Biosynthesis of Polyhydroxyalkanoate Terpolymer from Methanol via the Reverse β-Oxidation Pathway in the Presence of Lanthanide

Methylorubrum extorquens AM1 is the attractive platform for the production of value-added products from methanol. We previously demonstrated that M. extorquens equipped with PHA synthase with broad substrate specificity synthesized polyhydroxyalkanoates (PHAs) composed of (R)-3-hydroxybutyrate and small fraction of (R)-3-hydroxyvalerate (3HV) and (R)-3-hydroxyhexanoate (3HHx) units on methanol. This study further engineered M. extorquens for biosynthesis of PHAs with higher 3HV and 3HHx composition focusing on the EMC pathway involved in C1 assimilation. The introduction of ethylmalonyl-CoA decarboxylase, catalyzing a backward reaction in the EMC pathway, aiming to increase intracellular propionyl/butyryl-CoA precursors did not affect PHA composition. Reverse b-oxidation pathway and subsequent (R)-specific hydration of 2-enoyl-CoA were then enhanced by heterologous expression of four genes derived from Ralstonia eutropha for the conversion of propionyl/butyryl-CoAs to the corresponding (R)-3-hydroxyacyl-CoA monomers. The resulting strains produced PHAs with higher 3HV and 3HHx compositions, while the methylotrophic growth was severely impaired. This growth impairment was interestingly restored by the addition of La3+ without a negative impact on PHA biosynthesis, suggesting the activation of the EMC pathway by La3+. The engineered M. extorquens synthesized PHA terpolymer composed of 5.4 mol% 3HV and 0.9% of 3HHx with 41% content from methanol as a sole carbon source in the presence of La3+.

Draft Genome Sequences of Sulfurovum spp. TSL1 and TSL6, Two Sulfur-Oxidizing Bacteria Isolated from Marine Sediment

Sulfurovum spp. TSL1 and TSL6 are sulfur-oxidizing chemolithoautotrophic bacteria isolated from the tsunami-launched marine sediment in the Great East Japan earthquake. This announcement describes the draft genome sequences of the two isolates that possess the gene sets for the sulfur oxidation pathway.

Unraveling Fe(II)-oxidizing mechanisms in a facultative Fe(II)-oxidizer, Sideroxydans lithotrophicus ES-1 via culturing, transcriptomics, and RT-qPCR

Sideroxydans lithotrophicus ES-1 grows autotrophically either by Fe(II) oxidation or thiosulfate oxidation, in contrast to most other neutrophilic Fe(II)-oxidizing bacteria (FeOB) isolates. This provides a unique opportunity to explore the physiology of a facultative FeOB and constrain the genes specific to Fe(II) oxidation. We compared the growth of S. lithotrophicus ES-1 on Fe(II), thiosulfate, and both substrates together. While initial growth rates were similar, thiosulfate-grown cultures had higher yield with or without Fe(II) present, which may give ES-1 an advantage over obligate FeOB. To investigate the Fe(II) and S oxidation pathways, we conducted transcriptomics experiments, validated with RT-qPCR. We explored the long-term gene expression response at different growth phases (over days-week) and expression changes during a short-term switch from thiosulfate to Fe(II) (90 min). The dsr and sox sulfur oxidation genes were upregulated in thiosulfate cultures. The Fe(II) oxidase gene cyc2 was among the top expressed genes during both Fe(II) and thiosulfate oxidation, and addition of Fe(II) to thiosulfate-grown cells caused an increase in cyc2 expression. These results support the role of Cyc2 as the Fe(II) oxidase and suggest that ES-1 maintains readiness to oxidize Fe(II) even in the absence of Fe(II). We used gene expression profiles to further constrain the ES-1 Fe(II) oxidation pathway. Notably, among the most highly upregulated genes during Fe(II) oxidation were genes for alternative complex III, reverse electron transport and carbon fixation. This implies a direct connection between Fe(II) oxidation and carbon fixation, suggesting that CO 2 is an important electron sink for Fe(II) oxidation. Importance Neutrophilic FeOB are increasingly observed in various environments, but knowledge of their ecophysiology and Fe(II) oxidation mechanisms is still relatively limited. Sideroxydans are widely observed in aquifers, wetlands, and sediments, and genome analysis suggests metabolic flexibility contributes to their success. The type strain ES-1 is unusual amongst neutrophilic FeOB isolates as it can grow on either Fe(II) or a non-Fe(II) substrate, thiosulfate. Almost all our knowledge of neutrophilic Fe(II) oxidation pathways comes from genome analyses, with some work on metatranscriptomes. This study used culture-based experiments to test the genes specific to Fe(II) oxidation in a facultative FeOB and refine our model of the Fe(II) oxidation pathway. We gained insight into how facultative FeOB like ES-1 connect Fe, S, and C biogeochemical cycling in the environment, and suggest a multi-gene indicator would improve understanding of Fe(II) oxidation activity in environments with facultative FeOB.

