hydroxybutyrate (2HB) monomer by direct fermentation of a metabolically engineered E. coli strain. The recombinant E. coli ldhA mutant XLdh strain expressing PhaC1( Ps6-19) and Pct(Cp) was developed and cultured in a chemically defined medium containing 20 g/L of glucose and varying concentrations of 2HB and 3HB. PHAs consisting of 2HB, 3HB, and a small fraction of lactate were synthesized. Their monomer compositions were dependent on the concentrations of 2HB and 3HB added to the culture medium. Even though the ldhA gene was completely deleted in the chromosome of E. coli, up to 6 mol% of lactate was found to be incorporated into the polymer depending on the culture condition. In order to synthesize PHAs containing 2HB monomer without feeding 2HB into the culture medium, a heterologous metabolic pathway for the generation of 2HB from glucose was constructed via the citramalate pathway, in which 2-ketobutyrate is synthesized directly from pyruvate and acetyl-CoA. Introduction of the Lactococcus lactis subsp. lactis Il1403 2HB dehydrogenase gene (panE) into E. coli allowed in vivo conversion of 2-ketobutyrate to 2HB. The metabolically engineered E. coli XLdh strain expressing the phaC1437, pct540, cimA3.7, and leuBCD genes together with the L. lactis Il1403 panE gene successfully produced PHAs consisting of 2HB, 3HB, and a small fraction of lactate by varying the 3HB concentration in the culture medium. As the 3HB concentration in the medium increased the 3HB monomer fraction in the polymer, the polymer content increased. When Ralstonia eutropha phaAB genes were additionally expressed in this recombinant E. coli XLdh strain, P(2HB-co-3HB-co-LA) having small amounts of 2HB and LA monomers could also be produced from glucose as a sole carbon source. The metabolic engineering strategy reported here should be useful for the production of PHAs containing 2HB monomer.

The site-specific mutagenesis for PHA synthase PhaC2Ps1317 from Pseudomonas stutzeri 1317 was conducted for optimizing production of short-chain-length and medium-chain-length polyhydroxyalkanoates (scl-mcl PHA). Recombinant Ralstonia eutropha PHB-4 harboring double mutated phaC2 Ps1317 gene (phaC2 Ps QKST) produced 42 wt.% PHA content in the cell dry weight (CDW) with 93 mol% 3-hydroxybutyrate (HB) as monomer in the PHA copolymer. Compared to that of wild-type phaC2 Ps1317 , the higher PHA content indicated the effectiveness of the specific point mutations for improvement on PhaC2Ps1317 activity and PHA production. The physical characterization revealed that the PHA produced by the recombinant strain was scl-mcl PHA copolymers with molecular weights and polydispersity reasonable for practical applications. Recombinant R. eutropha PHB-4 containing mutated phaC2 Ps1317 termed phaC2 Ps QKST was demonstrated to be able to produce scl-mcl PHA copolymers consisting of even-numbered, odd-numbered, or a combination of even- and odd-numbered monomers covering the carbon chain lengths from C4 to C12 when related substrates were provided. Recombinant R. eutropha PHB-4 containing phaC2PsQKST could be used as a strain for production of copolymers consisting of dominated HB and medium-chain-length 3-hydroxyalkanoates (HA) with better application properties.

Poly(hydroxyalkanoates) (PHAs) constitute biodegradable polyesters and are considered among the most promising candidates to replace common petrochemical plastics in various applications. To date, all commercial processes for PHA production employ microbial discontinuous fed-batch fermentations. These processes feature drawbacks such as varying product quality and the inevitable periods of downtime for preparation and post-treatment of the bioreactor equipment. An unprecedented approach to PHA production was chosen in the presented work using a multistage system consisting of five continuous stirred tank reactors in series (5-SCR), which can be considered as a process engineering substitute of a continuous tubular plug flow reactor. The first stage of the reactor cascade is the site of balanced bacterial growth; thereafter, the fermentation broth is continuously fed from the first into the subsequent reactors, where PHA accumulation takes place under nitrogen-limiting conditions. Cupriavidus necator was used as production strain. The focus of the experimental work was devoted to the development of a PHA production process characterized by high productivity and high intracellular polymer content. The results of the experimental work with the reactor cascade demonstrated its potential in terms of volumetric and specific productivity (1.85 g L⁻¹ h⁻¹ and 0.100 g g⁻¹ h⁻¹, respectively), polymer content (77%, w/w) and polymer properties (M (w) = 665 kg/mol, PDI = 2.6). Thus, implementing the technology for 5-SCR production of PHB results in an economically viable process. The study compares the outcome of the work with literature data from continuous two-stage PHA production and industrial PHA production in fed-batch mode.

Recombinant Cupriavidus necator H 16 with a novel metabolic pathway using a cobalamin-dependent mutase was exploited to produce 2-hydroxyisobutyric acid (2-HIBA) from renewable resources through microbial fermentation. 2-HIBA production capacities of different strains of C. necator H 16 deficient in the PHB synthase gene and genetically engineered to enable the production of 2-HIBA from the intracellular PHB precursor (R)-3-hydroxybutyryl-CoA were evaluated in 48 parallel milliliter-scale stirred tank bioreactors (V = 11 mL). The effects of media composition, limitations, pH, and feed rate were studied with respect to the overall process performances of the different recombinant strains. 2-HIBA production was at a maximum at nitrogen limiting conditions and if the pH was controlled between 6.8 and 7.2 under fed-batch operating conditions (intermittent fructose addition). The final concentration of 2-HIBA was 7.4 g L(-1) on a milliliter scale. Best reaction conditions identified on the milliliter scale were transferred to a laboratory-scale fed-batch process in a stirred tank bioreactor (V = 2 L). Two different process modes for the production of 2-HIBA, a single-phase and a dual-phase fermentation procedure, were evaluated and compared on a liter scale. The final concentration of 2-HIBA was 6.4 g L(-1) on a liter scale after 2 days of cultivation.

