Abstract:A one-pot enzymatic synthesis of 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase was established. Glycolysis by baker's yeast (Saccharomyces cerevisiae) generated ATP which was used to produce D: -glyceraldehyde 3-phosphate production from glucose via fructose 1,6-diphosphate. The D: -glyceraldehyde 3-phosphate produced was transformed to 2'-deoxyribonucleoside via 2-deoxyribose 5-phosphate and then 2-deoxyribose 1-phosphate in the presence of acetaldehyde and a nucleobase by deoxyriboaldol… Show more
“…However, prior to the discovery of ribonucleotide reduction (Reichard and Rutberg 1960 ), the reverse DERA pathway was considered the most likely cellular route for deoxyribonucleotide synthesis (Racker 1951 , 1952 ). That said, owing to low intracellular levels of acetaldehyde, the reaction is only known to proceed in the degradative direction in vivo, and synthesis is only observed in cell extracts (Horinouchi et al 2006c ; Racker 1952 ). Fig.…”
Section: A Bottom-up Perspective On the Origin Of Dnamentioning
confidence: 99%
“…The subsequent action of phosphopentomutase (PPMase), phosphorylases (PPases) and deoxynucleotide kinases (dNKases) generates dNTPs. Modern enzymes can drive the synthesis of deoxyribonucleosides but have thus far only been shown to do so in vitro (Horinouchi et al 2006c ) …”
Section: A Bottom-up Perspective On the Origin Of Dnamentioning
confidence: 99%
“…That said, there is an important parallel between SELEX experiments to test the feasibility of the individual steps in this pathway in a hypothetical RNA world (Burton and Lehman 2009 ) and microbial process engineering. Here, screening has yielded novel natural versions of these enzymes that, together, can improve the synthetic yield of the desired product (Horinouchi et al 2006a , b , c , 2003 , 2012 ; Ogawa et al 2003 ). Thus, pathway optimisation may be rapidly achieved through creation of a patchwork of enzymes from different species.…”
Section: Towards Testing Deep Evolution In a Cellular Experimental Cmentioning
All life generates deoxyribonucleotides, the building blocks of DNA, via ribonucleotide reductases (RNRs). The complexity of this reaction suggests it did not evolve until well after the advent of templated protein synthesis, which in turn suggests DNA evolved later than both RNA and templated protein synthesis. However, deoxyribonucleotides may have first been synthesised via an alternative, chemically simpler route—the reversal of the deoxyriboaldolase (DERA) step in deoxyribonucleotide salvage. In light of recent work demonstrating that this reaction can drive synthesis of deoxyribonucleosides, we consider what pressures early adoption of this pathway would have placed on cell metabolism. This in turn provides a rationale for the replacement of DERA-dependent DNA production by RNR-dependent production.Electronic supplementary materialThe online version of this article (doi:10.1007/s00239-014-9656-6) contains supplementary material, which is available to authorized users.
“…However, prior to the discovery of ribonucleotide reduction (Reichard and Rutberg 1960 ), the reverse DERA pathway was considered the most likely cellular route for deoxyribonucleotide synthesis (Racker 1951 , 1952 ). That said, owing to low intracellular levels of acetaldehyde, the reaction is only known to proceed in the degradative direction in vivo, and synthesis is only observed in cell extracts (Horinouchi et al 2006c ; Racker 1952 ). Fig.…”
Section: A Bottom-up Perspective On the Origin Of Dnamentioning
confidence: 99%
“…The subsequent action of phosphopentomutase (PPMase), phosphorylases (PPases) and deoxynucleotide kinases (dNKases) generates dNTPs. Modern enzymes can drive the synthesis of deoxyribonucleosides but have thus far only been shown to do so in vitro (Horinouchi et al 2006c ) …”
Section: A Bottom-up Perspective On the Origin Of Dnamentioning
confidence: 99%
“…That said, there is an important parallel between SELEX experiments to test the feasibility of the individual steps in this pathway in a hypothetical RNA world (Burton and Lehman 2009 ) and microbial process engineering. Here, screening has yielded novel natural versions of these enzymes that, together, can improve the synthetic yield of the desired product (Horinouchi et al 2006a , b , c , 2003 , 2012 ; Ogawa et al 2003 ). Thus, pathway optimisation may be rapidly achieved through creation of a patchwork of enzymes from different species.…”
Section: Towards Testing Deep Evolution In a Cellular Experimental Cmentioning
All life generates deoxyribonucleotides, the building blocks of DNA, via ribonucleotide reductases (RNRs). The complexity of this reaction suggests it did not evolve until well after the advent of templated protein synthesis, which in turn suggests DNA evolved later than both RNA and templated protein synthesis. However, deoxyribonucleotides may have first been synthesised via an alternative, chemically simpler route—the reversal of the deoxyriboaldolase (DERA) step in deoxyribonucleotide salvage. In light of recent work demonstrating that this reaction can drive synthesis of deoxyribonucleosides, we consider what pressures early adoption of this pathway would have placed on cell metabolism. This in turn provides a rationale for the replacement of DERA-dependent DNA production by RNR-dependent production.Electronic supplementary materialThe online version of this article (doi:10.1007/s00239-014-9656-6) contains supplementary material, which is available to authorized users.
