Increasing the availability of acetyl-CoA stimulated growth and ethanol production from xylose by prolonging the growth phase of ethanologenic E. coli. The resulting increase in biocatalyst rather than an increase in cellular activity was responsible for the increased rate of ethanol production (Table ). Similar benefits were obtained by minimizing the loss of acetyl-CoA as acetate (ackA mutation) and by increasing intracellular levels of acetate (supplementing with acetate, pyruvate, or acetaldehyde). Inactivation of the native E. coli alcohol-aldehyde dehydrogenase gene (adhE) had little effect, indicating that this pathway has limited function in ethanologenic KO11.
Inactivation of ackA rather than pta was chosen to minimize the potential problems associated with global regulation. Acetyl-P is proposed to serve as an important global regulator in E. coli (, , ), affecting gene expression and fundamental processes such as the turnover of RpoS. During oxidative metabolism, inactivation of the acetate pathway is detrimental to growth (, , ). Although not fully understood, this detrimental effect has been attributed to the depletion of free CoA due to low rates of acetyl-CoA turnover (). In contrast to that found in previous studies concerning oxidative metabolism, inactivation of ackA (SU102) stimulated growth and ethanol production during the fermentation of xylose (Fig. ). An adhE mutation in strain KO11 (strain SU104) was of no benefit during xylose fermentation. Together, these results suggest that ADH contributes little to metabolism in KO11. The beneficial effect of inactivating ackA is presumed to result from a decrease in the production of acetate, increasing the availability of acetyl-CoA for biosynthesis. With regard to biosynthesis, the ackA mutation is a genetic equivalent of adding acetate, pyruvate, or acetaldehyde.
The introduction of ethanol metabolism is closely related to the metabolic pathways of carbohydrates and glucose, the metabolite of ethanol, acetyl CoA, enters in the process of glycolysis.
This is important because it allows you to convert almost all chemical substances into energy. The reaction proceeds in , characterized by a different production / energy expenditure, according to the .
If the metabolism of ethanol is complete, we will have an exothermic event with release of 1325 kJ / mol of energy. If the reaction stops early due to urinary elimination of acetic acid, the energy derived from alcohol is only 215.1 kJ / mol. It is important to remember that the first step involves an energy consumption of about 3ATP for each ethanol molecule. Our body produces everyday about 3 g of ethanol by fermentation processes for the synthesis of fatty acids, glycerolipid metabolism and biosynthesis of bile acids. It is therefore appropriate to consider as normal the presence of catabolism systems in our body able to prevent accumulation and toxicity typical of alcohol. Enzymes involved in ethanol metabolism can be found in various tissues and organs, but primarily, they are located in the liver and kidneys, and for this reason appear to be the main metabolic sites, as well as major damaged from toxic metabolites (eg. acethaldeide).
The production of ethanol from xylose by ethanologenic Escherichia coli strain KO11 was improved by adding various medium supplements (acetate, pyruvate, and acetaldehyde) that prolonged the growth phase by increasing cell yield and volumetric productivity (approximately twofold). Although added pyruvate and acetaldehyde were rapidly metabolized, the benefit of these additives continued throughout fermentation. Both additives increased the levels of extracellular acetate through different mechanisms. Since acetate can be reversibly converted to acetyl coenzyme A (acetyl-CoA) by acetate kinase and phosphotransacetylase, the increase in cell yield caused by each of the three supplements is proposed to result from an increase in the pool of acetyl-CoA. A similar benefit was obtained by inactivation of acetate kinase (ackA), reducing the production of acetate (and ATP) and sparing acetyl-CoA for biosynthetic needs. Inactivation of native E. coli alcohol-aldehyde dehydrogenase (adhE), which uses acetyl-CoA as an electron acceptor, had no beneficial effect on growth, which was consistent with a minor role for this enzyme during ethanol production. Growth of KO11 on xylose appears to be limited by the partitioning of carbon skeletons into biosynthesis rather than the level of ATP. Changes in acetyl-CoA production and consumption provide a useful approach to modulate carbon partitioning. Together, these results demonstrate that xylose fermentation to ethanol can be improved in KO11 by redirecting small amounts of pyruvate away from fermentation products and into biosynthesis. Though negligible with respect to ethanol yield, these small changes in carbon partitioning reduced the time required to complete the fermentation of 9.1% xylose in 1% corn steep liquor medium from over 96 h to less than 72 h.
