Data sheets are arranged in their EC-Number sequence and the volumes themselves are arranged according to enzyme class Analytical chemistry.
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Analytical Chemistry. Temperature 2. Value of pH 2.
Activation 2. Inhibition 2. Allostery 2. Biogenic Regulation of Activity 2. Enzyme Assays 2. Reaction Rate as a Measure of Catalytic Activity 2. Definition of Units 2. Absorption Photometry 2.
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Fluorometry 2. Luminometry 2. Radiometry 2. Potentiometry 2.
Conductometry 2. Calorimetry 2. Polarimetry 2. Manometry 2. Viscosimetry 2. Turbidimetry 2. Immobilized Enzymes 2. Electrophoresis 2. Quality Evaluation of Enzyme Preparations 2. Quality Criteria 2. Specific Activity 2. Protein Determination 2. Contaminating Activities 2.
Class 1 Oxidoreductases VII
Electrophoretic Purity 2. Performance Test 2. Stability 2. Formulation of Enzyme Preparations 2.
Overview of Industrial Enzyme Applications 3. Enzyme Safety and Regulatory Considerations 3. Safe Handling of Enzymes 3. Following from FIG. Methanol is a relatively inexpensive organic feedstock that can be derived from synthesis gas components, CO and H 2 , via catalysis. Specifically, acetogens such as Moorella thermoacetica formerly, Clostridium thermoaceticum use syngas via the Wood-Ljungdahl pathway. For example, the direct conversion of synthesis gas to acetate is an energetically neutral process see FIG.
ATP consumption can be circumvented by ensuring that the methyl group on the methyl branch product, methyl-THF, is obtained from methanol rather than CO 2.
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The result is that acetate formation has a positive ATP yield that can help support cell growth and maintenance. A non-naturally occurring microbial organism of the present invention, engineered with these capabilities, that also naturally possesses the capability for anapleurosis e. This electron acceptor is used to accept electrons from the reduced quinone formed via succinate dehydrogenase. A further use of adding an external electron acceptor is that additional energy for cell growth, maintenance, and product formation can be generated from respiration of acetyl-CoA.
In some embodiments, engineering a pyruvate ferredoxin oxidoreductase PFOR enzyme into a non-naturally occurring microbial organism allows the synthesis of biomass precursors in the absence of an external electron acceptor. Specifically, the combination of certain syngas-utilization pathway components with the acetyl-CoA to 1,3-butanediol, isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathways results in high yields of these products from carbohydrates by providing an efficient mechanism for fixing the carbon present in carbon dioxide, fed exogenously or produced endogenously, into acetyl-CoA as shown below.
The enzymatic transformations for carbon fixation are shown in FIGS. The maximum theoretical yields of isopropanol, 4-hydroxybutyrate, and 1,4-butanediol from synthesis gases or carbohydrates can be further enhanced by the addition of methanol in different ratios of methanol to glucose. Exemplary flux distributions showing improvements in yields of 1,3-butanediol and isopropanol via carbohydrate-based carbon feedstock when carbon can be fixed via the Wood-Ljungdahl pathway using syngas components with and without methanol are shown in FIGS.
Thus, the non-naturally occurring microbial organisms and conversion routes described herein provide an efficient means of converting carbohydrates to products such as isopropanol, 4-hydroxybutyrate, or 1,4-butanediol.
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Additional product molecules that can be produced by the teachings of this invention include but are not limited to ethanol, butanol, isobutanol, isopropanol, 1,4-butanediol, succinic acid, fumaric acid, malic acid, 4-hydroxybutyric acid, 3-hydroxypropionic acid, lactic acid, adipic acid, 6-aminocaproic acid, hexamethylenediamine, caprolactam, 3-hydroxyisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid, acrylic acid, glycerol, 1,3-propanediol, and long chain hydrocarbons, alcohols, acids, and esters.
Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon.
Exemplary metabolic polypeptides include enzymes or proteins within a 1,4-butanediol, 4-hydroxybutyrate, 1,3-butanediol, isopropanol, 6-aminocaproic acid, hexamethylene diamine, caprolactam, glycerol, or 1,3-propanediol biosynthetic pathway. A metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring microorganisms can have genetic modifications to nucleic acids encoding metabolic polypeptides, or functional fragments thereof.
Exemplary metabolic modifications are disclosed herein. The term includes a microbial organism that is removed from some or all components as it is found in its natural environment. The term also includes a microbial organism that is removed from some or all components as the microbial organism is found in non-naturally occurring environments. Therefore, an isolated microbial organism is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments.
Specific examples of isolated microbial organisms include partially pure microbes, substantially pure microbes and microbes cultured in a medium that is non-naturally occurring. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical. Coenzyme A functions in certain condensing enzymes, acts in acetyl or other acyl group transfer and in fatty acid synthesis and oxidation, pyruvate oxidation and in other acetylation.
The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism.
The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid. It is understood that when more than one exogenous nucleic acid is included in a microbial organism that the more than one exogenous nucleic acids refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed above.
It is further understood, as disclosed herein, that such more than one exogenous nucleic acids can be introduced into the host microbial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof, and still be considered as more than one exogenous nucleic acid. For example, as disclosed herein a microbial organism can be engineered to express two or more exogenous nucleic acids encoding a desired pathway enzyme or protein.
In the case where two exogenous nucleic acids encoding a desired activity are introduced into a host microbial organism, it is understood that the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids.