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One of the advantages of microbial synthesis of polyhydroxyalkanoates (PHAs) is the production of diverse polymers with different properties by the addition of different monomers, such as 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx). Considering the number of possible variables, terpolymers can have more variations than copolymers. In this study, we aimed to synthesize the terpolymer P(3HB-co-3HV-co-3HHx) from volatile fatty acids such as propionate and butyrate using the recombinant Ralstonia eutropha strain (Re2133/pCB81), containing deletions in the phaB1, phaB2, and phaB3 genes, and overexpression of synthetic PHA operon (phaC2, phaA, phaJ). This strain produced terpolymers depending on the ratio of two different carbon sources, namely, propionic and butyric acids; however, wild type R. eutropha could not produce the same type of polymer. The incorporation of 3-hydroxyvalerate and 3-hydroxyhexanoate monomers was confirmed by gas chromatography and H-nuclear magnetic resonance spectroscopy, and the parameters affecting the terpolymer composition were obtained based on regression. In addition, the thermal analysis showed that this terpolymer has properties different from those of the copolymer, obtained from the same composition of volatile acids. Depending on the ratio of two volatile acids, the composition of the terpolymer can be regulated resulting in different properties.

With global CO2 emissions at their highest in several years, mitigation and possibly reduction of greenhouse gas buildup and concomitant production of renewable fuel molecules for growing transportation fuel needs are urgent challenges for renewable energy scientists and engineers. Knallgas bacteria provide a biocatalyst platform for utilization of CO2 and production of diverse and some high-energy density biofuel molecules, requisite for drop-in transportation fuels. The most well-studied Knallgas bacterium, Ralstonia eutropha, has been engineered to produce n-butanol, isobutanol, and terpene molecules under chemolithoautotrophic conditions. There are other representatives of this group of bacteria that potentially have the capabilities for CO2-based fuel molecule synthesis. In principle, fermentative production of biofuel from CO2 could rival the "power-to-gas" (non-biological production of fuels using CO2 and H2) production methods. However, challenges remain for both methods in order to compete with currently priced petroleum-based fuels. With continued streamlining of processes and attention to Industrial Ecology principles, biofuel synthesis by Knallgas bacteria could represent a viable part of a nation's energy portfolio.

As a potential feedstock for biofuel production, a high-cell-density continuous culture for the lipid production by Cryptococcus albidus was investigated in this study. The influences of dilution rates in the single-stage continuous cultures were explored first. To reach a high-cell-density culture, a single-stage continuous culture coupled with a membrane cell recycling system was carried out at a constant dilution rate of 0.36/h with varied bleeding ratios. The maximum lipid productivity of 0.69 g/L/h was achieved with the highest bleeding ratio of 0.4. To reach a better lipid yield and content, a two-stage continuous cultivation was performed by adjusting the C/N ratio in two different stages. Finally, a lipid yield of 0.32 g/g and lipid content of 56.4% were obtained. This two-stage continuous cultivation, which provided a higher lipid production performance, shows a great potential for an industrial-scale biotechnological production of microbial lipids and biofuel production.

For decades, polyhydroxyalkanoates (PHA) have been touted as a bio-based, biodegradable alternative to “traditional” chemical-based plastics. PHA are synthesized and degraded by a variety of different microorganisms, and many have been found to possess thermal and mechanical properties similar to some types of widely used synthetic plastics. However, one of the main hurdles for large-scale PHA production and subsequent use in day-to-day applications has been the price of feedstocks (raw materials) required for synthesis. Suitable carbon and energy sources must be used for optimal microbial growth and polymer production. Refined carbon sources tend to be very costly, especially in quantities that would be required for large-scale PHA syntheses. In recent years, PHA synthesis efforts have turned to waste streams as sources of carbon and energy for microbial growth and polymer synthesis. Industries, such as agriculture and food processing, produce a large amount of waste that is not fit for human consumption but can be made bioavailable for microbial activities. This chapter discusses efforts in using available industrial waste materials as inexpensive feedstocks for PHA production, the types of polymers made as a result of these efforts, and the possibility of scaling-up these efforts to make large-scale PHA production feasible.

Once cells have been used to produce biochemicals, there are only a few effective ways to utilize the residual cell mass, even though the utilization of leftover cells would aid in decreasing production costs. Here, a polyhydroxybutyrate (PHB) and isobutanol co-production system was designed to address this challenge. The addition of the PHB operon into Escherichia coli conferred a metabolic advantage for alcohol production, generating 1.14-fold more isobutanol. Furthermore, following nitrogen source optimization and cofactor engineering, the engineered E. coli strain produced 2-fold more isobutanol and 0.25 g/L PHB. Moreover, E. coli cells showed higher tolerance to isobutanol with the overexpression of PHB biosynthesis genes. This co-production system resulted in an increased biomass, higher glucose utilization, and lower acetate maintenance, leading to higher productivity regarding PHB and isobutanol yield. Thus, this novel system is applicable to future fermentation studies for the co-production of PHB and isobutanol.

