Summary of Invention

An enzymatic process is described for the production of chemicals from carbon sources In particular according to one aspect a process for the production of a target organic compound from a carbon source by a cell-free enzyme system is disclosed

Background of the Invention

The invention is directed to a process for the bioconversion of a carbon source, which is preferably a carbohydrate or another carbon containing compound into a target organic compound by an enzymatic process, preferably in the absence of productive living ceils The target organic compound is preferably a hydrophobic, a hydrophilic or an intermediate chemical compound

Hydrophobic chemicals according to the invention comprise, without limitation C4 alcohols such as n-butaπol, 2-butanol, and isobutanol, and other chemicals that have a limited miεcibility with water Limited miscibility means that at room temperature not more than 20% (w/w) can be rmxed with water without phase separation Hydrophilic and intermediate chemicals according to the invention comprise, without limitation ethanol and other chemicals

n-Butanol is a colorless, neutral liquid of medium volatility with restricted miεcibility (about 7- 8% at RT) in water n-Butanol is used as an intermediate in the production of chemicals, as a solvent and as an ingredient in formulated products such as cosmetics n-Butano! is used in the synthesis of acrylate/methacrylate esters glycol ethers n-butyl acetate ammo resins and n-butylamines n-Butanol can also be used as a fuel in combustion engines due to low vapor pressure high energy content and the possibility to be blended with gasoline at high concentrations

2-Butano! is a colorless, neutrai liquid of medium volatility with restricted misαbihty (about 12 % at RT) in water 2~Butanol is used as solvent for paints and coatings as weli as food ingredients or in the production of 1-buten isobutanol is a colorless, neutral liquid of medium volatility with restricted miscibility (about 9- 10 % at RT) in water. Isobutanol is used as solvent or as plasticizer. It is also used in the production of isobuten which is a precursor for the production of MTBE or ETBE.

n-Butanol can be produced using solventogenic Clostridia, such as C, acetobuylicum or C. beijerinckii, typically producing a mixture of n-butanoi, acetone and ethanol. Butanoi production using solventogenic Clostridia has several drawbacks: (i) Product isolation from dilute aqueous solution is very expensive as it is either elaborate (e.g. using membrane processes) or energy consuming (e.g. using distiilation). (ii) The yield is Sow as significant parts of the substrate go into the formation of byproducts such as acetone, ethanol, hydrogen and biomass. (iii) The productivity of butanoi production is low due to Simited cell titers, (iv) The complex metabolism limits meiaboltca! engineering for higher productivity and yield, (v) Limited process stability often leads to production losses and sterility is difficult to maintain, (vi) The biphasic nature of clostridial growth limits process flexibility and productivity.

Several approaches exist to overcome the limitations of classical fermentative butanoi production. For example, WO2008/052596 describes recombinant modification of Clostridia for improved yield. Selection or engineering of variants for higher Butanoi resistance is, for example, described in WO 2008/006038.

The cell-free production of chemicals has been shown as early as 1897 when Eduard Buchner used a lysate of yeast cells to convert glucose to ethanol. Later Welch und Scopes, 1985 demonstrated cell free production of ethanol, a process which, however, was technically not useful. The system Jacked specificity (side reaction of enzymes, unwanted activities in the lysate) and a maximum of 9 % ethanol was obtained.

A number of technical processes have been described that use isolated enzymes for the production of chemicals. For example, alcohol dehydrogenases are used in the production of chiral alcohols from ketonε. Such processes require cofactor (NAD) regeneration which can be achieved, for example, by adding glucose and glucose dehydrogenase. Such processes have been designed to produce high-value chemicals but not to provide enzyme system comprising multiple enzyme reactions that convert carbohydrates into chemicals with high energy and carbon efficiency,

Zhang et a!., 2008 describe the idea for cell free enzymatic conversion of glucose to n- butanol. The concept includes a minimum of 18 enzymes, several different cofactors and coenzymes (e.g. ATP, ADP, NADH, NAD ₁ ferredoxin and coenzyme A). In addition the postulated process results in a net-production of ATP so that it requires in addition an ATPase enzyme to remove the ATP. Under practical terms control of ATPase addition while maintaining a balanced ATP level is very difficult to achieve. In summary, the described process would be expensive, technicaliy instable and would give only low butanol yields.

In summary there is a need for a cost effective process for the production of chemicals from renewable resources, in particular ethanol and C4 alcohols such as n-butanol and its isomers.

According to one aspect, the present invention addresses this need through a cell free enzymatic system, using only a limited number of enzymes and a limited set of cofactors. In particular, according to a preferred aspect, the inventive process does not lead to net ATP production, and/or does not use phosphorylative enzyme reactions, and/or uses only enzymes that withstand the inactivating presence of the produced chemicals.

Definitions

A hydrophobic chemical is a chemical which is only partially soluble in water and which resides in the solid or liquid state at ambient pressure and temperature. Hydrophobic chemicals have a limited miscibiSiy with water of not more than 20% (w/w) without phase separation. Particular examples of hydrophobic chemicals according to the present invention include n-butanol, 2-butanσl and isobutanoi.

Carbon source can be any material which can be utilized by microorganisms for growth or production of chemicals. These include carbohydrates and derivatives: polyoses such as cellulose, hemicellulose, starch; bioses such as sucrose, maltose, lactose; hexoses such as glucose, mannose, galactose; pentoses such as xylose, arabinose; uronic acids, glucosamines etc.; polyols such as sorbitol, glycerol; lipids and derivatives, lignin and derivatives. Particularly preferred carbon sources are glucose, a glucose-containing oligomer or polymer, a non-glucose monomeric hexose, or a polymeric sugar derivative, or mixtures thereof. Detailed Description of invention

The present invention is directed to a cell-free process for the biotechnological production of chemicals from carbon sources, in particular of hydrophobic chemicais such as C4 alcohols including n-butaπol, isobutanol or 2-butanol, and of hydrophiiic and intermediate chemicals such as ethanoi.

According to a preferred aspect, the invention discloses and claims a process for the production of a target organic compound from a carbon source by a cell-free enzyme system, comprising the conversion of glucose to pyruvate as an intermediate product wherein no net production of ATP occurs. Preferably, no net production of ATP occurs in the conversion of glucose to pyruvate as an intermediate product. More preferably, no net production of ATP occurs in the overall conversion of the carbon source to the target organic compound.

According to a preferred aspect, the carbon source compound is glucose, a glucose- containing oligomer or polymer, a non-glucose monomeric hexose, or a polymeric sugar derivative.

According to a further preferred aspect, the target organic compound is ethanol, a four- carbon mono-alcohol, in particular n-butanol, iso-butanol, 2-butanol, or another organic compound derivable from pyruvate, preferably by an enzymatic pathway.

According to a preferred embodiment of the invention, the conversion of giucose to pyruvate as an intermediate product works completely without ATP and/or ADP as cofactors. More preferably, the overall conversion of the carbon source to the target organic compound (the reaction pathway) works completely without ATP and/or ADP as cofactors.

According to a further preferred embodiment of the invention and as further described herein, the production process is performed in a ϋquid system comprising two separate phases, and the target organic compound is mainly present in or forms one of the separate phases, and the target organic compound is collected from the separate phase.

According to a further preferred embodiment of the invention and as further described herein, an organic solvent is added to establish the two separate phases. According to a further preferred embodiment of the invention and as further described herein, the carbon source compound is continuously fed to the process and the target organic compound is continuously removed.

According to a further preferred embodiment of the invention and as further described herein, the enzyme system comprises the following enzymes or enzyme activities, preferably for the conversion from glucose to pyruvate:

glucose dehydrogenase (EC 1.1.1.47), gluconolactonase {EC 3.1.1.17), gluconate dehydratase (EC 4.2.1.39),

2-keto-3-deoxy gluconate aldolase (EC 4.1.2.14), aldehyde dehydrogenase (EC 1.2.1.3), giycerate dehydrogenase (EC 1.1.1.29) or Hydroxypyruvate reductase

(EC 1.1.1.81 ), serine-pyruvate transaminase (EC 2.6.1.51 ),

• L-serine ammonia-lyase (EC 4.3.1.17) and alanine dehydrogenase (EC 1.4.1.1 ).

According to a further preferred embodiment of the invention and as further described herein, the enzyme system comprises the following enzymes or enzyme activities, preferably for the conversion from glucose to pyruvate:

glucose dehydrogenase (EC 1.1.1.47),

• gluconolactonase (EC 3.1.1 .17), gluconat dehydratase (EC 4.2.1.39) 2-keto-3-deoxy gluconate aldolase (EC 4.1.2.14) aldehyde dehydrogenase (EC 1.2.1.3) and giycerate dehydratase (EC 4.2.1.).

According to a further preferred embodiment of the invention and as further described herein, ethanol is produced from glucose by an enzyme system comprising 1 1 , 10, 9, 8, 7 or less enzymes.

According to a further preferred embodiment of the invention and as further described herein, n-butanol is produced from glucose by an enzyme system comprising 17, 16, 15 or less enzymes.

According to a further preferred embodiment of the invention and as further described herein, iso-butanoS is produced by an enzyme system comprising 14, 13 or less enzymes. According to a further preferred embodiment of the invention and as further described herein, 2-butanol is produced by an enzyme system comprising 13 or less enzymes.

According to one preferred aspect, the inventive production process comprises the following 4 steps:

I. Production of enzymes (the "target enzymes") for the conversion of a carbon source into a chemical (also herein referred to as the "target chemical" or "target organic compound") using microbial cells;

IL Release of the target enzymes from the microbial cells used in step I ₁ preferably combined with release of cofactors and with inactivation of further, non-target enzyme activities; MI. Bringing the target enzymes of step il in contact with the carbon source under conditions suitable for the conversion of the carbon source into the target chemical; iV. Separating the target chemical from the reaction mixture.

Figure 1 further illustrates a possible implementation of the concept of the invention according to a preferred embodiment.

Step i:

In step I the target enzymes are produced using microbial cells.

Sn one embodiment of the invention, enzyme production is done in two or more different microbial cell lines, such that the entire production route or major parts of it are not reconstituted in one microorganism. This avoids the unwanted initiation of substrate conversion towards the chemical and leads to a more efficient enzyme production. Enzyme production can be intracellular or extracellular, recombinant or non-recombinant. if enzyme production is recombinant it can be homologous or heterologous.

In a further embodiment of the invention, the target enzymes are selective for one substrate and one reaction. Preferably, the target enzymes have a substrate selectivity (kcat/kM) of at least 10 fold compared to any other naturally present substance and a reaction selectivity of at least 90%. More preferably, the target enzymes have a substrate selectivity of at least 20 fold and a reaction selectivity of at least 95%. Even more preferably, the target enzymes have a substrate selectivity of at least 100 fold and a reaction selectivity of at ieast 99%.