We employed systematic mixture analysis to determine optimal levels of acetate, propionate, and butyrate for cell growth and polyhydroxyalkanoate (PHA) production by Ralstonia eutropha H16. Butyrate was the preferred acid for robust cell growth and high PHA production. The 3-hydroxyvalerate content in the resulting PHA depended on the proportion of propionate initially present in the growth medium. The proportion of acetate dramatically affected the final pH of the growth medium. A model was constructed using our data that predicts the effects of these acids, individually and in combination, on cell dry weight (CDW), PHA content (%CDW), PHA production, 3HV in the polymer, and final culture pH. Cell growth and PHA production improved approximately 1.5-fold over initial conditions when the proportion of butyrate was increased. Optimization of the phosphate buffer content in medium containing higher amounts of butyrate improved cell growth and PHA production more than 4-fold. The validated organic acid mixture analysis model can be used to optimize R. eutropha culture conditions, in order to meet targets for PHA production and/or polymer HV content. By modifying the growth medium made from treated industrial waste, such as palm oil mill effluent, more PHA can be produced., Malaysia. Ministry of Science, Technology and Innovation (MOSTI)

Nine anaerobic promoters were cloned and constructed upstream of PHB synthesis genes phbCAB from Ralstonia eutropha for the micro- or anaerobic PHB production in recombinant Escherichia coli. Among the promoters, the one for alcohol dehydrogenase (PadhE) was found most effective. Recombinant E. coli JM 109 (pWCY09) harboring PadhE and phbCAB achieved a 48% PHB accumulation in the cell dry weight after 48 h of static culture compared with only 30% PHB production under its native promoter. Sixty-seven percent PHB was produced in the dry weight (CDW) of an acetate pathway deleted (Deltapta deletion) E. coli JW2294 harboring the vector pWCY09. In a batch process conducted in a 5.5-l NBS fermentor containing 3 l glucose LB medium, E. coli JW2294 (pWCY09) grew to 7.8 g/l CDW containing 64% PHB after 24 h of microaerobic incubation. In addition, molecular weight of PHB was observed to be much higher under microaerobic culture conditions. The high activity of PadhE appeared to be the reason for improved micro- or anaerobic cell growth and PHB production while high molecular weight contributed to the static culture condition.

The binding preference of a polyhydroxyalkanoate (PHA) biosynthesis-related multifunctional protein from Ralstonia eutropha (PhaMRe) was characterized. In vitro activity assay showed that PHA synthase from R. eutropha (PhaCRe) was activated by the presence of PhaMRe but PHA synthase from Aeromonas caviae (PhaCAc) was not. Additionally, in vitro assays of protein-protein interactions demonstrated that PhaMRe interacted with PhaCRe directly, but did not interact with PhaCAc. These results suggest that the protein-protein interaction is important for the activation of PhaC by PhaMRe. Further analyses indicated that PhaMRe has little or no direct interaction with the PHA polymer chain. Subsequently, PHA biosynthesis genes (phaA Re, phaB Re, and phaC Re/phaC Ac) and the phaM Re gene were introduced into recombinant Escherichia coli and cultivated for PHA accumulation. Contrary to our expectations, the expression of PhaMRe decreased PHA accumulation and changed the morphology of PHA granules to be microscopically obscure shape in PhaCRe-expressing E. coli. No change in the amount of P(3HB) or the morphology of granules by PhaMRe expression was observed in PhaCAc-expressing E. coli. These observations suggest that PhaMRe affects cellular physiology through the PhaM-PhaC interaction.

Poly(3-hydroxybutyrate) (PHB) is a common carbon- and energy-storage compound simultaneously produced and degraded into its monomer 3-hydroxybutyrate (3HB) by numerous bacteria and Archae in a metabolic pathway called the PHB cycle. We investigated 3HB as a chemical chaperone capable of protecting model enzymes, namely lipase and lysozyme, from adverse effects of high temperature and oxidation. Heat-mediated denaturation of lipase in the presence or absence of 3HB was monitored by dynamic light scattering (DLS) revealing a significant protective effect of 3HB which increased as its concentration rose. Furthermore, when compared at the same molar concentration, 3HB showed a greater protective effect than the well-known chemical chaperones trehalose and hydroxyectoine. The higher protective effect of 3HB was also confirmed when employing differential scanning calorimetry (DSC) and lysozyme as a model enzyme. Furthermore, 3HB was capable of protecting lipase not only against thermal-mediated denaturation but also against oxidative damage by Cu(2+) and H2O2; its protection was higher than that of trehalose and comparable to that of hydroxyectoine. Taking into account that the PHB-producing strain Cupriavidus necator H16 reveals a 16.5-fold higher intracellular concentration than the PHB non-producing mutant C. necator PHB(-4), it might be expected that the functional PHB cycle might be responsible for maintaining a higher intracellular level of 3HB which, aside from other positive aspects of functional PHB metabolism, enhances stress resistance of bacterial strains capable of simultaneous PHB synthesis and mobilization. In addition, 3HB can be used in various applications and formulations as an efficient enzyme-stabilizing and enzyme-protecting additive.