Ralstonia eutropha H16 is capable of growth and polyhydroxyalkanoate production on plant oils and fatty acids. However, little is known about the triacylglycerol and fatty acid degradation pathways of this bacterium. We compare whole-cell gene expression levels of R. eutropha H16 during growth and polyhydroxyalkanoate production on trioleate and fructose. Trioleate is a triacylglycerol that serves as a model for plant oils. Among the genes of note, two potential fatty acid ␤-oxidation operons and two putative lipase genes were shown to be upregulated in trioleate cultures. The genes of the glyoxylate bypass also exhibit increased expression during growth on trioleate. We observed that single ␤-oxidation operon deletion mutants of R. eutropha could grow using palm oil or crude palm kernel oil as the sole carbon source, regardless of which operon was present in the genome, but a double mutant was unable to grow under these conditions. A lipase deletion mutant did not exhibit a growth defect in emulsified oil cultures but did exhibit a phenotype in cultures containing nonemulsified oil. Mutants of the glyoxylate shunt gene for isocitrate lyase were able to grow in the presence of oils, while a malate synthase (aceB) deletion mutant grew more slowly than wild type. Gene expression under polyhydroxyalkanoate storage conditions was also examined. Many findings of this analysis confirm results from previous studies by our group and others. This work represents the first examination of global gene expression involving triacylglycerol and fatty acid catabolism genes in R. eutropha.

Improved production costs will accelerate commercialization of polyhydroxyalkanoate (PHA) polymer and PHA‐based products. Plant oils are considered favorable feedstocks, due to their high carbon content and relatively low price compared to sugars and other refined carbon feedstocks. Different PHA production strategies were compared using a recombinant strain of Ralstonia eutropha that produces high amounts of P(HB‐co‐HHx) when grown on plant oils. This R. eutropha strain was grown to high cell densities using batch, extended batch, and fed batch fermentation strategies, in which PHA accumulation was triggered by nitrogen limitation. While extended batch culture produced more biomass and PHA than batch culture, fed batch cultivation was shown to produce the highest levels of biomass and PHA. The highest titer achieved was over 139 g/L cell dry weight (CDW) of biomass with 74% of CDW as PHA containing 19mol% HHx. Our data suggest that the fermentation process is scalable with a space time yield (STY) better than 1 g PHA/L/h. The achieved biomass concentration and PHA yield are among the highest reported for the fermentation of recombinant R. eutropha strains producing P(HB‐co‐HHx). Biotechnol. Bioeng. 2012;109: 74–83. © 2011 Wiley Periodicals, Inc.

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 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. Continuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this continuous 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.

We characterized the nanLET operon in Bacteroides fragilis, whose products are required for the utilization of the sialic acid N-acetyl neuraminic acid (NANA) as a carbon and energy source. The first gene of the operon is nanL, which codes for an aldolase that cleaves NANA into N-acetyl mannosamine (manNAc) and pyruvate. The next gene, nanE, codes for a manNAc/N-acetylglucosamine (NAG) epimerase, which, intriguingly, possesses more similarity to eukaryotic renin binding proteins than to other bacterial NanE epimerase proteins. Unphosphorylated manNAc is the substrate of NanE, while ATP is a cofactor in the epimerase reaction. The third gene of the operon is nanT, which shows similarity to the major transporter facilitator superfamily and is most likely to be a NANA transporter. Deletion of any of these genes eliminates the ability of B. fragilis to grow on NANA. Although B. fragilis does not normally grow with manNAc as the sole carbon source, we isolated a B. fragilis mutant strain that can grow on this substrate, likely due to a mutation in a NAG transporter; both manNAc transport and NAG transport are affected in this strain. Deletion of the nanE epimerase gene or the rokA hexokinase gene, whose product phosphorylates NAG, in the manNAc-enabled strain abolishes growth on manNAc. Thus, B. fragilis possesses a new pathway of NANA utilization, which we show is also found in other Bacteroides species.Many bacteria have the ability to release sialic acids from complex glycoproteins and oligosaccharides present in the media or on cell surfaces at sites of colonization or infection. To use the released sialic acids as a rich source of carbon and nitrogen for growth, bacteria must have the ability to transport these compounds into the cell and convert the nine carbon sugars into intermediates that enter the central glycolytic pathways. The utilization of N-acetyl neuraminic acid (NANA), one of the sialic acids, has been well studied in Escherichia coli (36, 37), Haemophilus spp. (1, 35), and Clostridium spp. (38), to name a few.In many microorganisms, the genes for NANA utilization are arranged in an operon that may be regulated by a repressor protein, termed NanR. A comprehensive review of the organization and composition of several prokaryotic operons involved in NANA utilization has been published (36). Many of these operons share common components, including a transport gene for NANA (nanT), a gene encoding an aldolase (nanA) that splits NANA into pyruvate and N-acetyl mannosamine (manNAc), a gene encoding a kinase activity (nanK) that phosphorylates manNAc to form manNAc 6-P and, finally, an epimerase gene (nanE) whose product converts manNAc 6-P to N-acetylglucosamine 6-P (NAG 6-P). NAG 6-P then enters the common pathway of aminosugar utilization (21). For a schematic of the NANA utilization pathway in E. coli, the current paradigm of prokaryotic NANA utilization, see Fig. 7A.Bacteroides fragilis possesses a neuraminidase activity, which can liberate free NANA from complex glycoproteins and oligosaccharides. Go...

The bio-based, biodegradable family of polymers, polyhydroxyalkanoates (PHA), is an attractive candidate for an environmentally friendly replacement of petroleum-based plastics in many applications. In the past decade, many groups have examined the biodegradability and biocompatibility of PHA in cell culture systems or in an animal host. Findings suggest that PHA is a suitable material for fabrication of resorbable medical devices, such as sutures, meshes, implants, and tissue engineering scaffolds. The degradation kinetics of some PHA polymers is also suggestive of drug release applications. In this review, we examine the progress, potential applications, challenges and outlook in the medical polyhydroxyalkanoate field.

bPoly(3-hydroxybutyrate) (PHB) production and mobilization in Ralstonia eutropha are well studied, but in only a few instances has PHB production been explored in relation to other cellular processes. We examined the global gene expression of wild-type R. eutropha throughout the PHB cycle: growth on fructose, PHB production using fructose following ammonium depletion, and PHB utilization in the absence of exogenous carbon after ammonium was resupplied. Our results confirm or lend support to previously reported results regarding the expression of PHB-related genes and enzymes. Additionally, genes for many different cellular processes, such as DNA replication, cell division, and translation, are selectively repressed during PHB production. In contrast, the expression levels of genes under the control of the alternative sigma factor 54 increase sharply during PHB production and are repressed again during PHB utilization. Global gene regulation during PHB production is strongly reminiscent of the gene expression pattern observed during the stringent response in other species. Furthermore, a ppGpp synthase deletion mutant did not show an accumulation of PHB, and the chemical induction of the stringent response with DL-norvaline caused an increased accumulation of PHB in the presence of ammonium. These results indicate that the stringent response is required for PHB accumulation in R. eutropha, helping to elucidate a thus-far-unknown physiological basis for this process.

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.

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