In a further embodiment of the invention, the target enzymes show no or low inhibition by the substrate or the product or other intermediates of the multistep reaction (no or low feedback inhibition) Preferably, the inhibition constants (K ₁ ) for any substrate, product or intermediate of the multtstep reaction are at least 10 fold higher than the K _M value for the respective enzyme and substrate More preferably, such inhibition constants are 100 fold higher than the respective K _M In a further, particularly preferred embodiment the target enzymes still have 50% of their maximum activity at concentrations of any substrate, intermediate or product of the multistep reaction of 100 mM or more

Preferably, the target enzymes have k _cat and K _M vaiues that are adjusted to the multistep nature of the enzymatic route

According to one embodiment of the process of the invention, the target enzymes tolerate elevated levels of the target chemical and, optionally, other organic solvents that are optionally added to support segregation of the target chemical into a separate phase Preferably the target enzymes tolerate concentrations of the target chemical of more than 2 wt%, more preferably more than 6 wt%, and most preferably more than 12 wt% In a particularly preferred embodiment, the target enzymes tolerate concentrations of the target chemicals up to the maximum solubility in water

In a preferred embodiment of the invention, the target enzymes tolerate elevated levels of chaotropic substances and elevated temperatures Preferably, the target enzymes tolerate concentrations of guanidinium chloride of more than 1 M, more preferably more than 3 M, and most preferably more than 6 M Alternatively or in combination, the target enzymes tolerate preferably temperatures of more than 50 ⁰ C, more preferably more than 70 °C, and most preferably more than 90 °C In such preferred embodiment, target enzyme production is done m a host organism whose endogenous enzyme activities are mostly inactivated at elevated levels of chaotropic substances and/or at elevated temperatures Preferably target enzyme production is done using of the following microbial species Escherichia coll, Pseudomonas fluorescence, Bacillus subtilis, Saccharomyces cerevisiae, Pichia pastons, Hansenuia polymorpha, Klyuveromyces iactiε, Trichoderma reeεes, Aspergillus niger More preferably target enzyme production is done using Escherichia coll as host organism

In a further preferred embodiment of the invention, the target enzymes tolerate elevated levels of oxygen Preferably, the target enzymes tolerate oxygen concentrations of more than 1 ppm, more preferably more than 7 ppm Most preferably the target enzymes are active and stable under aerobic conditions Preferably, the muitistep reaction does not require oxygen and is not inhibited by oxygen Thereby, no special precautions for oxygen exclusion have to be taken, making the process more stable with less effort on the production environment and/or equipment.

Preferably, enzyme production and cell growth are separated into separate phases. Thereby, no substrate is used for general metabolic activity.

Step II:

In step Il the target enzymes are released from the cells.

In a preferred embodiment, the target enzymes tolerate high temperatures and chaotropic conditions, whereas the background enzymes from the producing microorganism do not tolerate these conditions. According to this embodiment, the target enzymes are produced intracellular^ in microbial cells, the cells are lysed using high temperature and/or chaotropic conditions, thereby releasing the target enzymes in active form, optionally together with cofactors, while unwanted background enzyme activities are inactivated.

In another preferred embodiment, enzyme production is extracetiular, the target enzymes tolerate high temperatures and chaotropic conditions, and background enzyme activities (non-target enzymes) do not tolerate these conditions. According to this embodiment, the supernatant from extracellular production is treated under conditions such as high temperatures and/or chaotropic conditions. This leads to inactivation of unwanted background enzyme activities (non-target enzymes) while the target enyzmes remain active.

in one embodiment of the invention, cofactors are required for one or more of the multiple enzymatic conversion steps. In one aspect of the invention such cofactors are added to the enzyme mixture. In another, particularly preferred aspect of this embodiment such cofactors are also produced by the microbial cells intracellular^ and are released by the same treatment as to release the target enzymes. In another preferred embodiment of the invention, the microbial cells are engineered in order to optimize the level of cofactors produced.

Preferably, the microbial cells are inactivated during step II. Thereby, cell growth and enzyme activity are separated in the process, and no carbon source is consumed by undesirable cell growth. Step [It

in step II! the carbon source is converted by a mixture of enzymes in a multistep enzymatic reaction to the target chermcal

According to the inventive process, the enzymes are active under the denaturing activity of the target chemical Preferably, the microbial cells used in step I are inactive and/or are inactivated under the reaction conditions of step IM

Preferably, the concentration of each enzyme in the target enzyme mixture is adjusted to the optimal level under process conditions, in a particularly preferred embodiment one or more enzyme concentrations are increased above typical intracellular concentrations in order to improve the yield of the process (no limit by the maximal density of the microorganisms as in classical processes) in another particularly preferred embodiment, one or more enzymes are engineered for maximal catalytic efficiency (leading to lower reactor size and running costs compared to classical processes)

In a further preferred embodiment, the target chemical is added to the reaction mixture in step III at a concentration just above the maximum level that can be mixed in a single phase with water under process conditions According to this embodiment, the target chemical continuously segregates into the second phase during the process In a particular variant of this embodiment, a water soluble substance is added to the reaction mixture that leads to a phase separation of the target chemical at lower concentration than without the added substance Examples of such substances are salts and are known to the person skilled in the art In a particularly preferred variant of this embodiment, sodium chloride is added to lower the solubility of the target chemical in the water phase

in another preferred embodiment, an additional organic solvent is added to the process that forms its own phase and extracts the produced hydrophobic chemical from the water phase Preferred examples of such additional solvents comprise n-hexane, cyclohexane, octanol, ethylacetate, methylbutylketone, or combinations thereof

In another preferred embodiment, the yield is improved because the formation of side products is decreased by using target enzymes that are specific for the desired reactions in another preferred embodiment, host enzymes that would catalyse stde reactions are inactivated during step I! and/or are inactive under the reaction conditions of step 111 in yet another preferred embodiment of the invention, contamination of the process by microorganisms is avoided by adjusting reaction conditions in step Hl that are toxic for typical microbial contaminants Such conditions comprise elevated temperature, extreme pH, addition of organic chemicals In a particularly preferred embodiment of the invention, the target chemical itself is toxic at the concentration achieved in the process (more stable process with less effort on production enviroπmaent and/or equipment)

According to another preferred embodiment to the invention, no additional cofactors are added to the reaction mixture except for those cofactors that are produced by the microorganisms used in step ! and that are included in the cell lysate produced in step Il Examples of such cofactors are NAD/NADH, NADP/NADPH, ATP/ADP According to this embodiment, the cofactors that are required are produced by the microbial cells in step 1 and are regenerated during the process (NADH to NAD and vice versa, NADPH to NADP and vice versa, ADP to ATP and vice versa) In one embodiment of the invention, excess reduction equivalents (NADH, NADPH) or energy equivalents (ATP) are regenerated by additional enzymes (NADH oxidase for NADH, NADPH oxidase for NADPH, ATPase for ATP)

In a particularly preferred embodiment of the invention, neither ATP nor ADP ss involved as a cofactor in at least a substantial part of the multistep reaction process A substantial part of the multistep reaction pathway comprises preferably at least 20 % of the enzyme activities, and more preferably at least 50 % of the enzyme activities Most preferably, none of the enzyme activties of the target enzyme mixture (the enzyme mixture produced in step 1) comprises a phosphorylation step (non-phosρhorylatιve reaction pathway) In a particularly preferred embodiment of the invention the reaction pathway involves the conversion from glucose to pyruvate and none of the target enzymes involved in this conversion comprises a phosphorylation step (non-phosphorylative pyruvate production)

Step IV

In step IV the one or more target chemicals are separated from the reaction mixture

In a preferred embodiment of the invention the one or more target chemicals are hydrophobic and form a separate phase which preferably contains at least a substantial fraction of the produced chemicals In a particularly preferred embodiment the one or more target chemicals are continuousiy removed from the reaction mixture

In a further preferred embodiment of the invention, the carbon source is conttnuously fed to the reaction mixture to be converted into the target chemical Likewise the target chemical is preferably continuously removed as a separate phase and further purified by methods known in the art Thereby, product isolation is simplified as the product is collected in a separate phase from which it can be purified further Thereby, the yield is improved and product purification is simplified

A major problem of the described cell free enzymatic processes (Zhang et al , 2008, Welch and Scopes 1985) is the accumulation of ATP in the described processes this is circumvented by the addition of an ATPase To find the right concentration of ATPase, however, is difficult as it depends on the concentration of the substrate and different intermediates as well as on the activity of the enzymes With either too much ATPase or too little ATPase the conversion completely ceases (Welch and Scopes, 1985) In contrast, according to a particularly preferred aspect, the inventive cell-free process converts a carbon source such as glucose and its polymers to a target chemical such as ethanoi, butanol and/or isobutanol without net production of ATP and without using an ATPase

In a further preferred embodiment, the inventive process does not require ADP or ATP as cofactors Other processes (Welch and Scopes, 1985, Algar and Scopes, 1985) require cofactors such as ADP/ATP and NAD7NADH The postulated conversion of glucose to Butanol (Zhang et al, 2008} requires the cofactors ADP/ATP, NAD7NADH, Ferredoxin and Coenzyme A

As used herein, the term "enzyme" encompasses also term 'enzyme activity" and may be used interchangeably

Several preferred embodiments are hereinafter described regarding the production of n- butanol isobutanol, ethanol and 2-butanol Production of n-butanol

in one embodiment of the invention, the target chemical is n-butanol and the target enzyme mixture to produce n-butanol comprises less than 19 different enzyme activities. Preferably the target enzyme mixture comprises 17 or less different enzyme activities, more preferably 16 or less enzyme activities, even more preferably 15 or less enzyme activities, and most preferably only 12 different enzyme activities. Because production of enzymes is a major cost factor in the process this gives a major advantage over other processes.

In a particularly preferred embodiment, n-butanol is produced from glucose via the intermediates pyruvate and acetyl CoA. The production of n-butanol from glucose can be arbitrarily subdivided into three steps: (A) Conversion of one molecule giucose to two molecules of pyruvate. (B) Conversion of two molecules pyruvate to two moiecuies acetyl CoA. (C) Conversion of two molecules acetyl CoA to one molecule n-butanol. Step (A): Conversion of giucose to pyruvate.

This step is common in most if not ail organisms, even though different metabolic pathways exist (e.g. Embden-Meyerhof-Parnas way, Entner-Doudoroff-Way). A particular variant of step (A) uses enzymes from the Embden-Meyerhof-Parnas way, comprising the 10 enzyme activities as listed in Tab. 1 (enzyme combination A.1).. The reaction pathway catalysed by this enzyme combination includes phosphorylating enzymes and leads to net ATP production (2 molecules ATP are generated per molecule glucose).

Table 1 : Enzyme combination A.1 , comprising enzymes from the Embden-Meyerhof-Parnas way Another particular variant of step (A) uses enzyme activities of the Entner-Doudoroff pathway. According to a preferred embodiment of the invention, enzymes from the non- phosphorylative Entner-Doudoroff pathway known from archaea are used in step (A). This comprises the 8 enzyme activities listed in Tab. 2 (enzyme combination A.2). This enzyme combination comprises phosphoryiating enzymes and, therefore, requires ATP and/or ADP as cofactors, but the conversion of glucose to pyruvate does not lead to net production of ATP.

