The Aging Metabolome-Biomarkers to Hub Metabolites.

Aging biology is intimately associated with dysregulated metabolism, which is one of the hallmarks of aging. Aging-related pathways such as mTOR and AMPK, which are major targets of anti-aging interventions including rapamcyin, metformin, and exercise, either directly regulate or intersect with metabolic pathways. In this review, numerous candidate bio-markers of aging that have emerged using metabolomics are outlined. Metabolomics studies also reveal that not all metabolites are created equally. A set of core "hub" metabolites are emerging as central mediators of aging. The hub metabolites reviewed here are nicotinamide adenine dinucleotide, reduced nicotinamide dinucleotide phosphate, α-ketoglutarate, and ß-hydroxybutyrate. These "hub" metabolites have signaling and epigenetic roles along with their canonical roles as co-factors or intermediates of carbon metabolism. Together these hub metabolites suggest a central role of the TCA cycle in signaling and metabolic dysregulation associated with aging.

Chromosome engineering of the TCA cycle in Halomonas bluephagenesis for production of copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV).

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a promising biopolyester with good mechanical properties and biodegradability. Large-scale production of PHBV is still hindered by the high production cost. CRISPR/Cas9 method was used to engineer the TCA cycle in Halomonas bluephagenesis on its chromosome for production of PHBV from glucose as a sole carbon source. Two TCA cycle related genes sdhE and icl encoding succinate dehydrogenase assembly factor 2 and isocitrate lysase were deleted, respectively, in H. bluephagenesis TD08AB containing PHBV synthesis genes on the chromosome, to channel more flux to increase the 3-hydroxyvalerate (3HV) ratio of PHBV. Due to a poor growth behavior of the mutant strains, H. bluephagenesis TY194 equipped with a medium strength Pporin-194 promoter was selected for further studies. The sdhE and/or icl mutant strains of H. bluephagenesis TY194 were constructed to show enhanced cell growth, PHBV synthesis and 3HV molar ratio. Gluconate was used to activate ED pathway and thus TCA cycle to increase 3HV content. H. bluephagenesis TY194 (ΔsdhEΔicl) was found to synthesize 17mol% 3HV in PHBV. Supported by the synergetic function of phosphoenolpyruvate carboxylase and Vitreoscilla hemoglobin encoded by genes ppc and vgb inserted into the chromosome of H. bluephagenesis TY194 (ΔsdhE) serving to enhance TCA cycle activity, a series of strains were generated that could produce PHBV containing 3-18mol% 3HV using glucose as a sole carbon source. Shake flask studies showed that H. bluephagenesis TY194 (ΔsdhE, G7::Pporin-ppc) produced 6.3 g/L cell dry weight (CDW), 65% PHBV in CDW and 25mol% 3HV in PHBV when grown in glucose and gluconate. 25mol% 3HV was the highest reported via chromosomal expression system. PHBV copolymers with different 3HV molar ratios were extracted and characterized. Next-generation industrial biotechnology (NGIB) based on recombinant H. bluephagenesis grown under unsterile and continuous conditions, allows production of P(3HB-0∼25mol% 3HV) in a convenient way with reduced production complexity and cost.

Metabolic gene expression and epigenetic effects of the ketone body ß-hydroxybutyrate on H3K9ac in bovine cells, oocytes and embryos.

The rapid decline in fertility that has been occurring to high-producing dairy cows in the past 50 years seems to be associated with metabolic disturbances such as ketosis, supporting the need for research to improve our understanding of the relations among the diet, metabolism and embryonic development. Recently, the ketone body ß-hydroxybutyrate (BOHB) was demonstrated to be a potent inhibitor of histone deacetylases (HDACs). Herein, we performed a series of experiments aiming to investigate the epigenetic effects of BOHB on histone acetylation in somatic cells, cumulus-oocyte complexes (COCs) and somatic cell nuclear transfer (SCNT) embryos. Treatment with BOHB does not increase histone acetylation in cells but stimulates genes associated with ketolysis and master regulators of metabolism. We further demonstrated that maturing COCs with high levels of BOHB does not affect their maturation rate or histone acetylation but increases the expression of PPARA in cumulus cells. Treatment of somatic cell nuclear transfer zygotes with BOHB causes hyperacetylation, which is maintained until the blastocyst stage, causing enhanced FOXO3A expression and blastocyst production. Our data shed light on the epigenetic mechanisms caused by BOHB in bovine cells and embryos and provide a better understanding of the connection between nutrition and reproduction.