Complications arising from antibiotic-resistant bacteria are becoming one of the key issues in modern medicine. Members of drug-resistant Enterobacteriaceae spp. include opportunistic pathogens (e.g., Salmonella spp.) that are among the leading causes of morbidity and mortality worldwide. Overgrowth of these bacteria is considered a hallmark of intestinal dysbiosis. Microcins (small antimicrobial peptides) produced by some gut commensals can potentially cure these conditions by inhibiting these pathogens and have been proposed as a viable alternative to antibiotic treatment. In this proof-of-concept work, we leverage this idea to develop a genetically engineered prototype probiotic to inhibit Salmonella spp. upon exposure to tetrathionate, a molecule produced in the inflamed gut during the course of Salmonella infection. We developed a plasmid-based system capable of conferring the ability to detect and utilize tetrathionate, while at the same time producing microcin H47. We transferred this plasmid-based system to Escherichia coli and demonstrated the ability of the engineered strain to inhibit growth of Salmonella in anaerobic conditions while in the presence of tetrathionate, with no detectable inhibition in the absence of tetrathionate. In direct competition assays between the engineered E. coli and Salmonella, the engineered E. coli had a considerable increase in fitness advantage in the presence of 1 mM tetrathionate as compared to the absence of tetrathionate. In this work, we have demonstrated the ability to engineer a strain of E. coli capable of using an environmental signal indicative of intestinal inflammation as an inducing molecule, resulting in production of a microcin capable of inhibiting the organism responsible for the inflammation.

The polysaccharides chitin and chitosan are made up of monomer units of the amino sugars D-glucosamine and N -acetyl-D-glucosamine. The ratio of these two monomers dictates whether the polysaccharide is considered chitin or chitosan. Both polymers have unique properties and have uses in several diverse applications. In nature, chitin and chitosan primarily play a structural role. When purified from their producing organism, these polymers exhibit useful structural, chemical and biological properties. Chitin and chitosan have been used in several applications including biomedicine, food additives, cosmetics, and more. The charged chitosan polymer is especially effective in biomedical applications, as it has been demonstrated to possess antimicrobial properties. This review explores the properties of chitin and chitosan and how these biopolymers are used in a variety of healthcare and other applications.

Polyhydroxyalkanoates (PHAs) are a family of biodegradable and biocompatible polymers produced by several species microorganisms that possess favorable mechanical properties (e.g. strength and elongation properties). Different types of PHA polymers have been used in medical applications. However, in order to better understand the use of this polymer in the different applications, a thorough understanding of the kinetics of in vivo degradation is one of the major requirements. In this study, poly(3-hydroxybutyrate) (PHB) was subcutaneously implanted in mice and incubated for 2, 4, 8, or 16 weeks. After removal from the animal, the strength, elongation, mass loss, and enthalpy of the PHB were tested for each time point. From these data, a mathematical model was generated by Rayleigh's method of dimensional analysis, where polymer strength over tissue contact time could be predicted. To prove the model, previous data obtained by our group were used: poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] incubation in the presence of human embryonic kidney cells (HEK). It was found that the developed model was aligned with experimental results, could predict the strength of the polymer when in contact with cells, and the predicted strength follows the trend of the experimental data. Also, the dimensionless constant (K) value associated with the model is different for both experiments, where this constant, produced via experimental data, is used for construction of a homogeneous equation. Copyright © 2017 John Wiley & Sons, Ltd.

Biofilms consist of groups of microorganisms that adhere to surfaces, such as wound and implant surfaces, making it difficult to prevent or remove their formation by antibiotic treatment, due to the innate resistance of the biofilm. Effective treatments of medical biofilms are limited. Polyhydroxyalkanoate (PHA) is a biodegradable and biocompatible polymer that is a suitable alternative to petroleum based polymers for use as a raw material for medical applications. In this study, membranes of the copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] containing different HHx monomer contents were used due to its porosity and flexibility, and different sheets were prepared by solvent-casting and electrospinning methods. The sheets were loaded with lysozyme in order to measure the maximum amount of protein adsorption and to examine the ability of immobilized enzyme to inhibit biofilm formation and detach previously established biofilms. Our results have shown maximum loading of 16.1 µg enzyme per 9.5 mm3 discs, and these sheets are effective for inhibiting biofilm formation. Also, lysozyme loaded, eletrospun sheets were observed to more effectively inhibit biofilm formation, as compared to solvent-cast sheets. Based on this study, P(HB-co-HHx) sheets are a suitable material for being used as a potential raw material for fabrication of wound dressings to be used in anti-biofilm treatments.

Many poultry eggs are discarded worldwide because of infection (i.e., avian flu) or presence of high levels of pesticides. The possibility of adopting egg yolk as a source material to produce polyhydroxyalkanoate (PHA) biopolymer was examined in this study. Cupriavidus necator Re2133/pCB81 was used for the production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) or poly(3HHx), a polymer that would normally require long-chain fatty acids as carbon feedstocks for the incorporation of 3HHx monomers. The optimal medium contained 5% egg yolk oil and ammonium nitrate as a nitrogen source, with a carbon/nitrogen (C/N) ratio of 20. Time course monitoring using the optimized medium was conducted for 5 days. Biomass production was 13.1 g/l, with 43.7% co-polymer content. Comparison with other studies using plant oils and the current study using egg yolk oil revealed similar polymer yields. Thus, discarded egg yolks could be a potential source of PHA.

Halomonas sp. strain SL1, a halophilic gammaproteobacterium, was isolated from samples from the Great Salt Lake in Utah. We report here the draft genome sequence of SL1, which has an estimated total sequence length of 3.6 Mb.

Studies have shown that the copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] possesses favorable mechanical properties for use in medical supplies and products (e.g., sutures, scaffolds, bone plates). One of the major under-addressed issues associated with the use of biodegradable, bio-based PHA polymers in resorbable medical products is the correlation between the mechanical properties and the in vivo material degradation over time. In this study, P(HB-co-17 mol% HHx) matrices were mechanically tested after either incubation in cultures of human embryonic kidney cells (HEK) for in vitro degradation studies for up to 4 weeks, or inserted into Danio rerio (zebrafish) tissues for in vivo degradation studies for up to 7 weeks. The mechanical properties and scanning electron microscopy (SEM) images of the degraded materials were examined and later correlated to understand the degradation phenomenon. Our results show that Young’s modulus of P(HB-co-17 mol%HHx) during in vitro studies decreased from 3.26 to 2.42 GPa within 4 weeks, and in vivo breakdown resulted in a significant decrease in Young’s modulus with a decrease from 3.26 to 0.51 GPa and a mass loss of 59 % within 7 weeks. SEM images showed the development of pores and cracks on the surface of the material over time. Plasticization and recrystallization were observed and likely play a role in the alteration of mechanical properties.