Table 2: Enzyme combination A.2, comprising enzymes from the non-phosphorylative Entner-Doudoroff pathway

Another particularly preferred variant of step (A) uses an enzyme combination that neither leads to net production of ATP nor does it require ATP or ADP as cofactors. The respective enzyme combination comprises the 9 different enzyme activities as listed in Tab. 3 (enzyme combination A.3).

Table 3: Enzyme combination A.3, comprising enzyme activities from various pathways

A further, particularly preferred variant of step (A) uses another enzyme combination that also neither leads to net production of ATP nor does it require ATP or ADP as cofactors. The respective enzyme combination comprises the 5 different enzyme activities as listed in Tab. 4 (enzyme combination A.4).

Table 4: Enzyme combination A.4, comprising enzyme activities from various pathways

Step (B): Conversion of pyruvate to acetyϊ CoA

Various options exist for the conversion of pyruvate to acetyl CoA.

in one embodiment of the invention, one or more of the following enzymes is used for the conversion: (i) pyruvate oxidoreductase using ferredoxin as cofactor; (ii) pyruvate dehydrogenase using NAD(P)H as cofactor; (ϋi) pyruvate formate lyase; (iv) pyruvate dehydrogenase enzyme complex.

In a peferred embodiment, pyruvate dehydrogenase is used as the enzyme for this conversion, using NADH as cofactor (as fisted in Tab. 5).

Table 5: Enzyme combination B.1

Pyruvate dehydrogenases are usually part of a multi enzyme complex (Pyruvate dehydrogenase complex, PDC) which consists of three enzymatic activities and has a molecular weight of ca. 1 Mio Da. For application in a cell-free reaction system it is beneficial to have small and robust non-complexed enzymes. It has been found that the pyruvate dehydrogenase from Euglena gracilis can be used in the enzyme mixture B.1. This enzyme is singular and complex-free. Furthermore it uses NADH as cofactor which is compared to NADPH the more suitable cofactor (available in higher quantities; easier to regenerate). Alternatively, pyruvate formate lyase can be combined with a formate dehydrogenase using NADH as cofactor.

Step (C): Conversion of acetyl CoA to n-butano!

Various options exist for the conversion of acetyl CoA to n-butanol, as several microorganisms produce n-butanol via this pathway (e.g. C. acetobutylicum, C. beijeήnckii).

In a preferred variant of step (C), the enzymes as listed in Tab. 6 are used for the conversion. Depending on the enzymes (source organisms) different cofactors are used.

Table 6: Enzyme combination C.1 for the conversion of Acetyl CoA to n-Butanol

In another preferred variant the reaction pathway from the intermediate Butyryi CoA to Butanoi does not involve butyrate but butyryl phosphate. The listed pathways and the enzymes are not exclusive. Alternative enzymes and routes known in the art can also be used.

When all reaction steps as described above are combined (enzyme combination A.1 , A.2, A.3 or A.4; plus enzyme combinations B.1 and C.1) a net conversion of one moiecule glucose to two molecules of CO ₂ , one moiecule of water and one moiecule of n-butanol is achieved. None of the enzyme combinations leads to a net production of reduction equivalents (4 molecules H ₂ are released and subsequently used again). Depending on the route from glucose to pyruvate none (A.2, A.3 or A.4) or up to 2 molecules of ATP (A.1 ) are generated from ADP and phosphate.

In a particuiarly preferred embodiment of the invention, enzyme combinations A.4, B.1 and C.1 are combined. This target enzyme mixture comprises only 12 different enzyme activities, does not require ADP/ATP as cofactor and, consequently, does not lead to a net ATP production. In another particularly preferred embodiment of the invention, enzyme combinations A.3, B.1 and C.1 are combined. This target enzyme mixture comprises 16 different enzyme activities, does not require ADP/ ATP as cofactor and, consequently, does not lead to a net ATP production.

In another preferred embodiment of the invention, enzyme combinations A.2, B.1 and C.1 are combined. This target enzyme mixture comprises 15 different enzyme activities, requires ADP/ATP as cofactors, but does not lead to a net ATP production.

Production of isobutano!

In another embodiment of the invention the target chemical is isobutanol.

In a particularly preferred embodiment, isobutanol is produced from glucose via the intermediate pyruvate. The production of isobutanol from glucose can be arbitrarily subdivided in two steps: (A) Conversion of one molecule glucose to two molecules pyruvate; and (D) Conversion of two molecules pyruvate to one molecule iso-butanoi.

Step (A): Conversion of glucose to pyruvate

This step is equivalent to step (A) in the production of n-butanol (see above).

Step (D): Conversion of pyruvate to isobutanol

in a preferred embodiment of the invention, the enzyme activities as listed in Table 7 are used in Step (D) for the cell free production of isobutanol.

Table 7: Enzyme combination D.1 , comprising enzyme activities for the conversion from pyruvate to isobutanol When all reactions as described above are combined (enzyme combinations A.1 , A.2, A.3 or A.4; plus enzyme combination D.1 ) a net conversion of one molecule glucose to two molecuies CO ₂ , one molecule water and one molecule isobutanol is achieved. None of the enzyme combinations leads to a net production of reduction equivalents (4 molecules H ₂ are formed in the formation of two molecules pyruvate and are subsequently used up in the formation of isobutanol). Depending on the route from glucose to pyruvate none (A.2, A.3 or A.4) or up to 2 molecules of ATP (A.1 ) are generated from ADP and phosphate.

In a particularly preferred embodiment of the invention, enzyme combinations A.4 and D.1 are combined. This target enzyme mixture comprises only 10 different enzyme activities, does not require ADP/ ATP as cofactor and, consequently, does not Sead to a net ATP production.

in another particularly preferred embodiment of the invention, enzyme combinations A.3 and D.1 are combined. This target enzyme mixture comprises 14 different enzyme activities, does not require ADP/ATP as cofactor and, consequently, does not lead to a net ATP production.

In another preferred embodiment of the invention, enzyme combinations A.2 and D.1 are combined. This target enzyme mixture comprises 13 different enzyme activities, requires ADP/ATP as cofactors, but does not lead to a net ATP production.

Production of ethanol

In another embodiment of the invention, the target chemical is ethanol. Ethanol is completely soluble in water at any ratio and does not allow easy purification by formation of a separate organic phase. The inventive process for the cell-free production nevertheless provides a cost-efficient way to produce ethanol from renewable carbon sources.

In a particuiarly preferred embodiment, ethanol is produced from glucose via the intermediate pyruvate. The production of ethanol from glucose can be arbitrarily subdivided in two steps: (A) Conversion of one molecule glucose to two molecuies pyruvate; and (E) Conversion of two molecules pyruvate to two molecules ethanol.

Step (A): Conversion of glucose to pyruvate

This step is equivalent to step (A) in the production of n-butanol (see above). Step (E): Conversion of pyruvate to ethanol

In a preferred embodiment of the invention, the enzyme activities as listed in Table 8 are used for the conversion of pyruvate to ethanol.

Table 8: Enzyme combination E.1 , comprising enzyme activities for the conversion from pyruvate to ethanol

When aSI reactions as described above are combined (enzyme combinations A.1, A.2, A.3 or A.4; plus enzyme combination E.1 ) a net conversion of one molecule giucose to two molecules CO ₂ and two molecules ethanol is achieved. None of the enzyme combinations leads to a net production of reduction equivalents. Depending on the route from glucose to pyruvate none (A.2, A.3 or A.4) or up to 2 molecuies of ATP (A.1 ) are generated from ADP and phosphate.

in a particularly preferred embodiment of the invention, enzyme combinations A.4 and E.1 are combined. This target enzyme mixture comprises only 7 different enzyme activities, does not require ADP/ATP as cofactor and, consequently, does not lead to a net ATP production.

In another particularly preferred embodiment of the invention, enzyme combinations A.3 and E.1 are combined. This target enzyme mixture comprises 1 1 different enzyme activities, does not require ADP/ATP as cofactor and does not lead to a net ATP production.

in another preferred embodiment of the invention, enzyme combinations A.2 and E.1 are combined. This target enzyme mixture comprises 10 different enzyme activities, requires ADP/ATP as cofactors, but does not lead to a net ATP production.

Production of 2-butanol

In another embodiment of the invention the target chemical is 2-butanol.

In a particularly preferred embodiment, 2-butanol is produced from giucose via the intermediate pyruvate. The production of 2-butanol from glucose can be arbitrarily subdivided in two steps: (A) Conversion of one molecule glucose to two molecuies pyruvate; and (F) Conversion of two molecules pyruvate to one molecule 2-butanol. Step (A): Conversion of glucose to pyruvate

This step is equivalent to step (A) in the production of n-butanol (see above).

Step (F): Conversion of pyruvate to 2-butanoi

In a preferred embodiment of the invention, the enzyme activities as listed in Table 9 are used in Step (F) for the eel! free production of 2-butanol. In a preferred embodiment an aicohole dehydrogenase is used that uses acetoin as well as 2-butanon as substrate.

Table 9: Enzyme combination F.1 , comprising enzyme activities for the conversion from pyruvate to 2-butanol

When all reactions as described above are combined (enzyme combinations A.1 , A.2, A.3 or A.4; pius enzyme combination F.1 ) a net conversion of one molecule glucose to two molecules CO ₂ , one molecule water and one molecule 2-butanoi is achieved. None of the enzyme combinations leads to a net production of reduction equivalents (4 molecules H ₂ are formed in the formation of two moiecuies pyruvate and are subsequently used up in the formation of 2-butanol). Depending on the route from glucose to pyruvate none (A.2, A.3 or A.4) or up to 2 molecules of ATP (A.1 ) are generated from ADP and phosphate.

In a particularly preferred embodiment of the invention, enzyme combinations A.4 and F.1 are combined. This target enzyme mixture comprises only 9 different enzymes, does not require ADP/ATP as cofactor and, consequently, does not lead to a net ATP production.

In another particularly preferred embodiment of the invention, enzyme combinations A.3 and F.1 are combined. This target enzyme mixture comprises 14 different enzyme activities, does not require ADP/ATP as cofactor and, consequently, does not lead to a net ATP production. in another preferred embodimenl of the invention, enzyme combinations A.2 and F.1 are combined. This target enzyme mixture comprises 13 different enzyme activities, requires ADP/ ATP as cofactors, but does not lead to a net ATP production.

Other substrates than glucose

The described routes of the invention for the production of ethanoi, n-butanol, isobutanol, 2- butanol or other chemicals are not limited to the use of giucose as substrate. Depending on the selectivity of the glucose dehydrogenase applied in the process e.g. also other C6-sugars can be used as substrate, in addition starch can be used as substrate in combination with an amylase/glucoamylase activity. Ceflulosic material can be used as substrate together with an endoceliulase/exoceiluiase/glucosidase activity. Also lactose, sucrose and other oligomeric or poiymeric sugar derivatives can be used together with the corresponding enzymes that convert these to monomeric hexoεes.