Rediverting carbon flux in Clostridium ljungdahlii using CRISPR interference (CRISPRi).

Clostridium ljungdahlii has emerged as an attractive candidate for the bioconversion of synthesis gas (CO, CO2, H2) to a variety of fuels and chemicals through the Wood-Ljungdahl pathway. However, metabolic engineering and pathway elucidation in this microbe is limited by the lack of genetic tools to downregulate target genes. To overcome this obstacle, here we developed an inducible CRISPR interference (CRISPRi) system for C. ljungdahlii that enables efficient (> 94%) transcriptional repression of several target genes, both individually and in tandem. We then applied CRISPRi in a strain engineered for 3-hydroxybutyrate (3HB) production to examine targets for increasing carbon flux toward the desired product. Downregulating phosphotransacetylase (pta) with a single sgRNA led to a 97% decrease in enzyme activity and a 2.3-fold increase in titer during heterotrophic growth. However, acetate production still accounted for 40% of the carbon flux. Repression of aldehyde:ferredoxin oxidoreductase (aor2), another potential route for acetate production, led to a 5% reduction in acetate flux, whereas using an additional sgRNA targeted to pta reduced the enzyme activity to 0.7% of the wild-type level, and further reduced acetate production to 25% of the carbon flux with an accompanying increase in 3HB titer and yield. These results demonstrate the utility of CRISPRi for elucidating and controlling carbon flow in C. ljungdahlii.

A balanced ATP driving force module for enhancing photosynthetic biosynthesis of 3-hydroxybutyrate from CO2.

Using engineered photoautotrophic microorganisms for the direct chemical synthesis from CO2 is an attractive direction for both sustainability and CO2 mitigation. However, the behaviors of non-native metabolic pathways may be difficult to control due to the different intracellular contexts between natural and heterologous hosts. While most metabolic engineering efforts focus on strengthening driving forces in pathway design to favor biochemical production in these organisms, excessive driving force may be detrimental to product biosynthesis due to imbalanced cellular intermediate distribution. In this study, an ATP-hydrolysis based driving force module was engineered into cyanobacterium Synechococcus elongatus PCC 7942 to produce 3-hydroxybutyrate (3HB), a valuable chemical feedstock for the synthesis of biodegradable plastics and antibiotics. However, while the ATP driving force module is effective for increasing product formation, uncontrolled accumulation of intermediate metabolites likely led to metabolic imbalance and thus to cell growth inhibition. Therefore, the ATP driving force module was reengineered by providing a reversible outlet for excessive carbon flux. Upon expression of this balanced ATP driving force module with 3HB biosynthesis, engineered strain produced 3HB with a cumulative titer of 1.2 g/L, a significant increase over the initial strain. This result highlighted the importance of pathway reversibility as an effective design strategy for balancing driving force and intermediate accumulation, thereby achieving a self-regulated control for increased net flux towards product biosynthesis.

Evaluation of gene expression cassettes and production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with a fine modulated monomer composition by using it in Cupriavidus necator.

BACKGROUND: Cupriavidus necator has attracted much attention as a platform for the production of polyhydroxyalkanoate (PHA) and other useful materials. Therefore, an appropriate modulation of gene expression is needed for producing the desired materials effectively. However, there is insufficient information on the genetic engineering techniques required for this in C. necator. RESULTS: We found that the disruption of a potential ribosome binding site (RBS) in the phaC1 gene in C. necator caused a small decrease in the PhaC1 expression level. We applied this result to finely regulate the expression of other genes. Several gene expression cassettes were constructed by combining three Escherichia coli derived promoters (PlacUV5, Ptrc and Ptrp) to the potential RBS of phaC1 or its disruptant, respectively. Their expression levels were then determined via a lacZ reporter assay in C. necator strains. The promoter strengths were both ranked similarly for the cells that were cultured with fructose or palm kernel oil as a sole carbon source (Ptrc ≥ PlacUV5 > Ptrp), both of which were much stronger than the phaC1 promoter. The disruption of RBS had minute attenuation effect on the expression level of these expression cassettes with E. coli promoters. Furthermore, they were used to finely regulate the (R)-3-hydroxyhexanoate (3HHx) monomer ratio in the production of poly[(R)-3-hydroxybutyrate-co-3-hydroxyhexanoate] (PHBHHx) via R-specific enoyl-CoA hydratases (PhaJs). The 3HHx composition in PHBHHx is crucial because it defines the thermal and mechanical properties of the resulting plastic material. The C. necator mutant strains, whose PhaJ expression was controlled under the gene expression cassettes, could be used to produce PHBHHx with various 3HHx compositions in the same culture conditions. CONCLUSIONS: We constructed and evaluated several gene expression cassettes consisting of promoters and RBSs that finely regulate transcription and translation. These were then applied to finely modulate the monomer composition in the production of PHBHHx by recombinant C. necator.