Isobutanol (IBT) has attracted much attention from researchers as a next generation drop-in biofuel. Ralstonia eutropha is a gram-negative bacterium which naturally produces polyhydroxybutyrate (PHB), and has been reported to produce IBT after metabolic engineering. Similar to other microbes, R. eutropha experiences toxicity from branched-chain alcohols and is unable to grow in the presence of IBT concentrations higher than 0.5% (v v(-1)). Such low tolerance greatly limits the ability of R. eutropha to grow and produce IBT. In order to study toxicity to the cells, IBT-tolerant strains were developed by experimental evolution, revealing that two genes, previously described as being related to IBT tolerance in Escherichia coli (acrA and acrA6), also presented mutations in R. eutropha evolved strains. The effect on the physiology of the cells of in-frame deletions of each of these genes was assessed in wild type and engineered IBT-producing strains in an attempt to reproduce a tolerant phenotype. The mutant strains' ability to tolerate, consume, and produce IBT were also analyzed. Although deletions of acrA6 and acrA did not significantly improve R. eutropha growth in the presence of IBT, these deletions improved cell survival in the presence of high concentrations of IBT in the extracellular milieu. Moreover, an in-frame acrA deletion in an engineered IBT-producing R. eutropha enhanced the strain's ability to produce IBT, which could potentially be associated with enhanced survival at high IBT concentrations.

Mixtures of crude palm kernel oil (CPKO) and oil palm tree trunk sap (OPTS) as carbon sources for the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)) by the recombinant Cupriavidus necator strain Re2058/pCB113 were investigated. Shake flask cultures using CPKO as the sole carbon source produced 5.82 g/L of cell dry weight (CDW) with 73 wt% of PHA content and 44 mol% of 3-hydroxyhexanoate (3HHx). When the total CPKO concentration in the cultures was increased from 2.5 to 7.5 g/L, a general pattern of decreasing 3HHx molar fraction (from 42 to 27 mol%) was observed in the resulting copolymer. In order to produce biopolymer with properties that resemble commodity plastics, the 3HHx molar fraction should be lowered to an optimal level of ∼12–20 mol%. The lowest 3HHx molar fraction (14 mol%) was obtained when a mixture of 4% (w/v) OPTS sugars and 1.5 g/L CPKO was used as the carbon source. The highest molar fraction of 3HHx produced (42 mol%) was observed when the cells were cultivated in 5 g/L of CPKO. 1H-NMR and 13C-NMR analyses confirmed the structure and monomer fraction of the different P(3HB-co-3HHx) polymers. This study has established a method to control the monomer molar fractions in P(3HB-co-3HHx) produced using a mixture of CPKO and sugars from OPTS.

Triacylglycerols (TAGs) can be produced via bacterial fermentation by the oleaginous Gram-positive microorganism Rhodococcus opacus strain PD630 in regulated, nutrient-deprived conditions with sufficient carbon supply. Microbially produced TAGs may be further refined via transesterification into biodiesel and glycerol, with 3 mole of biodiesel and 1 mole of glycerol produced from every 1 mole of TAG by chemical conversion. Large-scale industrial production of biodiesel has been conducted for over a decade, yet microbially derived biodiesel has been, up to this point, absent from the biodiesel market. The use of a novel feedstock, chitin, from New England fishery waste may present a viable, cost-effective, unexplored carbon feedstock source for local biodiesel development. Availability and implementation of chitin as a feedstock, along with analysis of potential fuel characteristics, yield promising results for future industrial development of biodiesel production from R. opacus PD630 TAGs in regional locations with large lobster, shrimp, and crab harvesting operations around the world. With declining resources of fossil fuels and increased societal awareness of carbon emissions and climate change, an analytical review of this nature is critically relevant.

Wound healing is a complex and dynamic process that requires suitable environment to promote the healing process. Biomaterials have been used to augment and treat tissue wounds and injuries. In this regard, chitin-based materials are receiving increased attention due to their unique and appealing biological properties. Chitin membranes prepared by dissolution of the polymer in ionic liquid (IL) and subsequent freeze-drying were previously shown to be flexible, highly porous, and stable. In this work, these membranes were used as support substrates for growth of two mammalian cell types: NIH 3T3 and HEK293T. The membranes did not exhibit any obvious cytotoxicity and supported attachment and proliferation of these cells. Chitin membranes were also used as a matrix for loading and prolonged in vitro release of the antibiotic bacitracin as well as quorum-sensing (QS) inhibitory compounds. These results suggest that the chitin membranes are promising candidates for application in next-generation wound treatment systems and support the concept of using wound dressing materials for tissue engineering and drug delivery purposes, especially for patients suffering from chronic wounds.

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.

The application of green chemistry principles for the processing of biopolymers is a steadily increasing field of research. Chitin membranes were successfully prepared by using the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) as solvent media. The resulting materials were thoroughly characterized, revealing that freeze drying produced membranes that were highly porous. The drying methods and the concentration of chitin used defined many of the membrane properties, such as mechanical strength, porosity, and water absorbency. From these data, an empirical model was generated which could be used to correlate the different membrane properties. The model could be used to predict the properties of the chitin membrane made with different wt% of chitin-IL solutions, and the predicted values aligned with the experimental results. This allowed for prediction of the properties of the chitin membrane (e.g., tensile strength) and gives the ability to tune the properties of the biomaterial. The methods and structures described here provide a starting point for the design and fabrication of a family of polysaccharide-based sustainable materials with potentially broad applicability.