For one embodiment of the invention, lignoceiluiosic material is converted to ethanoi using only 10 enzymes in total (enzymes combinations A.4 and E.1 , endoceiluiase, exocellulase, beta-glucosidase).

Examples

The present invention is further defined in the following examples. It should be understood that these examples are given by way of illustration only and are not limiting the scope of the invention. From the above discussion and these examples, a person skilϊed in the art can ascertain the essential characteristics of this invention and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.

Example 1 : Conversion of glucose to n-butanol using enzyme mixtures from celi Ivsates of C. saccharobutylicum, S. cerevisae and Z, mobilis

S. cerevisae is grown anaerobically at 30 ⁰ C on YPD-medium containing 12 % giucose. The formation of ethanoi is monitored by sampling every hour and analysis by gas chromatography, in a phase of highest ethanoi productivity the cells are harvested and suspended with 4x volume of reaction buffer (20 mM potassium phosphate, pH 6.5; 5 mM mercaptoethanol, 10 mM NaCI ₁ 10 mM MgSO ₄ , 500 μM ZnSO ₄ , 500 μM CoCI ₂ , 200 μM MnCi ₂ ). Ceils are then lysed by disruption in a French press, the cell supernatant is clarified by centrifugation and fiStersterilized to remove any remaining cells.

Z. mobilis (ATCC10998) is grown at 35 ⁰ C on LB medium containing 12 % glucose. The formation of Ethanol is monitored by sampling every hour and analysis by gas chromatography. In a phase of highest ethanol productivity the cells are harvested and suspended with 4x volume of reaction buffer (20 mM potassium phosphate, pH 6,5; 5 mM mercaptoethanol, 10 mM NaCI ₅ 10 mM MgSO ₄ , 500 μM ZnSO ₄ , 500 μM CoCI ₂ , 200 μM MnCS ₂ ). Cells are then lysed by disruption in a French press and cell supernatant is clarified by centrifugation and filtersterilized to remove any remaining cells.

Clostridium saccharobutylicum DSMZ 13864 is grown on TYA medium (6 g/i Tryptone peptone, 2 g/l Yeast extract, 3 g/l NH ₄ CH ₃ COO, 0,3 g/l MgSO ₄ , 0,5 g/l KH ₂ PO ₄ , 1 mg/l Resazurin, 10 mg/l MnSO ₄ , 1 mg/l p-aminobenzoic acid, 1 mg/l thiamine chloride hydrochloride, 0,2 mg/i biotin) with 6% glucose concentration at 35 ⁰ C. The formation of butanol is followed by sampling every 2 h and analysis by GC. After 40 h the celis are in a solvent producing phase. The cells are harvested, lysed and the lysate is filter sterilized.

One millilitre of cell lysate from C. saccharobutylicum is incubated with 100 mM glucose with or without equal amount of the lysaie from S. cerevisae or Z. mobilis. In all reactions 10 mM ketomalonic acid (71740, Fluka) are added, which acts as inhibitor of pyruvate decarboxylase. After 2 h anaerobic incubation at 30 ⁰ C the produced n-butanol is quantified in all reactions.

Example 2: Conversion of glucose to n-butanol using isolated enzymes and enzymes from ceNjysates of C. saccharobutylicum

The eel! lysate of C. saccharobutyiicum from example 1 is incubated with the following enzymes: hexokinase (20 u/ml), phosphohexoisomerase (15 u/ml), phosphofructokinase (4 u/ml), aldolase (16 u/ml), phosphotrioseϊsomerase (300 u/ml), glycerinaldehyd-3-phosphat- dehydrogenase (60 u/ml), phoεphoglycerate kinase (120 u/ml), phosphoglycerate mutase (60 u/ml), enolase (11 u/ml), pyruvatkinase (15 u/ml), pyruvat dehydrogenase (20 u/ml), phosphotranεacetyiase (15 u/ml) (ail enzymes obtained from Sigma, see table below, only phosphoglycerate mutase obtained from USB, 261 11 Miles Road, Cleveland Ohio, Product number 261 18 100 UG, human enzyme recombinant^ expressed in E .cofi). incubation is done in 20 mM potassium phosphate, pH 6.5; 5 mM mercaptoethanol, 10 mM NaCI ₁ 10 mM MgSO ₄ , 500 μM ZnSO ₄ , 500 μM CoCI ₂ , 200 μM MnCI ₂ , 4 mM ATP ₁ 1 mM ADP, 1 mM NADH, 1 mM NAD 1mM Coenzyme A and 200 mM glucose at 30 ⁰ C under anaerobic conditions AN reagents used are oxygen depleted by standard methods After 10 mm ATPase (0 5 u/ml) is added to avoid early built up of ATP After 3 hours the produced n-butanoi is quantified

Table 10 Enzymes (Sigma-Aldπch) for the conversion of glucose to Acetyl CoA

Example 3 Conversion of glucose to n-butanol using isolated enzymes

The enzymes Thioiase (EC 2 3 1 16, Clostridium acetobutylicum, NCBI-GenlD NP_349476 1 ), 3-hydroxybutyryl-CoA dehydrogenase (EC 1 1 1 157, NPJ549314 1), Crotonase (EC 4 2 1 55, Clostridium acetobutylicum, NP__349318 1 ), Butyryl-CoA dehydrogenase (EC 1 3 99 2, Clostridium acetobutylicum, NCBI-GenlD NP_349317 1 ) Coenzyme A acylating aldehyde dehydrogenase (EC 1 2 1 57, Clostridium beijerinckii NCBI- GenlD AF132754J ) NADH-dependent butanol dehydrogenase B (BDH N) (EC 1 1 1 -, Clostridium acetobutylicum, NCBl-GeniD NPJ349891 1 } and an electron transfer flavoprotein (etfA und B, Clostridium acetobutylicum, NCBI-GenlD NP_349315 1 and NP_349316 1 ) are synthesized and recombinarrtly expressed tn E coll as described All enzymes are combined (1 mg each) in a 5 ml reaction solution (20 mM potassium phosphate, pH 6,5, 5 mM mercaptoethanol, 10 mM NaCI, 10 mM MgSO ₄ , 500 μM ZnSO ₄ , 500 μM CoCI ₂ , 200 μM MnCI ₂ , 4 mM ATP ₁ 1 mM ADP, 1 mM NADH, 1 mSVI NAD, 1 mM Coenzyme A) An equal volume of the enzyme mixture as listed in Tab 9 above (conversion of glucose to acetyl CoA) is added hexokinase (200 u/ml), phosphohexoisomerase (150 u/ml), phosphofrukto- kinase (40 u/ml), aldolase (160 u/ml), phosphotrioseisomerase (3000 u/ml) glyceπnaldehyd- 3-phosphat-dehydrogenase (600 u/ml), phosphoglycerate kinase (1200 u/ml), phosphoglycerate mutase (600 u/ml), enoiase (1 10 u/ml), pyruvatkinase (150 u/ml), pyruvatdehydrogenase (200 u/mi), phoεphotransacetylase (150 u/ml), ATPase (4,5 u/ml) combined in 20 mM potassium phosphate buffer (pH 6 5) including 500 mM glucose The reaction is stirred under anaerobic conditions at 30 ⁰ C After 3 hours the produced n-butanol is quantified

Alternatively, the reaction is performed under continuous addition of glucose at a rate of 120 mg/h (manual addition of 60 mg in 30 minutes intervals, beginning 2 h after start of reaction) After total reaction time of 20 hrs the produced n-butanol is quantified

Example 4 Conversion of glucose to isobutanol using isolated enzymes and a cell Ivsate from T maritima

T maritima is grown at 80 ⁰ C on 5 % glucose Before complete utilisation of glucose the cells are harvested, resuspended in 4x volume of reaction buffer (20 mM potassium phosphate, pH 6,5, 5 mM mercaptoethanol, 10 mM NaCI, 10 mM MgSO ₄ , 500 μM ZnSO ₄ , 500 μM CoCI ₂ , 200 μM MnCI ₂ ) and lysed by disruption in a French press The cell supernatant is clarified by ceπtnfugation and sterilized by filtration. The genes ilvlCD for valine biosynthesis (acetolacfate synthetase, EC 2 2 1 6, NCBI-GenelD NP_228358 1 , ketol-acid reductoisomerase, EC 1 1 1 86, NCBi-GenelD NP_228360 1 and dihydroxy-acsd dehydratase, EC 4 2 1 9, NCBi-GenelD NP_228361 1 ) are cloned from Thermotoga maritima and recombinant^ expressed in E coil by standard methods α-ketoιsovalerate decarboxylase from Lactococcus lactis (EC 4 1 1 -, NCBI-GenelD CAG34226 1 ) is cloned and recombinantiy expressed in E coll by standard methods Alcohol dehydrogenase (EC 1 1 1 1 ) from S cerevisiae is purchased from Stgma-Aϊdπch (A3263)

All enzymes are combined in 5 ml of reaction solution (20 mM potassium phosphate, pH 6 5, 5 mM mercaptoethanol, 10 mM NaCI, 10 mM MgSO ₄ , 500 μM ZnSO ₄ , 500 μM CoCl ₂ , 200 μM MnCI ₂ , 4 mM ATP, 1 mM ADP, 1 mM NADH, 1 mM NAD) at a concentration of 100 u/ml An equal volume of eel! lysate from T maritima is added and the combined sample is incubated at 50 ⁰ C with 250 mM glucose After 1 h the produced isobutanol is quantified Example 5: Conversion of glucose to ethanol using isolated enzymes

The genes for the enzymes: glucose dehydrogenase (EC 1.1.1.47, S. soifataricus, NCBI Gen ID: NP_344316.1 }, gluconoiactonase (EC 3.1.1 .17, Picrophilus torridus, NCBI Gen ID YPJ323685.1), gluconate dehydratase (EC 4.2.1.39, Sulfolobus solfataricus, NCBl-Gene ID: NP__344505, Mutation I9L), 2-keto-3-deoxy gluconate aldolase (EC 4.1.2.14, Sulflobus solfataricus, NCBI Gen ID NPJ344504.1 ), aldehyde dehydrogenase (EC 1.2.1.3, Fiavobacterium frigidimaris, NCBI Gen ID: BAB96577.1 ), glycerate kinase (EC 2.7.1.-, Sulfolobus solfataricus, NCBI Gen ID: NP_342180.1 ), enolase (EC 4.2.1.1 1 , Sulfolobus solfataricus, NCB! Gen ID: NP_342405.1) and pyruvate kinase (EC 2.7.1.40, Sulfoiobus solfataricus, NCBS Gen ID: NP_342465.1 ) are synthesized and cloned into expression vector pET3b by standard methods using Ndel and BamHI for cloning. Cell growth, protein expression and partial purification is done as described by Lamble et a!. (2003) but in addition supplying all buffers with 5 mM mercaptoethanol and using 80 ⁰ C as general temperature for heat precipitation (for the aldehyde dehydrogenase 60 °C is used). Protein production is usually between 5 and 50 mg/i. Pyruvate decarboxylase and aldehyde dehydrogenase from S. cerevisiae are obtained from Sigma-Aldrich (P# 29163 and 82884).

