Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels

The idea of renewable and regenerative resources has inspired research for more than a hundred years. Ideally, the only spent energy will replenish itself, like plant material, sunlight, thermal energy or wind. Biodiesel or ethanol are examples, since their production relies mainly on plant material. However, it has become apparent that crop derived biofuels will not be sufficient to satisfy future energy demands. Thus, especially in the last decade a lot of research has focused on the production of next generation biofuels. A major subject of these investigations has been the microbial fatty acid biosynthesis with the aim to produce fatty acids or derivatives for substitution of diesel. As an industrially important organism and with the best studied microbial fatty acid biosynthesis, Escherichia coli has been chosen as producer in many of these studies and several reviews have been published in the fields of E. coli fatty acid biosynthesis or biofuels. However, most reviews discuss only one of these topics in detail, despite the fact, that a profound understanding of the involved enzymes and their regulation is necessary for efficient genetic engineering of the entire pathway. The first part of this review aims at summarizing the knowledge about fatty acid biosynthesis of E. coli and its regulation, and it provides the connection towards the production of fatty acids and related biofuels. The second part gives an overview about the achievements by genetic engineering of the fatty acid biosynthesis towards the production of next generation biofuels. Finally, the actual importance and potential of fatty acid-based biofuels will be discussed

Heterologous Expression of Various PHA Synthase Genes in Rhodospirillum rubrum

The phototrophic non-sulfur purple bacterium Rhodospirillum rubrum is known for its metabolic versatility. Particularly, R. rubrum is able to synthesize PHA under heterotrophic or even autotrophic growth with carbon monoxide as carbon and energy source. R. rubrum has therefore become a promising candidate for future cheap PHA production. However, R. rubrum synthesizes lower amounts of PHAs in comparison to well-known PHA producers like Ralstonia eutropha H16 or recombinant Escherichia coli strains. Since the PHA synthase is the key enzyme of PHA biosynthesis, genes encoding for twelve different PHA synthases were heterologously expressed in two generated phaC deletion mutants of R. rubrum in this study. To clearly see the effect of the foreign PHA synthases, PHA-negative mutants were required. The single mutant R. rubrum ΔphaC2 showed a PHA-leaky phenotype (< 1 % PHA, wt/wt, of CDW), while the double mutant R. rubrum ΔphaC1ΔphaC2 accumulated no measurable PHA. Eight different PHA synthase genes of class I, and four of class IV were chosen for heterologous expression. All recombinant R. rubrum strains showed significant PHA synthesis and accumulation, although PHA contents in the recombinant strains of the single mutant R. rubrum ΔphaC2 were generally higher in comparison to those of the double mutant R. rubrum ΔphaC1ΔphaC2. Recombinant strains of the single mutant could be divided into two groups according to the accumulation of PHA in the cells. While recombinant strains dedicated to group one showed an increased PHA synthesis when compared to the wild type carrying an empty vector, strains of group two accumulated less PHA than the wild type. Finally, it was possible to increase the accumulation of PHA by up to 25 % due to heterologous expression of PHA synthase genes compared to the wild type

Strain and process development for poly(3HB-co-3HP) fermentation by engineered Shimwellia blattae from glycerol

Poly(3-hydroxybytyrate-co-3-hydroxypropionate), poly(3HB-co-3HP), is a possible alternative to synthetic polymers such as polypropylene, polystyrene and polyethylene due to its low crystallinity and fragility. We already reported that recombinant strains of Shimwellia blattae expressing 1,3-propanediol dehydrogenase DhaT as well as aldehyde dehydrogenase AldD of Pseudomonas putida KT2442, propionate-CoA transferase Pct of Clostridium propionicum X2 and PHA synthase PhaC1 of Ralstonia eutropha H16 are able to accumulate up to 14.5% (wtPHA/wtCDW) of poly(3-hydroxypropionate), poly(3HP), homopolymer from glycerol as a sole carbon source (Appl Microbiol Biotechnol 98:7409-7422, 2014a). However, the cell density was rather low. In this study, we optimized the medium aiming at a more efficient PHA synthesis, and we engineered a S. blattae strain accumulating poly(3HB-co-3HP) with varying contents of the constituent 3-hydroxypropionate (3HP) depending on the cultivation conditions. Consequently, 7.12, 0.77 and 0.32 gPHA/L of poly(3HB-co-3HP) containing 2.1, 8.3 and 18.1 mol% 3HP under anaerobic/aerobic (the first 24 hours under anaerobic condition, thereafter, aerobic condition), low aeration/agitation (the minimum stirring rate required in medium mixing and small amount of aeration) and anaerobic conditions (the minimum stirring rate required in medium mixing without aeration), respectively, were synthesized from glycerol by the genetically modified S. blattae ATCC33430 strains in optimized culture medium