“…c) An example of energy-requiring bioprocesses: Microbial production of dNS from glucose, acetaldehyde, and a nucleobase. In this process, FDP generated from glucose by baker’s yeast [4,9] serves as a substrate for fructose 1,6-diphosphate aldolase (FDP ALD) reaction in E. coli (DERA-PPMase−co-expressing E. coli ), and then the generated triose phosphates (DHAP and G3P) are converted to dNS through reactions catalyzed by triose phosphate isomerase (TPI) [10] and the enzymes involved in dNS metabolism (DERA-PPMase-NPase) [9,11,12]. This process is classified into type I in Figure 1a.…”
BackgroundReproduction and sustainability are important for future society, and bioprocesses are one technology that can be used to realize these concepts. However, there is still limited variation in bioprocesses and there are several challenges, especially in the operation of energy-requiring bioprocesses. As an example of a microbial platform for an energy-requiring bioprocess, we established a process that efficiently and enzymatically synthesizes 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase. This method consists of the coupling reactions of the reversible nucleoside degradation pathway and energy generation through the yeast glycolytic pathway.ResultsUsing E. coli that co-express deoxyriboaldolase and phosphopentomutase, a high amount of 2′-deoxyribonucleoside was produced with efficient energy transfer under phosphate-limiting reaction conditions. Keeping the nucleobase concentration low and the mixture at a low reaction temperature increased the yield of 2′-deoxyribonucleoside relative to the amount of added nucleobase, indicating that energy was efficiently generated from glucose via the yeast glycolytic pathway under these reaction conditions. Using a one-pot reaction in which small amounts of adenine, adenosine, and acetone-dried yeast were fed into the reaction, 75 mM of 2′-deoxyinosine, the deaminated product of 2′-deoxyadenosine, was produced from glucose (600 mM), acetaldehyde (250 mM), adenine (70 mM), and adenosine (20 mM) with a high yield relative to the total base moiety input (83%). Moreover, a variety of natural dNSs were further synthesized by introducing a base-exchange reaction into the process.ConclusionA critical common issue in energy-requiring bioprocess is fine control of phosphate concentration. We tried to resolve this problem, and provide the convenient recipe for establishment of energy-requiring bioprocesses. It is anticipated that the commercial demand for dNSs, which are primary metabolites that accumulate at very low levels in the metabolic pool, will grow. The development of an efficient production method for these compounds will have a great impact in both fields of applied microbiology and industry and will also serve as a good example of a microbial platform for energy-requiring bioprocesses.
“…Coupling the different reaction steps in a one-pot cascade allowed a 3-fold increase of 2'-deoxyribonucleoside production. [118] 3.3.4 Multi-Enzyme Cascades Involving l-Threonine Aldolases l-Threonine aldolases catalyze the reversible cleavage of l-threonine to glycine and acetaldehyde. They belong to the glycine-dependent aldolases and require pyridoxal 5'-phosphate.…”
Multi-enzymatic cascade reactions, i.e., the combination of several enzymatic transformations in concurrent one-pot processes, offer considerable advantages: the demand of time, costs and chemicals for product recovery may be reduced, reversible reactions can be driven to completion and the concentration of harmful or unstable compounds can be kept to a minimum. This review summarizes the developments in multi-enzymatic cascades employed for the asymmetric synthesis of chiral alcohols, amines and amino acids, as well as for C À C bond formation. In addition, a general classification of biocatalytic cascade systems is provided and bioprocess engineering aspects associated with the topic are discussed.
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