Compositional regulation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by replacement of granule-associated protein in Ralstonia eutropha.

BACKGROUND: Phasin (PhaP), a kind of polyhydroxyalkanoate (PHA) granule-associated proteins, has a role in controlling the properties of PHA granules surface, and is thought to have influence on PHA biosynthesis in PHA-producing bacteria. This study focused on the phaP1(Re) locus in Ralstonia eutropha as a site of chromosomal modification for production of flexible poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from soybean oil. RESULTS: Considering the high expression level of phaP1(Re), phaJ(Ac) [encoding (R)-specific enoyl-CoA hydratase from Aeromonas caviae] was inserted into the downstream of phaP1(Re) on chromosome 1 of R. eutropha strain NSDG harboring phaC(NSDG) (encoding PHA synthase with broad substrate specificity). The constructed strain efficiently accumulated P(3HB-co-3HHx) on soybean oil with higher 3HHx composition when compared to the previous strain having phaJ(Ac) within pha operon. Insertion of the second phaC(NSDG) along with phaJ(Ac) at the phaP1(Re) locus led to incorporation of much larger 3HHx fraction into PHA chains, although the molecular weight was markedly reduced. The R. eutropha strains were further engineered by replacing phaP1(Re) with phaP(Ac) (encoding phasin from A. caviae) on the chromosome. Interestingly, the phasin replacement increased 3HHx composition in the soybean oil-based PHA with keeping high cellular contents, nevertheless no modification was conducted in the metabolic pathways. Kinetic and Western blot analyses of PHA synthase using cellular insoluble fractions strongly suggested that the phasin replacement not only enhanced activity of PHA synthase from A. caviae but also increased affinity especially to longer (R)-3HHx-CoA. It was supposed that the increased affinity of PHA synthase to (R)-3HHx-CoA was responsible for the higher 3HHx composition in the copolyester. CONCLUSIONS: The downstream of phaP1(Re) was a useful site for integration of genes to be overexpressed during PHA accumulation in R. eutropha. The results also clarified that polymerization properties of PHA synthase was affected by the kind of phasin co-existed on the surface of PHA granules, leading to altered composition of the resulting P(3HB-co-3HHx). The phasin replacement is a novel engineering strategy for regulation of composition of PHA copolyesters.

Improved artificial pathway for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with high C6-monomer composition from fructose in Ralstonia eutropha.

Poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)], a flexible and practical kind of polyhydroxyalkanoates, is generally produced from plant oils and fatty acids by several wild and recombinant bacteria. This study established an improved artificial pathway for the biosynthesis of P(3HB-co-3HHx) with high 3HHx composition from structurally unrelated fructose in Ralstonia eutropha. Depression of (R)-specific reduction of acetoacetyl-CoA by the deletion of phaB1 was an effective modification for formation of the C6-monomer unit from fructose driven by crotonyl-CoA carboxylase/reductase (Ccr). Co-overexpression of phaJ4a, which encodes medium-chain-length (R)-enoyl-CoA hydratase, with ccr promoted the incorporation of both 3HB and 3HHx units. Further introduction of emdMm, a synthetic gene encoding ethylmalonyl-CoA decarboxylase derived from mouse, was remarkably effective for P(3HB-co-3HHx) biosynthesis, probably by converting ethylmalonyl-CoA generated by the reductive carboxylase activity of Ccr back into butyryl-CoA. A high cellular content of P(3HB-co-3HHx) composed of 22mol% 3HHx could be produced from fructose by the engineered strain of R. eutropha with ΔphaB1 genotype expressing ccr, phaJ4a, and emd.

Synthesis of polyhydroxyalkanoates from glucose that contain medium-chain-length monomers via the reversed fatty acid ß-oxidation cycle in Escherichia coli.