Biofuels come in a variety of forms: from wood, the classic biofuel, to liquid fuels like ethanol, higher alcohols, and the energy-dense biodiesel. Given the recent uncertainty of fuel prices and the certainty of global climate change, there has been much focus on fuels from renewable sources. Researchers and corporations are looking toward a fermentative route to produce fuels as well as chemicals that had been traditionally produced using petroleum feedstocks. The key challenge for effective biofuel production and distribution is controlling the cost of production. We as researchers can begin to address this by utilizing waste carbon as the feedstock for fermentative fuel production. Many different feedstocks, including lignocellulose and carbon dioxide, are being considered for production of alcohol fuels or lipids that would be used for production of biodiesel. This chapter is a survey of the most common liquid biofuels that are being produced by fermentative means using robust biocatalysts and renewable feedstocks.

For several thousand years, mankind has used microbial processes for food production. The microorganisms involved in these mixed cultivations may be active simultaneously or a typical sequence of prevalent microorganisms may develop dependent upon the conditions formed by the microorganisms. In several cases, the types of participating microorganisms are not even known completely. Nevertheless, traditionally fermented foods often exhibit good digestibility, pleasant sensorial properties, and widespread acceptance by the consumers. For these reasons, there is a good chance for the food industry to bring new, high value products to market, but traditional production is strongly affected by varying compositions of microorganisms on the substrate, microorganisms in vicinity of the production site, or even changing atmospheric conditions. Examples of the simultaneous utilization of different microorganisms in industrial processes and the development of controlled and reproducible coculture fermentation processes enabling the combination of traditional fermented food with industrial production processes are presented.

A variety of biologically derived polymers (termed “bio-based polymers” or “biopolymers”) have been examined for replacement of petrochemically derived polymers in many applications. In general, these polymers have either a structural or a nutritional role in the organisms that synthesize them. As biopolymers for our use, they have been removed from their biocatalyst organism and purified to assess their molecular properties. Biopolymers have a variety of structural characteristics that make them useful for different applications. In many cases, such as medical applications, biocompatibility is required of polymers; however, mechanical strength, flexibility, toughness, structural stability, and biodegradability may also be required. In this chapter, animal-based polymers (collagen, silk), plant-based polymers (cellulose, starch), and microbe-derived polymers (polyhydroxyalkanoate, cellulose) are examined in terms of their primary chemical structure, functions, and application as bio-based alternatives to petroleum-based polymers.

Aim This study aims to increase the 3-hydroxyvalerate (3HV) fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HB-co-HV)] using succinyl-CoA synthase. Methods and Results Escherichia coli YH090, a polyhydroxyalkonate (PHA)-producing strain, was further engineered for overexpression of succinyl-CoA synthase genes (sucCD), and examined for P(HB-co-HV) copolymer production in the presence of various precursor molecules using mixture analysis. Glycerol, succinate and propionate were screened as important factors for controlling intracellular PHA accumulation and monomer composition. Glycerol concentrations exerted the greatest influence on the overall biomass concentration and the intracellular PHA content, while propionate concentrations in the presence of succinate influenced the 3HV content of the copolymer. Mixture analysis also demonstrated that the engineered strain has the capacity to accumulate up to 80% of its cell dry weight (CDW) as PHA with a variable fraction of 3HV monomer (maximum of 72 wt %) depending on the controlled conditions. Conclusions Propionate is the principal precursor for 3HV monomer in P(HB-co-HV) biopolymer and its utilization requires conversion to propionyl-CoA. Engineered E. coli YHY99, overexpressing sucCD genes, leads to an increase of the succinyl-CoA pool, which enhances the conversion rate of propionate by providing a CoA supply to other acyltransferase enzymes that have a role in propionate utilization. Significance and Impact of the Study Engineered E. coli YHY99 was able to utilize propionate with a 4·5-fold increase in rate, as compared to the control strain, and resulted in the synthesis of a copolymer with high 3HV monomer content.

The utilization of inexpensive carbon sources, including waste streams, for production of value added products has been explored for over two decades. Palm oil mill effluent (POME), a waste water stream from the palm oil industry, can be used to produce organic acids and their salts by anaerobic digestion. These organic acids are suitable as the main carbon source for growth and production of polyhydroxyalkanoates (PHAs) by organisms like Ralstonia eutropha. The presence of propionate/propionic acid results in the biosynthesis of the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HB-co-HV)], which is desired for its favourable thermal and mechanical properties compared to the polyhydroxybutyrate (PHB) homopolymer. A specially-designed fed-batch strategy is necessary to decrease the toxic effects of organic acids in cultures of R. eutropha. A pH-stat fed-batch culture technique, using organic acids as the main carbon source, was adopted in combination with an additional pO2-dependent feed for delivering organic acid salts. The developed strategy is highly reproducible and shows a high productivity of >2 g PHA/L/h. After 44 h of fermentation, 112.4 ± 2.3 g/L cell dry weight (CDW) with 83.3 ± 1.1% P(HB-co-HV)/CDW containing 5.6 ± 0.4 mol% 3-hydroxyvalerate (3HV) were achieved. The described productivity, CDW and P(HB-co-HV)/CDW values are by far the highest reported results for R. eutropha cultivated on mixed organic acids as the main carbon source.