Formation and evolution of secondary organic aerosols derived from urban-lifestyle sources: vehicle exhaust and cooking emissions

Vehicle exhaust and cooking emissions are closely related to the daily life of city dwellers. Here, we defined the secondary organic aerosols (SOAs) derived from vehicle exhaust and cooking emissions as “urban-lifestyle SOAs” and simulated their formation using a Gothenburg potential aerosol mass reactor (Go:PAM). The vehicle exhaust and cooking emissions were separately simulated, and their samples were defined as “vehicle group” and “cooking group”, respectively. After samples had been aged under 0.3–5.5 d of equivalent photochemical age, these two urban-lifestyle SOAs showed markedly distinct features in the SOA mass growth potential, oxidation pathways, and mass spectra. The SOA/POA (primary organic aerosol) mass ratios of vehicle groups (107) were 44 times larger than those of cooking groups (2.38) at about 2 d of equivalent photochemical age, according to the measurement of scanning mobility particle sizer (SMPS). A high-resolution time-of-flight aerosol mass spectrometer was used to perform a deeper analysis. It revealed that organics from the vehicle may undergo the alcohol and/or peroxide and carboxylic acid oxidation pathway to produce abundant less and more oxidized oxygenated OAs (LO-OOAs and MO-OOAs), and only a few primary hydrocarbon-like organic aerosols (HOAs) remain unaged. In contrast, organics from cooking may undergo the alcohol and/or peroxide oxidation pathway to produce moderate LO-OOAs, and comparable primary cooking organic aerosols (COAs) remain unaged. Our findings provide an insight into atmospheric contributions and chemical evolutions for urban-lifestyle SOAs, which could greatly influence the air quality and health risk assessments in urban areas.

Mechanisms Mediating the Regulation of Peroxisomal Fatty Acid Beta-Oxidation by PPARα

In mammalian cells, two cellular organelles, mitochondria and peroxisomes, share the ability to degrade fatty acid chains. Although each organelle harbors its own fatty acid β-oxidation pathway, a distinct mitochondrial system feeds the oxidative phosphorylation pathway for ATP synthesis. At the same time, the peroxisomal β-oxidation pathway participates in cellular thermogenesis. A scientific milestone in 1965 helped discover the hepatomegaly effect in rat liver by clofibrate, subsequently identified as a peroxisome proliferator in rodents and an activator of the peroxisomal fatty acid β-oxidation pathway. These peroxisome proliferators were later identified as activating ligands of Peroxisome Proliferator-Activated Receptor α (PPARα), cloned in 1990. The ligand-activated heterodimer PPARα/RXRα recognizes a DNA sequence, called PPRE (Peroxisome Proliferator Response Element), corresponding to two half-consensus hexanucleotide motifs, AGGTCA, separated by one nucleotide. Accordingly, the assembled complex containing PPRE/PPARα/RXRα/ligands/Coregulators controls the expression of the genes involved in liver peroxisomal fatty acid β-oxidation. This review mobilizes a considerable number of findings that discuss miscellaneous axes, covering the detailed expression pattern of PPARα in species and tissues, the lessons from several PPARα KO mouse models and the modulation of PPARα function by dietary micronutrients.

Exploring functionality of the reverse β-oxidation pathway in Corynebacterium glutamicum for production of adipic acid

Background Adipic acid, a six-carbon platform chemical mainly used in nylon production, can be produced via reverse β-oxidation in microbial systems. The advantages posed by Corynebacterium glutamicum as a model cell factory for implementing the pathway include: (1) availability of genetic tools, (2) excretion of succinate and acetate when the TCA cycle becomes overflown, (3) initiation of biosynthesis with succinyl-CoA and acetyl-CoA, and (4) established succinic acid production. Here, we implemented the reverse β-oxidation pathway in C. glutamicum and assessed its functionality for adipic acid biosynthesis. Results To obtain a non-decarboxylative condensation product of acetyl-CoA and succinyl-CoA, and to subsequently remove CoA from the condensation product, we introduced heterologous 3-oxoadipyl-CoA thiolase and acyl-CoA thioesterase into C. glutamicum. No 3-oxoadipic acid could be detected in the cultivation broth, possibly due to its endogenous catabolism. To successfully biosynthesize and secrete 3-hydroxyadipic acid, 3-hydroxyadipyl-CoA dehydrogenase was introduced. Addition of 2,3-dehydroadipyl-CoA hydratase led to biosynthesis and excretion of trans-2-hexenedioic acid. Finally, trans-2-enoyl-CoA reductase was inserted to yield 37 µg/L of adipic acid. Conclusions In the present study, we engineered the reverse β-oxidation pathway in C. glutamicum and assessed its potential for producing adipic acid from glucose as starting material. The presence of adipic acid, albeit small amount, in the cultivation broth indicated that the synthetic genes were expressed and functional. Moreover, 2,3-dehydroadipyl-CoA hydratase and β-ketoadipyl-CoA thiolase were determined as potential target for further improvement of the pathway.