All enzymes are combined (1 mg each of the recombϊnantly produced enzymes and 2 u of the purchased enzymes) in 5 ml of reaction solution (20 mM potassium phosphate, pH 6.5; 5 mM mercaptoethanol, 10 mM NaCI, 10 mM MgSO ₄ , 500 μM ZnSO ₄ , 500 μM CoCI ₂ , 200 μM MnCI ₂ , 4 mM ATP, 1 mM ADP, 1 mM NADH, 1 mM NAD) containing 250 mM glucose and incubated at 47 ⁰ C. Every 30 min the two yeast enzymes are added to the solution to compensate for thermal inactivation. After 6 hrs the produced ethanoi is quantified. Ethanol is produced from glucose using only 10 enzymes. In addition no net ATP is produced.

Example 6: Conversion of glucose to ethanol using isolated enzymes

While it is of advantage to produce ethanol or chemicals and fuels from glucose without a net production of ATP or any other cofactor it is more beneficial when ATP or ADP as cofactor are eliminated entirely from the process. Therefore, the enzymes as described in example 5 but without the enzymes glycerate kinase (EC 2.7.1.-), enolase (EC 4.2.1.1 1 ) and pyruvate kinase (EC 2.7.1.40) are used. The conversion of glycerate to pyruvate is achieved using the enzymes glycerate dehydrogenase (alias hydroxypyruvate reductase (EC 1.1.1.29 / 1.1.1.81 )), serine-pyruvate transaminase (EC 2.6.1.51), L-serine ammonia-iyase (EC 4 3 1 17) and alanine dehydrogenase (EC 1 4 1 1 ) The genes for the enzymes glycerate dehydrogenase/hydroxypyruvate reductase (EC 1 1 1 29 / 1 1 1 81 , Picrophilus torπdus, NCBI Gen iD YP_023894 1), serine-pyruvate transaminase (EC 2 6 1 51 , Sulfolobus solfataricus, NCBI Gen ID NP_343929 1 ), L-seπne ammonta-lyase (EC 4.3.1 17, Thermus thermophiluε, YPJ 44295 1 and YP_144005 1 ) and alanine dehydrogenase (EC 1 4 1 1 , Thermus thermophilus, NCBI-Gen ID YPJJ05739 1 ) are synthesized (codons optimized for production in E cod) and cloned into expression vector pET3b using the Ndel and BamHI Protein expression is done in E coli

Al! enzymes are combined (1 mg each of the recombinantly produced enzymes and 2 u of the purchased enzymes) in 5 ml of reaction solution (20 mM Potassium phosphate, 10 mM ammoniumsulfate, pH 6 5, 5 mM mercaptoethanol, 10 mM NaCI, 10 mM MgSO ₄ , 500 μM ZnSO ₄ , 500 μM CoCI ₂ , 200 μM MnCI ₂ 1 mM NADH, 1 mM NAD, 1 mM serine, 1 mM alanine) containing 250 mM glucose and incubated at 47 ⁰ C Every 30 mm the two yeast enzymes are added again to the solution to compensate for thermal inactivation. After 6 hrs incubation the produced ethanol is quantified Ethanol is produced from glucose without involving phosphoryiation and without requiring ATP or ADP as cofactor

Example 7 Conversion of glucose to ethanol using isolated enzymes

The formation of pyruvate from giycerate ss a critical step in the production of ethanol or other derivatives of pyruvate Dihydroxyacid dehydratase (EC 4 2 1 9) from S solfataricus has been shown to accept different substrates (Kim and Lee, 2006) The gene for dihydroxyacid dehydratase (EC 4 2 1 9, S solfataπcus, NP_344419 1 ) is expressed as described by Kim and Lee (2006) The enzyme is able to convert giycerate to pyruvate (though at lower activity in comparison to the natural substrate) The enzyme is also active towards gluconate (though at lower activity in comparison to natural substrate}

The enzymes glucose dehydrogenase, giuconoiactonase, 2-keto-3-deoxy gluconate aldolase and aldehyde dehydrogenase from example 9 are combined with dihydroxyacid dehydratase (as described above) and pyruvate decarboxylase and aldehyde dehydrogenase from Sigma (see example 5) (1 mg each of the recombinantly produced enzymes and 2 u of the purchased enzymes) in 5 ml of reaction solution (20 mM potassium phosphate, 10 mM ammoniumsulfate, pH 6 5, 5 mM mercaptoethanol 10 mM NaCI, 10 mlVi MgSO ₄ , 500 μM ZnSO ₄ , 500 μM CoCI ₂ , 200 μM MnCI ₂ I mM NADH, 1 mM NAD) containing 250 mM glucose and incubated at 47 ⁰ C Every 30 mm the two yeast enzymes are added again to the solution to compensate for thermal inactivation. After 6 hrs incubation the produced ethanoi is quantified.

Example 8: Conversion of glucose to n-butanol using isolated enzymes without net production of ATP

The genes of the 4 units of pyruvate synthase (EC 1.2.7, 1., Suifoiobus solfataricυs, NCBI- GeniD: NP_342664.1 , NP_342663.1 , NP_342666.1 , NP_343517.1 ) and the gene of NADH ferrodoxin reductase (EC 1.18.1.3, NCBl-GenlD NP_342682.1 ) are synthesized and cloned in pET3b using Ndel and BamHI sites. The genes are anaerobically expressed in E. coli and partially purified as described. Glucose dehydrogenase, giuconoiactoπase, gluconate dehydratase, 2-keto-3-deoxy gluconate aldolase, aldehyde dehydrogenase, glycerate kinase, enolase and pyruvate kinase are obtained as described in example 5. Thiolase, β- hydroxybutyryiCoA dehydrogenase, butyrylCoA dehydrogenase, CoA acylating butanal dehydrogenase and butano! dehydrogenase with etfA and etfB are obtained as described in example 3.

Ail enzymes are combined (1 mg each) in 10 mi of reaction solution (20 mM potassium phosphate, pH 6,5; 5 mM mercaptoethanol, 10 mM NaCI, 10 mM MgSO ₄ , 500 μM ZnSO ₄ , 500 μM CoCI ₂ , 200 μM MnCI ₂ 4 mM ATP, 1 mM ADP, 1 mM NADH, 1 mM NAD, 1mM Coenzyme A, 1 mM FAD, 1 mM FADH ₂ ) containing 250 mM glucose. The solution is incubated at 45 ⁰ C. After 1 h the produced butanol is quantified.

Example 9: Conversion of glucose to n-butanol using isolated enzymes without net production of ATP

Glucose dehydrogenase, gluconolactonase, 2-keto-3-deoxy gluconate aldolase and aldehyde dehydrogenase are obtained as described in example 5. Dihydroxyacid dehydratase is obtained as described in example 9. NADH ferrodoxin reductase and pyruvate synthase are obtained as described in example 10. Thiolase, β-hydroxybutyrylCoA dehydrogenase, butyrylCoA dehydrogenase, CoA acylating butanal dehydrogenase and butanol dehydrogenase with etfA and etfB are obtained as described in example 3.

All enzymes are combined (1 mg each) in 10 ml of reaction solution (20 mM potassium phosphate, pH 6,5; 5 mM mercaptoethanol, 10 mM NaCl, 10 mM MgSO ₄ , 500 μM ZnSO ₄ , 500 μM CoCI ₂ , 200 μM MnCI ₂ I mM NADH, 1 mM NAD, 1 mM Coenzyme A, 1 mM FAD ₁ 1 mM FADH ₂ ) containing 250 mM glucose. The solution is incubated at 45 ⁰ C. After 1 h the produced n-butanol is quantified.

Example 10: Conversion of glucose to isobutanol using isolated enzymes without net production of ATP

The catalytic unit of acetoiactate synthase (EC 2.2.1.6, Sulfolobus soifataricus, NCBI-GenlD: NP_342102.1 ), ketol-acid reductoisomerase (EC 1.1.1.86, Sulfolobus solfataricuε, NCBI- GenlD: NP_342100.1 ), branched-chain-2-oxo acid decarboxylase (EC 4.1.1.72, Lactococcus lactis, NCBI-GenlD:) and an alcohol dehydrogenase (EC 1 .1.1.1 , Fiavobacterium frigidimaris, NCB!-Gen!D: BAB9141 1.1 ) are cloned in pET3b using Ndel and BamHi sites. The genes are expressed in E. coli. Together with dihydroxyacid dehydratase (see example 7) these enzymes constitute the pathway from pyruvate to isobutanol. Glucose dehydrogenase, gluconolactonase, gluconate dehydratase, 2-keto-3-deoxy gluconate aldolase, aldehyde dehydrogenase, glycerate kinase, enolase and pyruvate kinase are obtained as described in example 5. Dihydroxyacid dehydratase is obtained as described in example 5.

All enzymes are combined (1 mg each) in 10 mi of reaction solution (20 mM potassium phosphate, pH 6,5; 5 mM mercaptoethanol, 10 mM NaCI, 10 mM MgSO ₄ , 500 μM ZnSO ₄ , 500 μM CoCI ₂ , 200 μM MnCl ₂ 4 mM ATP ₁ 1 mM ADP, 1 mM NADH, 1 mM NAD) containing 250 mM glucose. The solution is incubated at 53 ⁰ C for 1 h. Every 10 min fresh branched- chain-2-oxo acid decarboxylase is added to compensate for thermal inactivation. After 1 h the produced isobutanol is quantified.

References

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Lamble, HJ. ; Heyer, N. I.; Bull, S.D.; Hough, D.W. and Danson MJ. (2003) J.

Biol.Chem. 36 (5) 34066-72.

Welch, P und Scopes R. K: (1985) Journal of Biotechnology 2(5) 257-273.

Zhang Y.-H.P., Evans B.R., Mielenz J.R., Hopkins R.C., Adams M.W.W. (2007) PLoS

ONE 2(5): e456.