In this study, we constructed a set of Ralstonia eutropha H16 strains with single, double, or triple deletions of the (p)ppGpp synthase/hydrolase ( spoT1 ), (p)ppGpp synthase ( spoT2 ), and/or polyhydroxybutyrate (PHB) depolymerase ( phaZa1 or phaZa3 ) gene, and we determined the impact on the levels of (p)ppGpp and on accumulated PHB. Mutants with deletions of both the spoT1 and spoT2 genes were unable to synthesize detectable amounts of (p)ppGpp and accumulated only minor amounts of PHB, due to PhaZa1-mediated depolymerization of PHB. In contrast, unusually high levels of PHB were found in strains in which the (p)ppGpp concentration was increased by the overexpression of (p)ppGpp synthase (SpoT2) and the absence of (p)ppGpp hydrolase. Determination of (p)ppGpp levels in wild-type R. eutropha under different growth conditions and induction of the stringent response by amino acid analogs showed that the concentrations of (p)ppGpp during the growth phase determine the amount of PHB remaining in later growth phases by influencing the efficiency of the PHB mobilization system in stationary growth. The data reported for a previously constructed Δ spoT2 strain (C. J. Brigham, D. R. Speth, C. Rha, and A. J. Sinskey, Appl Environ Microbiol 78:8033–8044, 2012, https://doi.org/10.1128/AEM.01693-12 ) were identified as due to an experimental error in strain construction, and our results are in contrast to the previous indication that the spoT2 gene product is essential for PHB accumulation in R. eutropha . IMPORTANCE Polyhydroxybutyrate (PHB) is an important intracellular carbon and energy storage compound in many prokaryotes and helps cells survive periods of starvation and other stress conditions. Research activities in several laboratories over the past 3 decades have shown that both PHB synthase and PHB depolymerase are constitutively expressed in most PHB-accumulating bacteria, such as Ralstonia eutropha . This implies that PHB synthase and depolymerase activities must be well regulated in order to avoid a futile cycle of simultaneous PHB synthesis and PHB degradation (mobilization). Previous reports suggested that the stringent response in Rhizobium etli and R. eutropha is involved in the regulation of PHB metabolism. However, the levels of (p)ppGpp and the influence of those levels on PHB accumulation and PHB mobilization have not yet been determined for any PHB-accumulating species. In this study, we optimized a (p)ppGpp extraction procedure and a high-performance liquid chromatography–mass spectrometry (HPLC-MS)-based detection method for the quantification of (p)ppGpp in R. eutropha . This enabled us to study the relationship between the concentrations of (p)ppGpp and the accumulated levels of PHB in the wild type and in several constructed mutant strains. We show that overproduction of the alarmone (p)ppGpp correlated with reduced growth and massive overproduction of PHB. In contrast, in the absence of (p)ppGpp, mobilization of PHB was dramatically enhanced.

Volatile fatty acids (VFAs) that can be derived from food wastes were used for microbial lipid production by Chlorella protothecoides in heterotrophic cultures. The usage of VFAs as carbon sources for lipid accumulation was investigated in batch cultures. Culture medium, culture temperature, and nitrogen sources were explored for lipid production in the heterotrophic cultivation. The concentration and the ratio of VFAs exhibited significant influence on cell growth and lipid accumulation. The highest lipid yield coefficient and lipid content of C. protothecoides grown on VFAs were 0.187 g/g and 48.7%, respectively. The lipid content and fatty acids produced using VFAs as carbon sources were similar to those seen on growth and production using glucose. The techno-economic analysis indicates that the biodiesel derived from the lipids produced by heterotrophic C. protothecoides with VFAs as carbon sources is very promising and competitive with other biofuels and fossil fuels.

Microbial fermentation for bio-based products is quickly becoming an integral component of the world infrastructure, as the processes encompassing the synthesis of these natural products becomes more efficient and cost effective to compete with existing commodities. Bioethanol is currently one of the most desired fermentation products, as this constituent can be applied to multiple uses in not only contributing to the more traditional routes of beer brewing and winemaking, but also in the foundation for green fuel sources. By optimizing yields, the innovative processes could be applied towards engineering more rapid and productive biomanufacturing. In order to achieve these goals, we as researchers must understand the underlying principles and intricate networks that play a role within the microenvironment and also on the cellular level in key fermentative microbes such as Saccharomyces cerevisiae and Zymomonas mobilis . In-depth pathway analysis could lead to the development of more favorable metabolic outcomes. This review focuses on the key metabolic networks and cellular frameworks in these model organisms, and how biosynthesis of ethanol yields can be optimized throughout the fermentation 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.

Branched-chain alcohols are considered promising green energy sources due to their compatibility with existing infrastructure and their high energy density. We utilized a strain of Ralstonia eutropha capable of producing branched-chain alcohols and examined its production in flask cultures. In order to increase isobutanol and 3-methyl-1-butanol (isoamyl alcohol) productivity in the engineered strain, batch, fed-batch, and two-stage fed-batch cultures were carried out in this work. The effects of nitrogen source concentration on branched-chain alcohol production were investigated under four different initial concentrations in fermenters. A maximum 380 g m−3 of branched-chain alcohol production was observed with 2 kg m−3 initial NH4Cl concentration in batch cultures. A pH-stat control strategy was utilized to investigate the optimum carbon source amount fed during fed-batch cultures for higher cell density. In cultures of R. eutropha strains that did not produce polyhydroxyalkanoate or branched-chain alcohols, a maximum cell dry weight of 36 kg m−3 was observed using a fed-batch strategy, when 10 kg m−3 carbon source was fed into culture medium. Finally, a total branched-chain alcohol titer of 790 g m−3, the highest branched-chain alcohol yield of 0.03 g g−1, and the maximum branched-chain alcohol productivity of 8.23 g m−3 h−1 were obtained from the engineered strain Re2410/pJL26 in a two-stage fed-batch culture system with pH-stat control. Isobutanol made up over 95% (mass fraction) of the total branched-chain alcohols titer produced in this study.

