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Non-fermentative pathways for synthesis of branched-chain higher ..

Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels.

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Non-fermentative pathways for synthesis of ..

While fermentative pathways can be exploited for the synthesis of valuable fuel candidates, the use of non-fermentative pathways represents an invaluable opportunity to generate “designer” fuels with properties that make them compatible with current infrastructure for fuel transportation, storage and use. Such pathways include the fatty acid, polyketide, and isoprenoid biosynthesis pathways, as well as engineered keto-acid and reversal of the β-oxidation pathways ().

Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels

We clone the genes which will be translated into enzymes such as AlsS, IlvC, IlvD ,KivD, and assemble the genes into two circuits as following. The enzymes are crucial for producing butanol.(Reference: Atsumi, S.; T. Hanai and J.C. Liao (2008) Non-Fermentative Pathways for Synthesis of Branched-Chain Higher Alcohols as Biofuels, Nature, 451:86-89.)

Non-fermentative pathways for synthesis of branched-chain ..

Atsumi S, Hanai T, Liao JC (2008) Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels.

We clone the genes which will be translated into enzymes such as AlsS, IlvC, IlvD ,KivD, and assemble the genes into two circuits as following. The enzymes are crucial for producing butanol.(Reference: Atsumi, S.; T. Hanai and J.C. Liao (2008) Non-Fermentative Pathways for Synthesis of Branched-Chain Higher Alcohols as Biofuels, Nature, 451:86-89.)Following Figure 2 is about the strains where the alss, ilvC, ilvD and kivd genes are cloned from, along with their point mutation sites:

Liao (2008) Non-Fermentative Pathways for Synthesis of Branched-Chain Higher Alcohols as Biofuels, Nature, 451:86-89
Zhang, K., Sawaya, M.R., Eisenberg, D.S.

Non-Fermentative Pathways for Synthesis of Branched ..

pathways for synthesis of branched-chain ..

The environment within the mitochondrial matrix differs from the cytoplasm, including higher pH, lower oxygen concentration, and a more reducing redox potential-. This environment may more closely match the optimal for maximal activity of many enzymes such as the iron-sulfur clusters (ISC), which are essential cofactors of enzymes in diverse pathways including branched chain amino acid and isoprenoid biosynthetic pathways, and which are synthesized exclusively in mitochondria. Although ISCs can be exported to the cytoplasm, the molecular machinery that loads ISCs onto extramitochondrial enzymes is likely to be incompatible with most exogenous ISC-apoenzymes, especially those of bacterial, or archaeal origin ,. The smaller volume of mitochondria, could concentrate substrates favoring faster reaction rates and productivity and confine metabolic intermediates avoiding repressive regulatory responses, diversion of intermediates into competing pathways or even toxic effects of intermediates to cytoplasmic or nuclear processes.

A multitude of concerns that include climate change, political instability, and depletion of petroleum resources has recently ignited renewed interest in fossil fuel alternatives (). As a result, microbial systems have been extensively explored and successfully used for the biosynthesis of some biofuels, most notably ethanol (, ). Higher-chain alcohols, however, offer several advantages compared with ethanol, such as higher energy density and lower water solubility (). Despite this, the biosynthesis of such alcohols remains a daunting task, with the possible exception of 1-butanol. A new paradigm is now emerging, however, as evidenced in a recent issue of PNAS by the work of Liao and coworkers at University of California, Los Angeles (). Their work demonstrates the construction of nonnatural metabolism that allows the biosynthesis, for the first time, of an array of alcohols not readily produced by microorganisms. In the past, metabolic engineering efforts have exclusively focused on rewiring native metabolic pathways toward a metabolic pathway of interest (). At the same time, protein engineering, in general, and directed evolution, in particular, have mainly focused on the improvement of single-protein functions () with few examples of creating single proteins with novel functions (, ). The high evolutionary potential of biosynthetic enzymes has therefore remained largely untapped and their substrate promiscuity has been exploited for the generation of novel compounds mainly through feeding experiments (, ).

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  • Non-Fermentative Pathways for Synthesis of Branched-Chain Higher …

    Atsumi S, Hanai T, Liao JC (2008) Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels

  • Non-fermentative pathways for synthesis of branched ..

    Citations (2008). Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels

  • Lactic acid fermentation - Wikipedia

    (2008) Non-Fermentative Pathways for Synthesis of Branched-Chain Higher ..

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Semester Paper | Biochemistry for Medics – Lecture Notes

is an important substance for industry. No known organism can produce isobutanol or other branched-chain alcohols. presented a metabolic pathway to produce isobutanol in . The pathway is shown in Figure 1. The shown pathway starts with pyruvate and results in isobutanol. We also start with pyruvate which is generated from 3-phosphogylcerate in the glycolysis of the cell. For this 3-phosphogylcerate is required which is generated in the Calvin cycle of the CO2 fixation in . The steps in the conversion of pyruvate to 2-ketoisovalerate can be executed by proteins existing in (IlvIH, IlvC and IlvD). Since also has an alcohol dehydrogenase (AdhE), the only required protein for the isobutanol production is a ketoisovalerate decarboxylase. This protein (KivD) can be received from . The pathway shown in Figure 1 is already an improvement of the described way. The native protein IlvIH is replaced by the AlsS from to increase the isobutanol production. ()
As we want to integrate this pathway in we used and improved existing BioBricks from the iGEM team NCTU Formosa 2011/2012. We used gene coding sequences of four out of five required proteins for the isobutanol production.
These genes are The coding sequence of the gene of Adh (alcohol dehydrogenase), the fifth required protein, was not available as a BioBrick but because of 's own AdhE the pathway works (Atsumi et al., 2008).

As you can see in Figure 2 we have two approaches for our producing system. We want to reproduce the pathway from iGEM team NCTU Formosa without their temperature system (). In their system the first three proteins (AlsS, IlvC and IlvD) were generated while is incubated in a 37°C environment. During this the non-toxic intermediate 2-ketoisovalerate is accumulated. By shifting the temperature to a 30°C environment the missing KivD can be generated because of the non-active repressor. Together with the AdhE from KivD converts 2-ketoisovalerate into isobutanol.

In Figure 2A you can find our first approach where we also used the AdhE from . We disclaim the temperature system and put all coding sequences in a row behind a promoter separated by the RBS in front of each gene. We used as the cloning method for the part . For cloning the upstream the part , the was used. This idea resulted in the part .

We found out, that the AdhA from is the best alcohol dehydrogenase in literature (). We wanted to increase the production of isobutanol by cloning the adhA gene downstream of our producing pathway. By we designed a new part () which contains the coding sequence of the adhA gene from and is combined with the RBS . Afterwards we did several for combining the parts , and . You can find a schematic illustration of our created BioBrick in Figure 2B.

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ATSUMI, S.; HANAI, T. and LIAO, J.C. (2008b). Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. , vol. 451, no. 7174, p. 86-89.

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