P-307 Fatty acid degradation during in vitro decidualization of human endometrial stromal cells

Study question Is the activity of the β-oxidation pathway, involved in the degradation of fatty acids, modified during in vitro decidualization of human endometrial stromal cells (HESC)? Summary answer The level of expression of fatty acid´s transporters suggests that the activity of the mitochondrial β-oxidation pathway is increased during in vitro decidualization of HESC. What is known already The differentiation of endometrial stromal cells (ESC), named decidualization, is essential for the proper formation of the materno-fetal interphase. One important feature of decidualization is the increased glucose consumption. In the endometrium, glucose is incorporated into ESC by glucose-transporters (GLUT). Fatty acids are another important energy source in living cells. Fatty acids are transported into mitochondria by the carnitine-palmitoyl-transferases 1 and 2 (CPT1 and 2) and are degraded there through the β-oxidation pathway. It has been described that the inhibition of CPT1 affects ESC decidualization. However, it is unknown whether the turn-over of fatty acids degradation is modified during decidualization. Study design, size, duration This study was performed using primary HESC. Endometrial biopsies (mid-late proliferative-phase) were obtained from healthy-regularly-cycling women (33.6±2.2 years-old) after written informed consent was obtained (protocol approved by Ethics committee no. S-239/2005). HESC were decidualized (D) in vitro with a decidualization-cocktail (containing: medroxyprogesterone acetate, estradiol and 8-Bromo-cyclic adenosine monophosphate) for 6 days. Non-decidualized (ND) controls were treated with vehicle solutions. Cell-culture supernatant and cell extracts were collected for the evaluation of protein/gene expression and metabolite content. Participants/materials, setting, methods Decidualization was evaluated by measuring prolactin (PRL) protein levels in cell-culture supernatant (mU/l). Changes in mRNA expression levels of GLUT1, CPT1A and CPT2 were evaluated by real-time polymerase chain reaction (RT-PCR). Analysis was performed by the ΔΔCt method (internal control: RPLP0) (fold change -FC- in D compared to ND cells). Contents of acylcarnitines were evaluated by Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS) (nmol/mg of total protein). N = 5, mean±SEM. Paired Student’s t-test was used for statistical analysis. Main results and the role of chance PRL protein levels in cell-culture supernatant were significative increased in HESC treated with the decidualization-cocktail compared to ND cells (ND 16.80±0.73 mU/l; D 684.20±219.80 mU/l, *p<0.05). This result confirmed the decidualized state of HESC upon in vitro treatment with the decidualization-cocktail. Additionally, the mRNA expression level of GLUT1 was highly upregulated in D compared to ND cells (FC 10.02±2.90, ***p<0.001), consistent with the increase in glucose consumption characteristic of decidualization. Once confirmed the decidualized state of HESC, the mRNA expression levels of CPTA1 and CPT2 were evaluated. The mRNA expression levels of both fatty acid´s transporters were upregulated in D compared to ND cells (CPTA1: FC 1.84±0.44, **p<0.01; CPT2: FC 2.04±0.49, **p<0.01). Finally, the content levels of different acylcarnitines, intermediate metabolites of the β-oxidation degradation of fatty acids, were evaluated. The concentrations of acetyl- (C2) and butyryl- (C4) acylcarnites were decreased in D compared to ND cells [(C2: ND 1.37±0.10 nmol/mg of total protein; D 1.06±0.20 nmol/mg of total protein, *p<0.05), (C4: ND 0.03±0.01 nmol/mg of total protein; D 0.01±0.00 nmol/mg of total protein, *p<0.05)]. The content levels of other intermediate acylcarnitines measured from cell extracts had no differences between D and ND cells (p > 0,05). Limitations, reasons for caution This study was performed in vitro using primary HESC treated with a decidualization-cocktail. The interconnection of different metabolic pathways within a living cell is very complex. Further studies are necessary to define whether the different intermediate metabolites of the mitochondrial β-oxidation pathway are being used by related-metabolic pathways during decidualization. Wider implications of the findings The regulation of the energy metabolism and its interconnection with other important intra-cellular metabolic pathways is of great importance for cellular function. Our results contribute to highlight the importance of the regulation of fatty acids degradation during decidualization. Further insights into HESC metabolism could facilitate the improvement of womeńs health. Trial registration number not applicable