Polyhydroxyalkanoate (PHA) is a family of biodegradable polymers, and incorporation of different monomers can alter its physical properties. To produce the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) containing a high level of 3-hydroxyvalerate (3HV) by altering acetyl-CoA pool levels, we overexpressed an acetyl-CoA acetyltransferase (atoAD) in an engineered E. coli strain, YH090, carrying PHA synthetic genes bktB, phaB, and phaC. It was found that, with introduction of atoAD and with propionate as a co-substrate, 3HV fraction in PHA was increased up to 7.3-fold higher than a strain without atoAD expressed in trans (67.9 mol%). By the analysis of CoA pool concentrations in vivo and in vitro using HPLC and LC-MS, overexpression of AtoAD was shown to decrease the amount of acetyl-CoA and increase the propionyl-CoA/acetyl-CoA ratio, ultimately resulting in an increased 3HV fraction in PHA. Finally, synthesis of P(3HB-co-3HV) containing 57.9 mol% of 3HV was achieved by fed-batch fermentation of YJ101 with propionate.

Ralstonia eutropha is capable of utilizing a plethora of simple and complex carbon-containing compounds. Growth has been demonstrated on carbon dioxide, glycerol, acetate, mixed organic acids, sugars, fatty acids, oils, and more. R. eutropha possesses oxygen-tolerant hydrogenases, which allow the bacterium to grow in aerobic lithoautotrophic conditions. When experiencing nutrient stress in the presence of excess carbon, R. eutropha can store carbon and energy in the form of polyhydroxyalkanoates (PHAs), a biodegradable and biocompatible plastic. Engineered R. eutropha strains are capable of producing PHAs with different chain lengths, resulting in different physical, biological, and chemical properties. These tailored PHAs, when depolymerized, can also serve as valuable chiral precursors for antibiotics, vitamins, perfumes, and pheromones. The native PHA carbon storage system can be genetically disabled and the carbon flux redirected to produce value-added chemicals such as biofuels and pharmaceuticals. Various strains of R. eutropha are also capable of bioremediation by breaking down phenol compounds and scavenging heavy metals in contaminated soils and water. Metabolic versatility and genetic tractability combined with its ability to convert a variety of carbon sources into storage molecules make R. eutropha an excellent platform organism for the production of value-added compounds.

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

Aims: Of the biodegradable polyhydroxyalkanoates (PHAs), poly (hydroxybutyrate-co-hydroxyvalerate) (P(HB-co-HV)) is often considered for fabrication of biocompatible and absorbable medical devices and other applications. Depending on the application, however, specific mechanical or processing properties must be improved. To address these required properties, we sought to alter the monomer composition of the copolymer by a combination genetic engineering in an Escherichia coli host and carbon substrate feeding. Methods and Results: We applied a new method of 3-hydroxyvalerate (3HV) monomer synthesis to produce a co-polymer by the introduction of a propionyl-CoA transferase gene (pct), along with PHA biosynthetic genes bktB, phaB and phaC from Ralstonia eutropha into engineered E. coli to produce P (HB-co-HV). The resulting strain successfully produced the copolymer containing an ultra-high 3HV monomer composition (over 80 wt%). Conclusions: To the best of our knowledge, the P(HB-co-HV) production strain constructed here synthesized polymer with the highest 3HV content of any engineered E. coli strain. This strain could also produce P(HB-co-HV) with the use of lower concentrations of propionate in the growth medium, compared to other reported strains, which could avoid the known growth inhibition from propionate in E. coli. Significance and Impact of the Study: Polyhydroxyalkanoates have been emphasized as a potential alternative for petroleum-based plastics by virtue of their physical properties and environmentally friendly characteristics. The copolymer produced in this work validates our genetic engineering approach and suggests that the Pct enzyme is a more efficient method for production of propionyl-CoA, the 3-hydroxyvaleryl-CoA precursor.

Class I polyhydroxybutyrate (PHB) synthase (PhaC) from Ralstonia eutropha catalyzes the formation of PHB from (R)-3-hydroxybutyryl-CoA, ultimately resulting in the formation of insoluble granules. Previous mechanistic studies of R. eutropha PhaC, purified from Escherichia coli (PhaC(Ec)), demonstrated that the polymer elongation rate is much faster than the initiation rate. In an effort to identify a factor(s) from the native organism that might prime the synthase and increase the rate of polymer initiation, an N-terminally Strep2-tagged phaC (Strep2-PhaC(Re)) was constructed and integrated into the R. eutropha genome in place of wild-type phaC. Strep2-PhaC(Re) was expressed and purified by affinity chromatography from R. eutropha grown in nutrient-rich TSB medium for 4 h (peak production PHB, 15% cell dry weight) and 24 h (PHB, 2% cell dry weight). Analysis of the purified PhaC by size exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel permeation chromatography revealed that it unexpectedly copurified with the phasin protein, PhaP1, and with soluble PHB (M(w) = 350 kDa) in a "high-molecular weight" (HMW) complex and in monomeric/dimeric (M/D) forms with no associated PhaP1 or PHB. Assays for monitoring the formation of PHB in the HMW complex showed no lag phase in CoA release, in contrast to M/D forms of PhaC(Re) (and PhaC(Ec)), suggesting that PhaC in the HMW fraction has been isolated in a PHB-primed form. The presence of primed and nonprimed PhaC suggests that the elongation rate for PHB formation is also faster than the initiation rate in vivo. A modified micelle model for granule genesis is proposed to accommodate the reported observations.