Zhang Y.H.P., Ye, X. And Wang, Y. (2008) Biotechnology: Research, Technology and Applications

The following are exemplary and non-limiting sequences useful in various embodiments of the invention: Acetyl-CoA acetyltransferase (Thiolase) (2.3.1.16)

>gi I 15896127 ! ref J NP_34947e . 1 I acecyl - CoA acetyltransf erase [Clostridium acetobutylicum ATCC 824 ]

MKEWIASAWTAIGSYGKΞLKDVPAVDLGATAIKEAVKJKAGIKPEDVNEVILGNVL QAGLGQNPARQAS FKAGL PVE I PAMT INKVCGS GLRTVΞ LAAQI I KAGDAD VI I AGGMENMS RAPYL ANHARWGYRMGKAKF V DEMITDGLWDAFND YHMGITAENIAΞRWNISREΞQDEFALASQKKAΞEAIKSGQFKDEIVPVVIKGRKGE

AGVD PAIMGYG P FYATKAAI EKAGWTVDELDLI ESNE AFAAQS L AVAKDLKFDMNKVNVNGG AI ALGHP I GASGARILVTLVHAMQKRDAKKGLATLCIGGGQGTAILLEKC

3-hydroxybutyryi-CoA dehydrogenase (1.1.1.157)

>gi I 15895965 | ref |NP_349314.1 | 3 -hydroxytautyryl-CoA dehydrogenase [Clostridium acetobutylicum ATCC 824]

MKKVCVIGAGTMGSGIAQAFAAKGFEWLRDIKDEFVDRGLDFINKNLSKLVKKGKIE ΞATKVEILTRIS GTVDLNMAADCDLVIEAAVERMDIKKQIFADLDNICKPETILAΞNTΞSLSITΞVASA TKRPDRVIGMHFF NPAPVMKLVEVIRGIATSQETFDAVKETSIAIGKDPVEVAEAPGFWKrRILIPMINEAVG ILAEGIASVE DIDKAMKLGANHPMGPLELGDFIGLDICLAIMDVLYSETGDSKYRPHTLLKKYVRAGWLG RKSGKGFYDY SK

3-hydroxybutyryS-CoA dehydratase (Crotonase) (4,2.1.55)

>gi | 158959S9 | ref JNP_349318.11 3 -hydroxybutyryl- CoA dehydratase [Clostridium acetobutylicum ATCC 824]

MELNNVILEKEGKVAWTINRPKALNALNSDTLKEMDYVIGEIΞNDSEVLAVILTGA GEKSFVAGADISΞ MKEMNTIEGRKFGILGNKVFRRLELLEKPVIAAVNGFALGGGCEIAMSCDIRIASΞNAR FGQPEVGLGIT PGFGGTQRLSRLVGMGMAKQLIFTAQNIKADEALRIGLVNKWΞPSELMNTAKEIANKIV SNAPVAVKLS KQAINRGMQCDIDTALAFESEAFGECFSTEDQKDAMTAFIEKRiCIEGFKNR butyryl-CoA dehydrogenase (1.3.99.2)

>gi 115895968 ! ref I NP_349317.1 J butyryl-CoA dehydrogenase [Clostridium acetobutylicum ATCC 824]

MDFNLTREQELVRQMVREFAENEVKPIAAEIDETERFPMENVKKMGQYGMMGIPFSK ΞYGGAGGDVLSYI lAVEELSKVCGTTGVILSAKTSLCASLINEHGTEEQKQKYLVPLAKGΞKIGAYGLTEPN AGTDSGAQQTV

AVLΞGDHYVINGSKIFITNGGVADTFVIFAMTDRTKGTKGISAFIIEKGFKGFSIG KVΞQKLGIRASSTT

ELVFEDMIVPVΞNMIGKEGKGFPIAMKTLDGGRIGIAAQALGIAEGAFNEARAYMK ERKQFGRSLDKFQG

LAWMMADMDVAIESARYLVYKAAYLKQAGLPYTVDAARAKLKAANVAMDVTTKAVQL FGGYGYTKDYPVE

RMMRDAKITΞIYΞGTSEVQKLVIΞGKIFR coenzyme A acylating aldehyde dehydrogenase (1.2.1.57)

>gi j 48S4855 I gta I AAD31841.11 AF132754__1 coenzyme A acyiating aldehyde dehydrogenase [Clostridium bei]erinckii]

MNKDTLIPTTKDLKLKTNVENINLKNYKDNSSCFGVFENVENAINSAVHAQKILSLH YTKEQREKIITEI

RKAALΞNKEVLATMILEETHMGRYEDKILKHELVAKYTPGTEDLTTTAWSGDNGLT WEMSPYGVIGAIT

PSTNPTETVICNSIGMIAAGNAWFNGHPGAKKCVAFAIEMINKAIIΞCGGPENLVT TIKNPTMESLDAI

VFVFENVADDLISNMLKINrNAVIINEDQVSKLIDLVLQKNNETQEyFINKKWVGKD AKLFSDEIDVESPSN IKCIVCEVNANHPFVMTELMMPILPIVRVKDIDΞAVKYTKIAEQNRKHSAYIYSKNIDN LNRFEREIDTT IFVKNAKSFAGVGYEAEGFTTFTIAGSTGEGITSARNFTRQRRCVLAG

NADH-dependent bufanol dehydrogenase B (1.1.1.-)

>gi ; 15896542 ; ref | NP_349891. I | NADK-dependent butanol dehydrogenase B (BDH II) [Clostridium acetobutylicum ATCC 824]

MVDFEYSIPTRIFFGKDKINVLGRELKKYGSKVLIVYGGGSIKRNGIYDKAVSILEK NSIKFYELAGVΞP NPRVTTVEKGVKICRENGVEWLAIGGGSAIDCAKVIAAACEYDGNPWDIVLDGΞKIKRV LPIASILTIA ATGSEMDTWAVINNMDTNEKLlAAHPDMAPKFSILDPTYTYTVPTNQTAAGTADIMSHIF EVYFΞNTKTA YLQDRMAEALLRTCIKYGGIALEKPDDYEARANLMWASSLAINGLLTYGKDTNWSVHLME HELSAYYDIT HGVGLAILTPNWMEYILNNDTVYKFVEYGVNVWGIDKEKNHYDIAHQAIQKTRDYFVNVL GLPSRLRDVG IEEEKLDIMAKESVKLTGGTIGNLRPVNASEVLQIFKKSV etfB _> gi > 15895967 | ref [ NP_349316 . 1 ] electron transfer f lavαprotein subunit beta [ Clostridium acetobutylicum ATCC 824 ]

MNIWCLKQVPDTAEWIDPVKGTLIREGVPSIINPDDKmLEEALVLKDNYGAHVTVI SMGPPQAKNAL VEALAMGADEAVLLTDRAFGGADTLATΞHTIAAGIKKLKYDIVFAGRQAirJGDTAQVG PEIAEHLGIPQV TYVE KVΞVDGDTLKI RKAWΞDG YE WEVKTP VLLT AI KΞLNVPRYMS VE KI FGAFDKE VKMWT ADD I DVD KANLGLKGΞ PTKVKKSSTKEVKGQGEVIDKPVKΞAAAYWSKLKΞΞKYI etfA

>gi ^! 15895966 j ref I NP_349315.1 j electron transfer flavoprotem subunit alpha [Clostridium acetobutylicum ATCC 824]

MNICADYKGVWVFAEQRDGΞLQKVSLELLGKGKEMAEKLGVELTAVLLGHNTEKMS KDLLSHGADKVLAAD NELLAHFSTDGYAKVICDLVNΞRKPEILFIGATFIGRDLGPRIAARLSTGLTADCTSLD IDVΞNRDLLAT RPAFGGNLIATIVCΞDHRPQMATVRPGVPEKLPVNDANVSDDKIEKVAIKLTAΞDIRT KVSKWKLAKDI ADIGEAKVLVAGGRGVGSKENFEKLEELΛΞLLGGTIAASRAAIΞKEWVDKDLQVGQT GKTVRPTLYIACG ISGAIQHLAGMQDSDY11AIKKDVEAPIMKVADLAIVGDVNKWPELIAQVKAANM

Acetolactate synthase (2.2.1.6)

>gi I 15643314 I ref | NP_2283 58 . 1 acetolactate synthase , large subunit [Thermotoga maritima MSB8 ]

MVKVKMKGSKMLFEALLKEGVDTIFGTPGGAI INVYDΞLCNYΞDKINFYLFRHΞQGATHAADGYARVTGK PGWIVTSGPGATNTVTGIATAYMDΞIPIWITGQVPTSFIGTDAFQEVDVTGITMPITKH NHLVTS TEE LPYAI KEMFYVATTGRPGPVLLDFPKDIQTAΞGΞFNYPDTVEIPGYKPTVKGHPKQIKKAVEL LKESKRP WIVGGGANLSGAMDLVNQFIDKFKVPAVSTLMGRGVNPSDEKLYYEGIGMHGTYYGNYAV AN ADLIIAL GVRFSDRILGNPRTFAKNARIVHVD IDPAΞIGKNVRVDVP IVGDLKSVLEΞFLKYEIETDFSDWIEELQE IKKKYPLTYKRDGKLIKPQYWEKVNE VFPDDTVWADVGQNQMWVAQFYKFKHQRΞFLCSGGLGTMGYA LPAGIGAKIGAPDKEVWFAGDGGFQMNIQELMTIKRYNLPVKI IVMDNKALGMVRQWQQLFPNCRYSAT ILSDNPDFAKIAEAVGIKAMRIEKPDQVDΞAIΞKLAKΞ KEPMLIHAWD PAENVLPMVPPGGDVGTPLIΞ APYDE TFVERVLKVI E E SRRGDER

Ketol-acid reductoisotmerase (1.1.1.86)

>gi I 15643316 ! ref NP_228360.l| ketol-acid reductoisomerase [Thermotoga maritima MSB8]

MAVIYYDKDADLKLIKDKKIAIIGYGSQGHAHALNLKDSGLNVWGLRΞGSKSWKKA ΞΞQGLTVKTIΞEA

AKEADIIMILIPDEHQPEIYKKYIEKHLTEGKMLMFAHGFNIKYHQIIPPKKVDVTM IAPKSPGHIVRRE

YVEGRGVPALVAVYQDYTGKAKDIALAYAKGIGVTRAGVIETTFKEΞTETDLFGEQ AVLCGGVTALIKAG

FΞTLVDAGYQPEIAYFECLNELKLIVDLIYEGGLSFMRYSVSNTAEYGDYISQEKI VTKEVRΞNMKQMLK

DIQTGKFAKDWILENQAGRPYFYTMRKKESEHLIEKVGKELRKI^MPWLKERtJVDE E

Dihydroxy-acid dehydratase (4.2.1.9)

>gi [ 15643317 I ref |NP_228361.1 J dihydroxy-acid dehydratase [Thermotoga maritima MSB8]

MRΞDVIKKGLERVPHRSLLKALGITDDΞMRRPFIGIVSSWNEIIPGHVHLDKWEA VKAGVRMAGGVPFV FPTIGICDGIAMDHRGMKFSLPSRΞLIADSIEIVASGPPFDGLVFVPNCDKITPGMMMA MGRLNIPSVLI SGGPMLAGRYNGRDIDLITVFEAVGGYKVGKVDEETLKAIEDLACPGAGSCAGLFTANTM NSLAEALGIA PRGNGTVPAVHAKRLRMAKEAGMLWELVKRDVKPRDIVTLDSFMNAVMVDLATGGΞTNT VLHLKAIAES FGIDFDIKLFDELSRKIPHICNISPVGPYHIQDLDDAGGIYAVMKRLQENGLLKEDVMTI YLRKIGDLVR ΞAKILNEDVIRPFDNPYHKEGGLGILFGNLAPEGAVAKLSGVPEKMMHHVGPAWFEDGE EATKAILΞGK IKKGDVWIRYEGPKGGPGMREMLSPTSAIVGMGLAEDVALITDGRFSGGSHGAVIGHVSP EAAEGGPIG

IVKDGDLIEIDFEKRTLNLLISDEEFERRMKEFTPLVKEVDSDYLRRYAFFVQSASK GAIFRKP Alpha-ketoisovalerate decarboxylase (4.1.1.72)