P–307 Fatty acid degradation during in vitro decidualization of human endometrial stromal cells

Study question Is the activity of the β-oxidation pathway, involved in the degradation of fatty acids, modified during in vitro decidualization of human endometrial stromal cells (HESC)? Summary answer The level of expression of fatty acid´s transporters suggests that the activity of the mitochondrial β-oxidation pathway is increased during in vitro decidualization of HESC. What is known already The differentiation of endometrial stromal cells (ESC), named decidualization, is essential for the proper formation of the materno-fetal interphase. One important feature of decidualization is the increased glucose consumption. In the endometrium, glucose is incorporated into ESC by glucose-transporters (GLUT). Fatty acids are another important energy source in living cells. Fatty acids are transported into mitochondria by the carnitine-palmitoyl-transferases 1 and 2 (CPT1 and 2) and are degraded there through the β-oxidation pathway. It has been described that the inhibition of CPT1 affects ESC decidualization. However, it is unknown whether the turn-over of fatty acids degradation is modified during decidualization. Study design, size, duration This study was performed using primary HESC. Endometrial biopsies (mid-late proliferative-phase) were obtained from healthy-regularly-cycling women (33.6±2.2 years-old) after written informed consent was obtained (protocol approved by Ethics committee no. S–239/2005). HESC were decidualized (D) in vitro with a decidualization-cocktail (containing: medroxyprogesterone acetate, estradiol and 8-Bromo-cyclic adenosine monophosphate) for 6 days. Non-decidualized (ND) controls were treated with vehicle solutions. Cell-culture supernatant and cell extracts were collected for the evaluation of protein/gene expression and metabolite content. Participants/materials, setting, methods Decidualization was evaluated by measuring prolactin (PRL) protein levels in cell-culture supernatant (mU/l). Changes in mRNA expression levels of GLUT1, CPT1A and CPT2 were evaluated by real-time polymerase chain reaction (RT-PCR). Analysis was performed by the ΔΔCt method (internal control: RPLP0) (fold change -FC- in D compared to ND cells). Contents of acylcarnitines were evaluated by Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS) (nmol/mg of total protein). N = 5, mean±SEM. Paired Student’s t-test was used for statistical analysis. Main results and the role of chance PRL protein levels in cell-culture supernatant were significative increased in HESC treated with the decidualization-cocktail compared to ND cells (ND 16.80±0.73 mU/l; D 684.20±219.80 mU/l, *p<0.05). This result confirmed the decidualized state of HESC upon in vitro treatment with the decidualization-cocktail. Additionally, the mRNA expression level of GLUT1 was highly upregulated in D compared to ND cells (FC 10.02±2.90, ***p<0.001), consistent with the increase in glucose consumption characteristic of decidualization. Once confirmed the decidualized state of HESC, the mRNA expression levels of CPTA1 and CPT2 were evaluated. The mRNA expression levels of both fatty acid´s transporters were upregulated in D compared to ND cells (CPTA1: FC 1.84±0.44, **p<0.01; CPT2: FC 2.04±0.49, **p<0.01). Finally, the content levels of different acylcarnitines, intermediate metabolites of the β-oxidation degradation of fatty acids, were evaluated. The concentrations of acetyl- (C2) and butyryl- (C4) acylcarnites were decreased in D compared to ND cells [(C2: ND 1.37±0.10 nmol/mg of total protein; D 1.06±0.20 nmol/mg of total protein, *p<0.05), (C4: ND 0.03±0.01 nmol/mg of total protein; D 0.01±0.00 nmol/mg of total protein, *p<0.05)]. The content levels of other intermediate acylcarnitines measured from cell extracts had no differences between D and ND cells (p > 0,05). Limitations, reasons for caution This study was performed in vitro using primary HESC treated with a decidualization-cocktail. The interconnection of different metabolic pathways within a living cell is very complex. Further studies are necessary to define whether the different intermediate metabolites of the mitochondrial β-oxidation pathway are being used by related-metabolic pathways during decidualization. Wider implications of the findings: The regulation of the energy metabolism and its interconnection with other important intra-cellular metabolic pathways is of great importance for cellular function. Our results contribute to highlight the importance of the regulation of fatty acids degradation during decidualization. Further insights into HESC metabolism could facilitate the improvement of womeńs health. Trial registration number Not applicable