Optimization of macroelement concentrations, pH and osmolarity for triacylglycerol accumulation in Rhodococcus opacus strain PD630

The refinement of biodiesel or renewable diesel from bacterial lipids has a great potential to make a contribution for energy production in the future. This study provides new data concerning suitable nutrient concentrations for cultivation of the Gram-positive Rhodococcus opacus PD630, which is able to accumulate large amounts of lipids during nitrogen limitation. Enhanced concentrations of magnesium have been shown to increase the final optical density and the lipid content of the cells. Elevated phosphate concentrations slowed down the onset of the accumulation phase, without a clear effect on the final optical density and the cell’s lipid content. A robust growth of R. opacus was possible in the presence of ammonium concentrations of up to 1.4 g l-1 and sucrose concentrations of up to 240 g l-1, with an optimum regarding growth and lipid storage observed in the range of 0.2 to 0.4 g l-1 ammonium and 20 to 40 g l-1 sucrose, respectively. Moreover, R. opacus showed tolerance to high salt concentrations

A Closer Look on the Polyhydroxybutyrate- (PHB-) Negative Phenotype of Ralstonia eutropha PHB-4

The undefined poly(3-hydroxybutyrate)- (PHB-) negative mutant R. eutropha PHB-4 was generated in 1970 by 1-nitroso-3-nitro-1-methylguanidine (NMG) treatment. Although being scientific relevant, its genotype remained unknown since its isolation except a recent first investigation. In this study, the mutation causing the PHA-negative phenotype of R. eutropha PHB-4 was confirmed independently: sequence analysis of the phaCAB operon identified a G320A mutation in phaC yielding a stop codon, leading to a massively truncated PhaC protein of 106 amino acids (AS) in R. eutropha PHB-4 instead of 589 AS in the wild type. No other mutations were observed within the phaCAB operon. As further mutations probably occurred in the genome of mutant PHB-4 potentially causing secondary effects on the cells' metabolism, the main focus of the study was to perform a 2D PAGE-based proteome analysis in order to identify differences in the proteomes of the wild type and mutant PHB-4. A total of 20 differentially expressed proteins were identified which provide valuable insights in the metabolomic changes of mutant PHB-4. Besides excretion of pyruvate, mutant PHB-4 encounters the accumulation of intermediates such as pyruvate and acetyl-CoA by enhanced expression of the observed protein species: (i) ThiJ supports biosynthesis of cofactor TPP and thereby reinforces the 2-oxoacid dehydrogenase complexes as PDHC, ADHC and OGDHC in order to convert pyruvate at a higher rate and the (ii) 3-isopropylmalate dehydrogenase LeuB3 apparently directs pyruvate to synthesis of several amino acids. Different (iii) acylCoA-transferases enable transfer reactions between organic acid intermediates, and (iv) citrate lyase CitE4 regenerates oxaloacetate from citrate for conversion with acetyl-CoA in the TCC in an anaplerotic reaction. Substantial amounts of reduction equivalents generated in the TCC are countered by (v) synthesis of more ubiquinones due to enhanced synthesis of MenG2 and MenG3, thereby improving the respiratory chain which accepts electrons from NADH and succinate

Design and application of genetically-encoded malonyl-CoA biosensors for metabolic engineering of microbial cell factories