Polyhydroxyalkanoates that contain the medium-chain-length monomers (mcl-PHAs) have a wide range of applications owing to their superior physical and mechanical properties. A challenge to synthesize such mcl-PHAs from unrelated and renewable sources is exploiting the efficient metabolic pathways that lead to the formation of precursor (R)-3-hydroxyacyl-CoA. Here, by engineering the reversed fatty acid ß-oxidation cycle, we were able to synthesize mcl-PHAs in Escherichia coli directly from glucose. After deletion of the major thioesterases, the engineered E. coli produced 6.62wt% of cell dry weight mcl-PHA heteropolymers. Furthermore, when a low-substrate-specificity PHA synthase from Pseudomonas stutzeri 1317 was employed, recombinant E. coli synthesized 12.10wt% of cell dry weight scl-mcl PHA copolymers, of which 21.18mol% was 3-hydroxybutyrate and 78.82mol% was medium-chain-length monomers. The reversed fatty acid ß-oxidation cycle offered an efficient metabolic pathway for mcl-PHA biosynthesis in E. coli and can be further optimized.

Impact of peripheral ketolytic deficiency on hepatic ketogenesis and gluconeogenesis during the transition to birth.

Preservation of bioenergetic homeostasis during the transition from the carbohydrate-laden fetal diet to the high fat, low carbohydrate neonatal diet requires inductions of hepatic fatty acid oxidation, gluconeogenesis, and ketogenesis. Mice with loss-of-function mutation in the extrahepatic mitochondrial enzyme CoA transferase (succinyl-CoA:3-oxoacid CoA transferase, SCOT, encoded by nuclear Oxct1) cannot terminally oxidize ketone bodies and develop lethal hyperketonemic hypoglycemia within 48 h of birth. Here we use this model to demonstrate that loss of ketone body oxidation, an exclusively extrahepatic process, disrupts hepatic intermediary metabolic homeostasis after high fat mother's milk is ingested. Livers of SCOT-knock-out (SCOT-KO) neonates induce the expression of the genes encoding peroxisome proliferator-activated receptor γ co-activator-1a (PGC-1α), phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase, and glucose-6-phosphatase, and the neonate's pools of gluconeogenic alanine and lactate are each diminished by 50%. NMR-based quantitative fate mapping of (13)C-labeled substrates revealed that livers of SCOT-KO newborn mice synthesize glucose from exogenously administered pyruvate. However, the contribution of exogenous pyruvate to the tricarboxylic acid cycle as acetyl-CoA is increased in SCOT-KO livers and is associated with diminished terminal oxidation of fatty acids. After mother's milk provokes hyperketonemia, livers of SCOT-KO mice diminish de novo hepatic ß-hydroxybutyrate synthesis by 90%. Disruption of ß-hydroxybutyrate production increases hepatic NAD(+)/NADH ratios 3-fold, oxidizing redox potential in liver but not skeletal muscle. Together, these results indicate that peripheral ketone body oxidation prevents hypoglycemia and supports hepatic metabolic homeostasis, which is critical for the maintenance of glycemia during the adaptation to birth.

Engineering cyanobacteria for photosynthetic production of 3-hydroxybutyrate directly from CO2.

(S)- and (R)-3-hydroxybutyrate (3HB) are precursors to synthesize the biodegradable plastics polyhydroxyalkanoates (PHAs) and many fine chemicals. To date, however, their production has been restricted to petroleum-based chemical industry and sugar-based microbial fermentation, limiting its sustainability and economical feasibility. With the ability to fix CO2 photosynthetically, cyanobacteria have attracted increasing interest as a biosynthesis platform to produce fuels and chemicals from alternative renewable resources. To this end, synthesis metabolic pathways have been constructed and optimized in cyanobacterium Synechocystis sp. PCC 6803 to photosynthetically produce (S)- and (R)-3HB directly from CO2. Both types of 3HB molecules were produced and readily secreted from Synechocystis cells without over-expression of transporters. Additional inactivation of the competing pathway by deleting slr1829 and slr1830 (encoding PHB polymerase) from the Synechocystis genome further promoted the 3HB production. Up to 533.4mg/L 3HB has been produced after photosynthetic cultivation of the engineered cyanobacterium Synechocystis TABd for 21 days. Further analysis indicated that the phosphate consumption during the photoautrophic growth and the concomitant elevated acetyl-CoA pool acted as a key driving force for 3HB biosynthesis in Synechocystis. For the first time, the study has demonstrated the feasibility of photosynthetic production of (S)- and (R)-3HB directly from sunlight and CO2.