The first biosynthetic system for lactate (LA)-based polyesters was previously created in recombinant Escherichia coli (Taguchi et al. 2008). Here, we have begun efforts to upgrade the prototype polymer production system to a practical stage by using metabolically engineered Gram-positive bacterium Corynebacterium glutamicum as an endotoxin-free platform. We designed metabolic pathways in C. glutamicum to generate monomer substrates, lactyl-CoA (LA-CoA), and 3-hydroxybutyryl-CoA (3HB-CoA), for the copolymerization catalyzed by the LA-polymerizing enzyme (LPE). LA-CoA was synthesized by D: -lactate dehydrogenase and propionyl-CoA transferase, while 3HB-CoA was supplied by β-ketothiolase (PhaA) and NADPH-dependent acetoacetyl-CoA reductase (PhaB). The functional expression of these enzymes led to a production of P(LA-co-3HB) with high LA fractions (96.8 mol%). The omission of PhaA and PhaB from this pathway led to a further increase in LA fraction up to 99.3 mol%. The newly engineered C. glutamicum potentially serves as a food-grade and biomedically applicable platform for the production of poly(lactic acid)-like polyester.

The copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)], is produced by engineered Ralstonia eutropha strains using lipids and fatty acids as carbon feedstocks. The presence of the 3-hydroxyhexanoate (HHx) monomer disrupts the packing of polymer chains, resulting in less numerous polymer crystals and producing a polymer with favorable thermal and mechanical properties. In this study, copolymers varying in HHx content were recovered from different R. eutropha strains. Tensile constitutive response of solvent-cast samples of PHB and P(HB-co-17 mol%HHx), P(HB-co-23 mol%HHx), and P(HB-co-30 mol%HHx) was investigated using a custom-built micro-tensile tester. Our results show that presence and quantity of HHx monomer has a noticeable impact on P(HB-co-HHx) mechanical properties, such as tensile strength, elongation at break, toughness and thermal properties. It was found that with higher content of HHx in the material, the more flexible and tougher the polymer is, and the greater the reduction of Tm and crystallinity.

Ralstonia sp. strain MD27, a novel biopolymer-degrading betaproteobacterium, was isolated from compost samples. This organism has been shown to utilize the biopolymer poly(3-hydroxybutyrate) [P(3HB)] as a carbon source for growth. We report the draft genome sequence of MD27 with an estimated total sequence length of 5.9 Mb.

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polyesters considered as alternatives to petroleum-based plastics. Ralstonia eutropha is a model organism for PHA production. Utilizing industrially rendered waste animal fats as inexpensive carbon feedstocks for PHA production is demonstrated here. An emulsification strategy, without any mechanical or chemical pre-treatment, was developed to increase the bioavailability of solid, poorly-consumable fats. Wild type R. eutropha strain H16 produced 79-82% (w/w) polyhydroxybutyrate (PHB) per cell dry weight (CDW) when cultivated on various fats. A productivity of 0.3g PHB/(L × h) with a total PHB production of 24 g/L was achieved using tallow as carbon source. Using a recombinant strain of R. eutropha that produces poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)], 49-72% (w/w) of PHA per CDW with a HHx content of 16-27 mol% were produced in shaking flask experiments. The recombinant strain was grown on waste animal fat of the lowest quality available at lab fermenter scale, resulting in 45 g/L CDW with 60% (w/w) PHA per CDW and a productivity of 0.4 g PHA/(L × h). The final HHx content of the polymer was 19 mol%. The use of low quality waste animal fats as an inexpensive carbon feedstock exhibits a high potential to accelerate the commercialization of PHAs.

Conventional solvent-based methods are still the most practical approaches for recovery of polyhydroxyalkanoate (PHA) polymer from cellular biomass, even though potential alternatives exist, including chemical, mechanical, and enzymatic methods. It is still necessary, however, to avoid dangerous and environmentally unfriendly solvents (e.g., chloroform and dichloromethane) in the polymer recovery process. In the work presented here, we applied various solvent systems to recover PHA from Ralstonia eutropha and recombinant Escherichia coli cells. It was demonstrated that methyl ethyl ketone (MEK) is a promising solvent for PHA recovery from bacterial cells, particularly for the copolymer poly(hydroxybutyrate-cohydroxyvalerate) [P(HB-co-HV)], exhibiting > 90% polymer recovery. Even though MEK did not solubilize PHAs to the same extent as chloroform, it can recover a comparable amount of polymer because of its processing advantages, such as the low viscosity of the MEK/PHA solution, and the lower density of MEK as compared to cellular components. MEK was found to be the best alternative, non-halogenated solvent among examined candidates for recovery of P(HB-co-HV) from cells. The MEK treatment of PHAcontaining cells further allowed us to eliminate several costly and lengthy steps in the extraction process, such as cell lysis, centrifugation, and filtration., Korea (South). Ministry of Education (Basic Science Research Program through the National Research Foundation of Korea (NRF- 2013R1A1A2A10004690)), Korea Polar Research Institute (PE14030)

Pseudomonas fluorescens SF4c and SF39a, strains isolated from wheat rhizosphere, have potential applications in plant growth promotion and biocontrol of fungal diseases of crop plants. We report the draft genome sequences of SF4c and SF39a with estimated sizes of 6.5 Mb and 5.9 Mb, respectively.

