Metabolome and proteome analyses reveal transcriptional misregulation in glycolysis of engineered E. coli

Synthetic metabolic pathways are a burden for engineered bacteria, but the underlying mechanisms often remain elusive. Here we show that the misregulated activity of the transcription factor Cra is responsible for the growth burden of glycerol overproducing E. coli. Glycerol production decreases the concentration of fructose-1,6-bisphoshate (FBP), which then activates Cra resulting in the downregulation of glycolytic enzymes and upregulation of gluconeogenesis enzymes. Because cells grow on glucose, the improper activation of gluconeogenesis and the concomitant inhibition of glycolysis likely impairs growth at higher induction of the glycerol pathway. We solve this misregulation by engineering a Cra-binding site in the promoter controlling the expression of the rate limiting enzyme of the glycerol pathway to maintain FBP levels sufficiently high. We show the broad applicability of this approach by engineering Cra-dependent regulation into a set of constitutive and inducible promoters, and use one of them to overproduce carotenoids in E. coli.

Endocannabinoids Produced by White Adipose Tissue Modulate Lipolysis in Lean but Not in Obese Rodent and Human

White adipose tissue (WAT) possesses the endocannabinoid system (ECS) machinery and produces the two major endocannabinoids (ECs), arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG). Accumulating evidence indicates that WAT cannabinoid 1 receptors (CB1R) are involved in the regulation of fat storage, tissue remodeling and secretory functions but their role in controlling lipid mobilization is unclear. In the present study, we used different strategies to acutely increase ECS activity in WAT and tested the consequences on glycerol production as a marker of lipolysis. Treating lean mice or rat WAT explants with JLZ195, which inhibits ECs degrading enzymes, induced an increase in 2-AG tissue contents that was associated with a CB1R-dependent decrease in lipolysis. Direct treatment of rat WAT explants with AEA also inhibited glycerol production while mechanistic studies revealed it could result from the stimulation of Akt-signaling pathway. Interestingly, AEA treatment decreased lipolysis both in visceral and subcutaneous WAT collected on lean subjects suggesting that ECS also reduces fat store mobilization in Human. In obese mice, WAT content and secretion rate of ECs were higher than in control while glycerol production was reduced suggesting that over-produced ECs may inhibit lipolysis activating local CB1R. Strikingly, our data also reveal that acute CB1R blockade with Rimonabant did not modify lipolysis in vitro in obese mice and human explants nor in vivo in obese mice. Taken together, these data provide physiological evidence that activation of ECS in WAT, by limiting fat mobilization, may participate in the progressive tissue remodeling that could finally lead to organ dysfunction. The present findings also indicate that acute CB1R blockade is inefficient in regulating lipolysis in obese WAT and raise the possibility of an alteration of CB1R signaling in conditions of obesity.

Redirection of the central metabolism of Klebsiella pneumoniae towards dihydroxyacetone production

Background Klebsiella pneumoniae is a bacterium that can be used as producer for numerous chemicals. Glycerol can be catabolised by K. pneumoniae and dihydroxyacetone is an intermediate of this catabolism pathway. Here dihydroxyacetone and glycerol were produced from glucose by this bacterium based a redirected glycerol catabolism pathway. Results tpiA, encoding triosephosphate isomerase, was knocked out to block the further catabolism of dihydroxyacetone phosphate in the glycolysis. After overexpression of a Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase (hdpA), the engineered strain produced remarkable levels of dihydroxyacetone (7.0 g/L) and glycerol (2.5 g/L) from glucose. Further increase in product formation were obtained by knocking out gapA encoding an iosenzyme of glyceraldehyde 3-phosphate dehydrogenase. There are two dihydroxyacetone kinases in K. pneumoniae. They were both disrupted to prevent an inefficient reaction cycle between dihydroxyacetone phosphate and dihydroxyacetone, and the resulting strains had a distinct improvement in dihydroxyacetone and glycerol production. pH 6.0 and low air supplement were identified as the optimal conditions for dihydroxyacetone and glycerol production by K, pneumoniae ΔtpiA-ΔDHAK-hdpA. In fed batch fermentation 23.9 g/L of dihydroxyacetone and 10.8 g/L of glycerol were produced after 91 h of cultivation, with the total conversion ratio of 0.97 mol/mol glucose. Conclusions This study provides a novel and highly efficient way of dihydroxyacetone and glycerol production from glucose.

Re-oxidation of cytosolic NADH is a major contributor to the high oxygen requirements of the thermotolerant yeast Ogataea parapolymorpha in oxygen-limited cultures

Thermotolerance is an attractive feature for yeast-based industrial ethanol production. However, incompletely understood oxygen requirements of known thermotolerant yeasts are incompatible with process requirements. To study the magnitude and molecular basis of these oxygen requirements in the facultatively fermentative, thermotolerant yeast Ogataea parapolymorpha, chemostat studies were performed under defined oxygen-sufficient and oxygen-limited cultivation regimes. The minimum oxygen requirements of O. parapolymorpha were found to be at least an order of magnitude larger than those of the thermotolerant yeast Kluyveromyces marxianus. This high oxygen requirement coincided with absence of glycerol formation, which plays a key role in NADH reoxidation in oxygen-limited cultures of other facultatively fermentative yeasts. Co-feeding of acetoin, whose reduction to 2,3-butanediol can reoxidize cytosolic NADH, supported a 2.5-fold higher biomass concentration in oxygen-limited cultures. The apparent inability of O. parapolymorpha to produce glycerol correlated with absence of orthologs of the S. cerevisiae genes encoding glycerol-3P phosphatase (ScGPP1, ScGPP2). Glycerol production was observed in aerobic batch cultures of a strain in which genes including key enzymes in mitochondrial reoxidation of NADH were deleted. However, transcriptome analysis did not identify a clear candidate for the responsible phosphatase. Expression of ScGPD2, encoding NAD+-dependent glycerol-3P dehydrogenase, and ScGPP1 in O. parapolymorpha resulted in increased glycerol production in oxygen-limited chemostats, but glycerol production rates remained substantially lower than observed in S. cerevisiae and K. marxianus. These results identify a dependency on aerobic respiration for reoxidation of NADH generated in biosynthesis as a key factor in the unexpectedly high oxygen requirements of O. parapolymorpha.