X. dendrorhous can develop two metabolic modes depending on the type of carbon source present in the medium. Glucose or other fermentable sugars are assimilated through the glycolytic pathway followed by alcoholic fermentation to produce ethanol, even in the presence of oxygen . However, non-fermentable carbon sources, such as ethanol or succinate, are transformed to acetyl-CoA and are processed through the citric acid cycle. Thus, energy is produced mainly through oxidative phosphorylation.
Histidine is special in that its biosynthesis is inherently linked to thepathways of nucleotide formation. Histidine residues are often found in enzymeactive sites, where the chemistry of the imidazole ring of histidine makes it anucleophile and a good acid/base catalyzer. We now know that RNA can havecatalytic properties, and there has been speculation that life was originallyRNA-based. Perhaps the transition to protein catalysis from RNA catalysisoccurred at the origin of histidine biosynthesis.
We will look at this pathway in a bit more detail, because it involves themolecule 5-phosphoribosyl--pyrophosphate (which wewill refer to as "PRPP" from now on). PRPP is also involved in thesynthesis of purines and pyrimidines, as we will soon see. In the first step ofhistidine synthesis, PRPP condenses with ATP to form a purine, N1-5'-phosphoribosylATP, in a reaction that is driven by the subsequent hydrolysis of thepyrophosphate that condenses out. Glutamine again plays a role as an amino groupdonor, this time resulting in the formation of 5-aminoamidazole-4-carboximideribonucleotide (ACAIR), which is an intermediate in purine biosynthesis.
Previous studies performed in our laboratory indicated that as X. dendrorhous cultures age, the proportion of carotenoid intermediates relative to astaxanthin decreases. This phenomenon is accompanied by an increase in the relative amount of astaxanthin, which was explained by the termination of the de novo synthesis of pigments and the conversion of all of the intermediates to the final product of the pathway. Therefore, de novo synthesis of pigments can be evaluated by determining the proportion of intermediates relative to the amount of the final product (astaxanthin) over the course of the experiment. Accordingly, an analysis of the composition of the carotenoids present in the previously analyzed samples was conducted using reverse phase liquid chromatography (RP-HPLC). We measured the relative content of astaxanthin with respect to the total amount of pigments detected in each sample (i.e., astaxanthin, phoenicoxanthin, canthaxanthin, 3-OH-ketotorulene, echinenone, 3-OH-echinenone, neurosporene and β-carotene) (Figure ). In the control condition, the amount of astaxanthin remained constant at approximately 75% over the 24-h period studied, indicating that there were no intermediates generated. A very similar situation was observed when glucose was added; the proportion of astaxanthin remained the same as in the control at each of the times analyzed. A completely different phenomenon was observed when ethanol was added to the medium. In this case, 24 h after the addition of the carbon source, a significant decrease in the relative amount of astaxanthin was observed. This observation can be explained by the generation of carotenoid intermediates as a result of the induction of pigment biosynthesis. These results indicate that the addition of ethanol caused an increase in the amount of total carotenoids by promoting the de novo synthesis of pigments. In contrast, when glucose was added to the medium, there was an inhibition of pigment synthesis that was maintained over the entire analyzed time period. Importantly, both effects were detectable as early as 24 h after the addition of the carbon source and the effects correlated temporally with changes in the mRNA levels of the carotenogenesis genes.
The yeast Xanthophyllomyces dendrorhous is one of the most promising and economically attractive natural sources of astaxanthin. The biosynthesis of this valuable carotenoid is a complex process for which the regulatory mechanisms remain mostly unknown. Several studies have shown a strong correlation between the carbon source present in the medium and the amount of pigments synthesized. Carotenoid production is especially low when high glucose concentrations are used in the medium, while a significant increase is observed with non-fermentable carbon sources. However, the molecular basis of this phenomenon has not been established.
Possible causes for the decreased enzyme levels could be a decrease of the translation efficiency of ACVS and IPNS during degeneration, or the presence of a culture variant impaired in the biosynthesis of functional proteins of these enzymes, which outcompeted the high producing part of the population.