Every year, the amount of chemosynthetic plastic accumulating in the environment is increasing, and significant time is required for decomposition. Bio-based, biodegradable plastic is a promising alternative, but its production is not yet a cost effective process. Decreasing the production cost of polyhydroxyalkanoate by utilizing renewable carbon sources for biosynthesis is an important aspect of commercializing this biodegradable polymer. An Escherichia coli strain that expresses a functional amylase and accumulate polyhydroxybutyrate (PHB), was constructed using different plasmids containing the amylase gene of Panibacillus sp. and PHB synthesis genes from Ralstonia eutropha. This engineered strain can utilize starch as the sole carbon source. The maximum PHB production (1.24 g/L) was obtained with 2% (w/v) starch in M9 media containing 0.15% (w/v) yeast extract and 10 mM glycine betaine. The engineered E. coli SKB99 strain can accumulate intracellular PHB up to 57.4% of cell dry mass.

Bacteroides fragilis , a human gastrointestinal commensal and an opportunistic pathogen, utilizes simple and complex sugars and polysaccharides for growth in the large intestine and at sites of infection. Because B. fragilis lacks transport-linked sugar phosphorylation systems, cytoplasmic kinase(s) was expected to be required for the phosphorylation of hexoses and hexosamines. We have now identified two hexose kinases that are important for growth of B. fragilis on glucose, mannose, and other sugars. One kinase (RokA), a member of the ROK family of proteins, was found to be the sole kinase for activation of N -acetyl- d -glucosamine (NAG). The other kinase (HexA) is responsible for the majority of the glucose kinase activity in the cell, although a hexA deletion mutant strain was not defective for growth on any substrate tested. Deletion of both the rokA and hexA kinase genes resulted in inability of the cell to use glucose, mannose, NAG, and many other sugars. We purified RokA and determined its approximate molecular mass to be 36.5 kDa. The purified RokA protein was shown to phosphorylate several substrates, including glucose, NAG, and mannose, but not N -acetylmannosamine or N -acetylneuraminic acid. Phylogenetic analysis of RokA showed that it is most similar to kinases from the Cytophaga-Flavibacterium-Bacteroides group, while HexA was most similar to other bacterial hexokinases and eukaryotic hexokinases.

BackgroundRalstonia eutropha is bacterium known to naturally pro-duce polyhydroxybutyrate (PHB) as carbon storage duringnutrient starvation. Previously studies [1] showed that it ispossible through the incorporation of an engineered bio-synthetic pathway, to redirect carbon flux from PHB tothe production of Isobutanol (IBT), a biofuel largelystudied to replace the current fossil fuels in existing auto-mobile engines. However R. eutropha,isunabletogrowinthe presence of IBT at concentrations above 0.2% (v v

2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD). In this study, enzymatic activities and kinetic parameters were determined for each of the three R. eutropha enzymes as heterologously purified proteins. AHAS, which serves as a gatekeeper for the biosynthesis of all three branched-chain amino acids, demonstrated the tightest regulation through feedback inhibition by l-valine (IC[subscript 50] = 1.2 mM), l-isoleucine (IC[subscript 50] = 2.3 mM), and l-leucine (IC[subscript 50] = 5.4 mM). Intermediates in the valine biosynthesis pathway also exhibit feedback inhibitory control of the AHAS enzyme. In addition, AHAS has a very weak affinity for pyruvate (K[subscript M] = 10.5 μM) and is highly selective towards 2-ketobutyrate (R = 140) as a second substrate. AHAIR and DHAD are also inhibited by the branched-chain amino acids, although to a lesser extent when compared to AHAS. Experimental evolution and rational site-directed mutagenesis revealed mutants of the regulatory subunit of AHAS (IlvH) (N11S, T34I, A36V, T104S, N11F, G14E, and N29H), which, when reconstituted with wild-type IlvB, lead to AHAS having reduced valine, leucine, and isoleucine sensitivity. The study of the kinetics and inhibition mechanisms of R. eutropha AHAS, AHAIR, and DHAD has shed light on interactions between these enzymes and the products they produce; it, therefore, can be used to engineer R. eutropha strains with optimal production of 2-ketoisovalerate for value-added materials.

Carbonic anhydrase (CA) enzymes catalyze the interconversion of CO2 and bicarbonate. These enzymes play important roles in cellular metabolism, CO2 transport, ion transport, and internal pH regulation. Understanding the metabolic role of CAs in the chemolithoautotropic bacterium Ralstonia eutropha is important for the development of high performance fermentation processes based on the bacterium’s capability to fix carbon using the Calvin-Benson-Bassham (CBB) cycle. Analysis of the R. eutropha H16 genome sequence revealed the presence of four CA genes: can, can2, caa and cag. We evaluated the importance of each of the CAs in the metabolism of R. eutropha by examination of growth and enzyme activity in gene deletion, complementation, and overexpression strains. All four purified CAs were capable of performing the interconversion of CO2 and HCO3–, although the equilibrium towards the formation of CO2 or HCO3– differs with each CA. Deletion of can, encoding a β-CA, affected the growth of R. eutropha; however the growth defect could be compensated by adding CO2 to the culture. Deletion of the caa, encoding an α-CA, had the strongest deleterious influence on cell growth. Strains with deletion or overexpression of can2 or cag genes exhibited similar behavior to wild type under most of the conditions tested. In this work, Caa was studied in greater detail using microscopy and complementation experiments, which helped confirm its periplasmic localization and determine its importance for robust growth of R. eutropha. A hypothesis for the coordinated role of these four enzymes in the metabolism of R. eutropha is proposed., United States. Defense Advanced Research Projects Agency, United States. Defense Advanced Research Projects Agency (—Energy (ARPA-E))