Interaction between S. cerevisiae MONA, PE-2, CAT-1 and ATCC in fermentation sugar cane broth

The diversity microbial in ethanolic fermentation generate different behavior metabolic that depended on the microorganisms present. Some kinetic parameters can tell how interactions between microorganisms are occurring in fermentation and can also predict your metabolic behaviors. However, there are little studys about the influence of interactions microbial on kinetic parameters in fermentation sugar cane. Therefore, this work aimed to understand the influence of the yeast strain Saccharomyces cerevisiae CAT-1, MONA, PE-2 and ATCC in the production of biomass, ethanol, glycerol and sugar consumption, as well as to evaluate the kinetic parameters by means of response surface methodology for mixing. From the biomass models generated, it was observed that the yeasts ATCC and MONA when in the presence of CAT-1 and PE-2 showed antagonisms. For the ethanol, the synergistic effect was verified for the mixture MONA/ATCC and CAT-1/PE-2 being that CAT-1 and PE-2 were the yeasts that strongly favored the ethanol production. It stands out yeast MONA due to having lower glycerol production, character desirable in the sugar and alcohol industry. Thus, it is clear that from the analysis employed it was possible to infer about the kinetic behavior of the yeasts in pure cultures as well as the effect of the interaction between them during the cultivation.

Reverse glycerol catabolism pathway for dihydroxyacetone and glycerol production by Klebsiella pneumoniae

Background: Klebsiella pneumoniae is a bacterium that can be used as producer for numerous chemicals. Glycerol can be catabolised by K. pneumoniae and dihydroxyacetone is an intermediate of this catabolism pathway. Here dihydroxyacetone and glycerol were produced from glucose by this bacterium based on a reverse glycerol catabolism pathway. Results: tpiA, encoding triosephosphate isomerase, was knocked out to block the further catabolism of dihydroxyacetone phosphate in the glycolysis. After overexpression of a Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase (hdpA), the engineering strain produced remarkable levels of dihydroxyacetone and glycerol from glucose. Further increase in product formation were obtained by knocking out gapA encoding an iosenzyme of glyceraldehyde 3-phosphate dehydrogenase. There are two dihydroxyacetone kinases in K. pneumoniae. They were both disrupted to prevent an inefficient reaction cycle between dihydroxyacetone phosphate and dihydroxyacetone, and the resulting strains had a distinct improvement in dihydroxyacetone and glycerol production. pH 6.0 and low air supplement were identified as the optimal conditions for dihydroxyacetone and glycerol production by K, pneumoniae ΔtpiA-ΔDHAK-hdpA. In fed batch fermentation 23.9 g/L of dihydroxyacetone and 10.8 g/L of glycerol were produced after 91 hours of cultivation, with the total conversion ratio of 0.97 mol/mol glucose.Conclusions: This study provides a novel and highly efficient way of dihydroxyacetone and glycerol production from glucose.

Kā raugs uzvedas, kad tam atņem būvelementus?

The study explored changes in carbon fluxes in the central metabolism of brewer’s yeast in the absence of building blocks such as adenine or nitrogen. These flows provide insight into changes in the central metabolism of brewer’s yeast. It was found that in the absence of a building block, the yeast mainly uses fermentation for growth, producing ethanol. Deletion of Δade1 in purine de novo synthesis reduces ethanol production, and decreased glycerol production in adenine starvation indicates a slowing of central metabolism.

Glycerol and Glycerol-Based Deep Eutectic Mixtures as Emerging Green Solvents for Polyphenol Extraction: The Evidence So Far

The acknowledgement that uncontrolled and excessive use of fossil resources has become a prime concern with regard to environmental deterioration, has shifted the orientation of economies towards the implementation of sustainable routes of production, through the valorization of biomass. Green chemistry plays a key role in this regard, defining the framework of processes that encompass eco-friendly methodologies, which aim at the development of highly efficient production of numerous bioderived chemicals, with minimum environmental aggravation. One of the major concerns of the chemical industry in establishing sustainable routes of production, is the replacement of fossil-derived, volatile solvents, with bio-based benign ones, with low vapor pressure, recyclability, low or no toxicity, availability and low cost. Glycerol is a natural substance, inexpensive and non-toxic, and it is a principal by-product of biodiesel industry resulting from the transesterification process. The ever-growing market of biodiesel has created a significant surplus of glycerol production, resulting in a concomitant drop of its price. Thus, glycerol has become a highly available, low-cost liquid, and over the past decade its use as an alternative solvent has been gaining unprecedented attention. This review summarizes the utilization of glycerol and glycerol-based deep eutectic mixtures as emerging solvents with outstanding prospect in bioactive polyphenol extraction.