_> gi ] 51870502 I emb|CAG34226.1] alpha-ketoisovalerate decarboxylase [Lactococcus lactis subsp. lactis]

MYTVGDYLLDRLHELGIΞEIFGVPGDYNLQFLDQIISHKDMKWVGHANELNASYMA DGYARTKKAAAFLT TFGVGELSAVNGLAGSYAENLPWEIVGSPTSKVQNEGKFVHHTLADGDFKHFMKMHEPVT AARTLLTAE NATVEIDRVLSALLKERKPVYINLPVDVAAAKAEKPSLPLKKENΞTSNTSDQEILNKIQ ESLKNAKKPIV ITGHΞIISFGLΞKTVTQFISKTKLPITTLNFGKSSVDEALPSFLGIYNGTLSEPNLKE FVΞSADFILMLG VKLTDSSTGAFTHHLNENKMISLNIDEGKIFNERIQNFDFESLISSLLDLΞEIΞYKGK YIDKKQEDFVPS NALLSQDRLWQAVENLTQSNETIVAEQGTSFFGAΞSIFLKΞKSHFIGQPLWGSIGYTF PAALGSQIADKE SRHLLFIGDGΞLQLTVQELGLAIREKINPICFIINNDGYTVEREIHGPNQSYHDIPMWN YΞKLPΞSFGAT EDRWSKIVRTEKEFVSVMKEAQADPHRMYWIELILAKEGAPKVLKKMGKLFAEQNKS

Glucose dehydrogenase (EC 1.1.1.47)

>gi J 15899711 1 ref j NP_344316 . 1 \ glucose 1 - dehydrogenase ( dhg- l ) [Sul foiobus sol f ataricus P2 ]

MKAI I VKPPNAGVQ VKDVDE KKLDS YGKI KIRTIYNGICGTDRΞ I VEfGKLTLS TLPKGKDFL VLGKΞ AI G WEESYHGFSQGDLVMPVNRRGCGICRNCLVGRPDFCETGEFGEAGIHKMDGFMREWWYI ⁾ DPKYL VKIPK SIΞDIGILAQPLADIEKΞIEEILΞVQKRVPVWTCDDGTLNCRKVL WGTGPIGVLFTLLFRTYGLEVWMA KrRREPTEVEQTVIEETKTNYYNS SNGYDKLKDSVGKFDVIIDATGADVNILGNVIPLLGRNGVLGLFGFS TSGSVPLDYKTLQEI VHTNKTIIGL VNGQKPHFQQAWHLASWKTLYPKAAKMLITKTVS INDEKELLKV LREKEHGEIKIRILWE

Glucoπolactonase (EC 3.1.1.17}

>gi I 48477979 [ ref I YP_023685.11 gluconolactonase [Picrophilus torridus DΞM 9790]

MELNIEPQRLGESPIYIRELDTFLWVDILNGDIFΞYNGNAKMΞMHVNDMITSISP YKGTEVIASLRDGIA 11DWKNKITNTLLKLDFPENIRFNDGRCDARGRFFIGTMDMNEKEPLGALYKFSGRKLER VLDNVTISNG lAWSLDSRFMYYIDSPRKΞVQVFDYDLSMGRITRHLYDIDLKNYSGVPDGMAIDINNNL WVAIHGSSLIΞ VIDPAKNEILNEVKIAAKKVTΞCTFGΞVNMDKLFVTSAYDGTGGIPFIIDTGΞRGVE LNRYIP

Gluconate dehydratase (EC 4.2.1.39)

>gi I 15899900 I ref iNP__344505.11 muconate cycloi somerase related protein [Sulfoiobus solfataricus P2]

MRIREIEPIVLTSKEKGSATWASIMIVTRVITENGEVGYGEAVPTL ₁ RVI SVYWΆIKQVSKAYIGKEVEEV EKNYHEWYKQDFYLARSFESATAVSAIDIASWDIIGKΞLGAPIHKLLGGKTRDRVPVYA NGWYQDCVTPE EFAΞ KAKD WKMGYKALKFDPFGPYYDWIDERGLRΞAΞERVKAVREAVGDNVDILIEHHGRFN ANSAIMI AKRLE KYNP GFMΞΞ PVHKEDVI GLRKYKAS THLR VALGERL I S E KE TAFYVE E GLVNI LQ PDLTN I GGVT VGRSVIKIAEANDVEVAFHNAFGS IQNAVEIQLSAVTQNLYLLENFYDWFPQWKRDLVYNETPVEGGHVK VPYKPGLGVSINEKI IBQLRAEPIPLDVIΞEPVWWKGTWKNYGV

2-keto-3-deoxy gluconate aldolase (EC 4.1.2.14)

>gi | 15899899 | re f | NP_344504 . l | 2 ~ keto ~ 3 - deoxy gluconate aldolase ( eda ) [ Sul foiobus solfataricus P2 ]

MGRQGNLEELWCLRMPEI ITPIITPFTKDNRIDKΞKLKIHAENLIRKGIDKLFVNGTTGLGPSLSPEEKL ENLKAVYDVTNKI I FQVGGLNLDDAIRLAKLS KDFDIVG I AS YAPYYYPRMS EKHL VKYFKTLCEVS PHP VYL YNYPTATGKDIDAKVAKEIGCFTGVKDTIΞNIIHTLDYKRLNPNML VYSGSDMLIATVAS TGLDGNV AAGSNYLPEVTVTIKKLAMERKIDEALKLQFLHDEVIEASRIFGSLSSNYVLTKYFQGYD LGYPRPPIFP LDDEEERQLIKKVEGIRAKLVELKILKE

Aldehyde dehydrogenase (EC 1.2.1.3)

>gi I 21238945 [dbj |BAB96577.11 aldehyde dehydrogenase [Flavobacterium frigiditπaris]

MSNTIQRPEFKAKYDNYINGKFTAPVKGEYFDVLSPIDGKVFTKAAHSGKEDLELAV DAAYEAFKTWGKT SVTERSILLNKIAQKIEDNLEYIATVETIDNGKPIRETLAADIPLAIDHFRYFAGVIRAE ΞSSIAΞLDSQ TVSIALSΞPLGWAQIIPWNFPILMAVWKIAPALAAGNTIVLKPAESTPIΞILVLMΞL IGDILPPGVLNI VNGFGAELGRPLVTNKKVAKAAFTGSTTTGRLVMQYATENIIPVTLELGGKSPNIFFPSV ADHDDDFFDK AIEGAVLFALNQGΞICTCPSRLLIHΞDIYEKFIARVIERTEAIIAGNPLDKSTMIGAQ TSLVQKEKIMSY IKLGKEΞGAELLTGGDEKfHLGGDLEGGYYIKPTLFKGHNKMRIFQΞEIFGPVLAVTT FKTTEΞAIEIAND TMYGLGAGVWTRDAHEIYQVPRAIQSGRVWINQYHSYPAGAPFGGYKQSGIGRENHKMML GQYRQTKNML ISYDKKKLGFF

Glycerate kinase (EC 2.7.1.-)

>gi [15897575 I ref [NP_342180.1 j glycerate kinase, putative [Sulfoiobus solfataricus P2]

MDIVDKILΞYTDPYKALQΞKVRVYNNILLFNNEKIPFKKPILISIGKASLPMARF FRERMELKAKLIVTP

KGTNGKENDVIEAGHPLPDENSIKAGKRMIELLANEDYDLVIFAIΞGGASALVEYS ΞIPLDELKIINKVL

VTSGLGINKINIVRKHLSKVKGGKILEYVKDKIPIVSFIVSDVPGNDIΞΞIGSGL TΞIDNSSNDDALEIL

KAIGLBKYSKYLTBTPKSFSRIVKNYIILDNMEVLRKLANTLVNSFILTSEIRGEAR DVGAIIASIYNSS ΞΞYNIPPRRPYYLLVGGΞPΞVTIQGKAGKGGRNGEVCLSFLKYAKKRNRFΞLLGF ATDGIDGNΞΞYAGCK VSSDMEIRSDEINNALETHNSYGLLΞSHKAVIKTGYTHTNVNNIYVLRAP

Enolase (EC 4.2.1.11)

>gi | 15897800 I ref |NP_342405.11 enolase [Ξulfolobus solfataricus P2] MINRFΞIEKVKGLΞIVDΞRGNPTIRVFIRTSDGVESFGDAPAGASKGTRΞAVEVRD ΞNGLTVKRAVDIVN YTIDPALHGIDVREQGIIDKLLKDIDSTENKΞKLGGNTIIATSIAALKTASKALGLΞV FKYISGPRLPKI

PIPLLNIINGGLHAGNKLKIQEFXIVPIKFNTFKEΆLFAΛIDVYRTLKGLITER� �GKIYTAVGDEGGFΞP PLEDTREALDLIYTSINNAGYEGKIYMGMDAAGSDFYDSKKEKYIIDGRELDPNQLLEFY LDLVKQYPIV YLEDPFEΞNSFDMFSQLQNKLSΞTIITGDDLYTTNIKYLKIGIEKRSTKGVIVKPNQV GTISETFEFTNL ARRNSMKLITΞHRSGETEDΪSΓFIADFAVGIESDFIKVGAPARGERTSKYNKLLEIE NKFGLEYEGKYFYL Pyruvate Kinase (EC 27140)

>gi 115897860 I ref |NP_342465.11 pyruvate kinase (pyK) [SuIfolobus solfataricus P2]

MRKTKIVATLGPSSEEKVKELAΞYVDVFRINFAHGDΞTSHRKYFDLIRTYAPESS IIVDLPGPKLRLGΞL KEPIEVKKGDKIVFSQKDGIPVDDELFYSAVKENSDILIADGTIRVRVKSKAKDRVΞGT VIEGGILLSRK GINIPNVNLKSGITDNDLKLLKRALDLGADYIGLSFVISENDVKKVKEFVGDEAWVIAKI EKSEALKNLT NIVNESDGIMVARGDLGVETGLEKLPLIQREIVRTSRVPGKPVILATQVLTΞMINSPIP TRAE11DISNS IMQGVDSIMLSDETAIGNYPVΞΞVRTLHNIISNVEKSVKHRPIGPLNSEΞDAIALAA VNAΞKVSKADVXV VYSRSGMΞILRVΞRLRPERNIIGVSPDPRLAKKFKLCYGVIPIΞINKKMQSIDΞII DVSAKLMQEKIKDL KFKKIVIVGGDPKQEAGKTNFVIVKTLEQQKK

Glycerate dehydrogenase/Hydroxypyruvate reductase (EC 1 1 1 29 bzw 1 1 1 81 )

>gi ] 48478188 J ref I YP_023894.11 glycerate dehydrogenase [Picropϊiilus torridus

DSM 9790]