Malonyl-CoA is the basic building block for synthesizing a range of important compounds including fatty acids, phenylpropanoids, flavonoids and non-ribosomal polyketides. Centering around malonyl-CoA, we summarized here the various metabolic engineering strategies employed recently to regulate and control malonyl-CoA metabolism and improve cellular productivity. Effective metabolic engineering of microorganisms requires the introduction of heterologous pathways and dynamically rerouting metabolic flux towards products of interest. Transcriptional factor-based biosensors translate an internal cellular signal to a transcriptional output and drive the expression of the designed genetic/biomolecular circuits to compensate the activity loss of the engineered biosystem. Recent development of genetically-encoded malonyl-CoA sensor has stood out as a classical example to dynamically reprogram cell metabolism for various biotechnological applications. Here, we reviewed the design principles of constructing a transcriptional factor-based malonyl-CoA sensor with superior detection limit, high sensitivity and broad dynamic range. We discussed various synthetic biology strategies to remove pathway bottleneck and how genetically-encoded metabolite sensor could be deployed to improve pathway efficiency. Particularly, we emphasized that integration of malonyl-CoA sensing capability with biocatalytic function would be critical to engineer efficient microbial cell factory. Biosensors have also advanced beyond its classical function of a sensor actuator for in situ monitoring of intracellular metabolite concentration. Applications of malonyl-CoA biosensors as a sensor-invertor for negative feedback regulation of metabolic flux, a metabolic switch for oscillatory balancing of malonyl-CoA sink pathway and source pathway and a screening tool for engineering more efficient biocatalyst are also presented in this review. We envision the genetically-encoded malonyl-CoA sensor will be an indispensable tool to optimize cell metabolism and cost-competitively manufacture malonyl-CoA-derived compounds

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

The H2-oxidizing lithoautotrophic bacterium Ralstonia eutropha H16 is a metabolically versatile organism capable of subsisting, in the absence of organic growth substrates, on H2 and CO2 as its sole sources of energy and carbon. R. eutropha H16 first attracted biotechnological interest nearly 50 years ago with the realization that the organism’s ability to produce and store large amounts of poly[R-(–)-3-hydroxybutyrate] and other polyesters could be harnessed to make biodegradable plastics. Here we report the complete genome sequence of the two chromosomes of R. eutropha H16. Together, chromosome 1 (4,052,032 base pairs (bp)) and chromosome 2 (2,912,490 bp) encode 6,116 putative genes. Analysis of the genome sequence offers the genetic basis for exploiting the biotechnological potential of this organism and provides insights into its remarkable metabolic versatility

Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha

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 EnergyUnited States. Advanced Research Projects Agency-Energ

Differences between Men and Women in Treatment and Outcome after Traumatic Brain Injury

Traumatic brain injury (TBI) is a significant cause of disability, but little is known about sex and gender differences after TBI. We aimed to analyze the association between sex/gender, and the broad range of care pathways, treatment characteristics, and outcomes following mild and moderate/severe TBI. We performed mixed-effects regression analyses in the prospective multi-center Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study, stratified for injury severity and age, and adjusted for baseline characteristics. Outcomes were various care pathway and treatment variables, and 6-month measures of functional outcome, health-related quality of life (HRQoL), post-concussion symptoms (PCS), and mental health symptoms. The study included 2862 adults (36% women) with mild (mTBI; Glasgow Coma Scale [GCS] score 13-15), and 1333 adults (26% women) with moderate/severe TBI (GCS score 3-12). Women were less likely to be admitted to the intensive care unit (ICU; odds ratios [OR] 0.6, 95% confidence interval [CI]: 0.4-0.8) following mTBI. Following moderate/severe TBI, women had a shorter median hospital stay (OR 0.7, 95% CI: 0.5-1.0). Following mTBI, women had poorer outcomes; lower Glasgow Outcome Scale Extended (GOSE; OR 1.4, 95% CI: 1.2-1.6), lower generic and disease-specific HRQoL, and more severe PCS, depression, and anxiety. Among them, women under age 45 and above age 65 years showed worse 6-month outcomes compared with men of the same age. Following moderate/severe TBI, there was no difference in GOSE (OR 0.9, 95% CI: 0.7-1.2), but women reported more severe PCS (OR 1.7, 95% CI: 1.1-2.6). Men and women differ in care pathways and outcomes following TBI. Women generally report worse 6-month outcomes, but the size of differences depend on TBI severity and age. Future studies should examine factors that explain these differences.</p