MKVSVQVNSYYNNEMSKKLLELCRDITGLDAVPGFDDDAEIILFΞGRPVPGKKTKF MQSLSAGVNHLDFS

KIPDNIIIASNADAYSIPVAETAIGLMLAWARKICISNYKTIHNNNΥKRLDYKEYV SLYNKSLGILGYGGI

GRRTALIAKSFGMNIYAYSRSYKNDGISSYMEPEDIMKKSDFVLIΞLPLTKETANS INDKMLSLFRGLAI

INVGRAGWDRNΞMLNFLRNHNDKYYLTDVWWNΞPIINENIPDNVIITPHSAGMSD NIYQPAVAAIENIK

NYINGKPKNIVKRSDYI

Serine-pyruvate transaminase (EC 2 6 1 51 )

>gi I 15899324 ' ref NP_343929.11 Serine -pyruvate aminotransferase (agxT) [Sulfolobus solfataricus P2]

MDKLLLHVGPTTIKEDVLVAGLENNVGFTSKEFVEALAYSLKGLRYVMGASKNYQPL IIPGGGTSAMESV TSLLKPNDKILWSNGVFGDRWEQIFKRYPVNVKVLRPSPGDYVKPGEVEESVRKSEYKLV AL ₁ THVETST GVREPVKDVIKKIRKYVΞLIWDGVSSVGAEEVKAEEWNVDVYLTASQKALGΞAAGLGL LLLΞPKALSIL DSQNSlAGYYLDLRNWLPVMRGAEEGKAAYFATPPVHVILQLAΞAFRLIEKEGIΞNRI KRHTMVASAIRA GLEALGLEIVARRPEΞYSNTVTGVILKVADPQKVLAGTVNEGVEFAPGVHPAFKYFRIG HMGWVTPNDAI lAISVIΞRTLRKLGEPrRFGEGVKAVEEVLFΞAR

L-Seπne ammonia-lyase (EC 4 3 1 17)

>gi I 55980998 _f ref YP_144295 l| L-seπne dehydratase, alpha subumt [Thermus thermophilus HE8]

MPLTLNQLAELSGRASEHVLAEΞVEETGTPAEΞILARLRΞRLAVMRDSVRRGLA SDAPSVAGLVGKNAKT

LWEAPDPLQDPLLKRVQAYAMAVNEENARMGRIVAAPTAGSAGTLPGALLGVADHLG IPDEELLMPLVLA

GGVAKMIGRVIHIAGASGGCQAEIGSΞAAMAAAAVTELLGGTPEACAHAAALALQN TLGLVCDPVGGFVE

VPCVMRNGFYAVHAVΞAASMALAGIRSVIPPDEWLAMAGIGRLLPLELKETGLGGL ADTPTGRRLAEEA

LKKT

>gi I 55980708 J ref i YP_144005 l | L- serine dehydratase , beca subumt [Thermus thermophilus HB8 ]

MGLLDMIGPVMVGPSSSHTAGACRLALLZI ₁ RHLLGEKPKRVEPGLHGS FAKTGKGHGTHLALAAGVLGLTP

DDERL KESLΞLASREGVEWFKEVELGDVHPNTVRMVLEGEKERLAVTGΞSLGGGLVRVFDVDG FEVRIT

GΞAPTLVIΪ^n/DTPGVVARVARILADDΞVNIAYLTVΞRKKRGGΞAMMSIEV DRPLSEVPLRYLEHLSYIL

WVRQIPPVMG

Alanine dehydrogenase (EC 1.4 1 1 ) >gi j 46200072 | re f | YP__005739 . 1 alanine dehydrogenase [Thermus thermophilics HB27 ]

MVI GVPREIKTLENRVALTPGGVESLVRRGKTVL VERGACΞGΞGLSDAEYARAGAΞLVGREΞAWGABMW KVKEPLPEΞYGFLREGLILFTYLHLAADRGLTΞAMLRSGVTG IAYE TVQLPDGTLPLLVPMSEVAGRMAP QVGAQFLΞ K P KGGRGVLLGGVPGVAP ASWILGGGTVGTNAAKIALGMGAQVTILDVNHKRLQYLDDVFG GRWT LT ATE AMI KKS VQH ADLL I GAVL VPG AKAPKL VTRDMLS LMKEGAVI VDVAVDQGGC VE TIRPTT HAE PTYVVDG WHYG VANMPGAVPRTS T FALTNQTL PYVL KLAEKGLDALLEDAALLKGLNTHKGRLTH P GVAEAFGLP YTP PE EALRG

Dihydroxyacid dehydratase {EC 4.2.1.9)

>gi I 15899814 | ref |NP_344419.1 dihydroxy-acid dehydratase [SuIfolobus solfataricus P2]

MPAKLNSPSRYHGIYNAPHRAFLRSVGLTDΞEIGKPLVAIATAWSEAGPCNFHTLA LARVAKEGTKEAGL SPLAFPTMWNDNIGMGSEGMRYSLVSRDLIADMVEAQFNAHAFDGLVGIGGCDKTTPGIL MAMARLNVP SIYIYGGΞAEPGYFMGKRLTIEDVHEAIGAYLAKRITENELYEIEKRAHPTLGTCSGLF TAKTTMGSMSEA LGMALPGSASPTATSΞRRVMYVKETGKALGΞLIENGΣKSREILTFEAFΞNAITTLM AMGGSTNAVLHLLA IAYEAGVKLTLDDFNRISRRTPYIASMKPGGDYVMADLDEVGGVPWLKKLLDAGLLHGDV LTVTGKTMK QNLEQYKYPNVPHSHIVRDVKNPIKPRGGIVILKGSLAPEGAVIKVAATNWKFEGKAKVY NΞEDDAFKG VQSGEVSEGEWIIRYEGPKGAPGMPEMLRWAAIMGAGLNNVALVTDGRFSGATRGPMVGH VAPEAMVG

GPiAiVEDGDT IVIDVΞSERLDLKLSEEEIKNRLKRWSPPΞPRYKSGLLAKYAΞLVSQASMGΆVTRP A Pyruvate Synthase (1.2.7.1)

>gi j 15898059 ; ref ' NP_342664 . 1 1 pyruvate synthase alpha chain ( Pyruvic - ferredoxin oxidoreductase alpha chain) (porA- l ) [Sulfolobus solf ataricus

P2 ]

MQVLKRKVLALVGNKAVAYAVKQAKPKVLAVFPITPQTTMLEKLSEYIΞΞEELKA ELIKVESEHSALASI

YGAALAGARVFTATSSQGLLYMTΞMIYWAGGQRVPIVAAVATRAIAEPWSIWDDHQ DFVSKRDAIWIQIM

AΞNVQΞAYDMTIQAFRISEDΞRVILPVMMGFDGFILTHTMERIEVLEDNEVDNF LPPRQFNLIDFSDPIA

IGPIATPEEYIKYRYEAMKAMERAKGVIEEIMGEYERISGRKQHGLVECYKCEDAKY VFVTMGAWSGDGK

AAVDRLRDSGVKTGLLKIRVFRPFPKΞKVEEYLRSMKGVWFDRAYSYGYGGILVNE IKAALYGYRVPVY

SWAGIGGKDVRPRHFQKVIEDLINDNLEEERWLF

>gi | 15898058 [ ref |NP_342663.11 2-ketoisovalerate ferredoxin oxidoreductase subunit beta EΞulfoiobus solfataricus P2]

MAVLSSQVTPKRMPKFYRGNAACPGCPIPKELDVALΞVLGNKTVLWPASCTTIIMG DTNGMPSTVPWH

SAFGAAAAIGSGIVRSLRMRGDNDAIVAVWAGDGSTGDIGFAAVSGAAERKFEDILY ICYDNΞAYMNTGIQ

RSGLTPKGAWTTTTPEGKRΞVKKPLPFIIAEHKVPYVATASIAYIYDYEAKMRKAK QIRGFRYIHLLSPC

PPGWRFDSKLTIDIAKLAVETGVWPLFΞIΞNGΞFKLTSVSKTLVEKKNRKPVAE YLKLQGRFKQLTEEQI

KGIQEEIDEMWEEIKRLIKK

>gi j 15898061 J ref JNP_342666.1 I pyruvate synthase gamma chain (Pyruvic- ferredoxin oxidoreductase gamma chain) (porG-1) [Sulfolobus solfataricus

P2]

ME YYNCDKMTLIΞLALRGRGGQGIVTAGELMT KAMVLEDKYAQS I PFFGGERRGAPWSFVRLSDKPILL

HREVYKPDGVAI FDVSMI QLIWTEGLKENGFLLLNTNTPKRIW:mEYVVDATNIAKELGLIVAGWAIVKr

TAMIGALARILGQPΞLHΞLEEAVKEEFPGKIGELNAΞAVEIGYKEVKRVD

>gi 1 15898912 I ref j NP_343517 , 1 ! pyruvate synthase delta chain ( Pyruvic - ferredoxin oxidoreductase delta chain) (porD- Iike ) [Sulfolobus solfataricus

P2 ]

MGVKDEVKYVEIVYRGIFQKRLAKYIAEGIVYTAREMGRPALSFGRYGDSPERNGVP AKYYVGIGDGVNE

EDLIGYSTRVEPDLVDVIIVLDDTLLKGVEΞWAWQGVQPINLKLKSKTGTMLVTST KRINELLKMIPKKDF

NWTLGVIKTEPSFSGLWAFKDDLTMEKVWGGLAKLRPDIIDLDHLLKYVTKKQDANK RVSAVREAYSSVD

YRTVMKGEGIDFVYNPPRLLTWQEMLEGTVIPAVPRGKRNELFKRGTTKFΞRPTVD FDTCIKCKLCWVYC

PDECFDETPDGYYDIAYDYCVGCGICAEVCPVKDCIVMVDΞSMFTDYRRPYEMWKE DKAKYKEWLKTVRQ

ARKΞRVYVPGLG5.

NADH ferrodoxin reductase (1.1 S, 1.3)

>gi I 15398077 I ref iNP_3426S2.11 toluene 1 , 2 -dioxygenase system ferredoxin-- NAD (+) reductase component, (todA) [Sulfolobus solfataricus P2] MKCEYLIIGSGIAGYNALKELLQIKPNSRIIMITSDKYYPYDRPPLSKDYLKGKLEKDML ₁ FFESr)DFYKR DNLEVMLNKSVΞRIDANLKEAILNDGSVISFDKALISTGGRPRRLNIPGSΞNALYLRS LDDADRIRΞAAS KGKNALIIGAGFIGVEVASSLITLGVKTTWEVMPYIWNTFVDEKVSRVIQQYLESKGINF ILNΞSVKΞI QGKIATTΞΞGRKIEADMFLIAVGISPNVELAQRSGMQVDNGIWNΞYLETSARDIYAA GDIANIFDPREG KRKRIEHWNNAEYTGKLAARNMAGSRΞAYNFIΞSIWSDIFDLHIESAGETRNYDΞYI IRGKFΞLERPRFS VIYLKGGIIKGYLAINRNVKEIIALKKLIQKQADVSSKRDKLADESFDLQKLLI

Acetoiactate Synthase (2.2.1.6) (large subunit)

>gi I 15897497 I ref lMP_342102.11 acetoiactate synthase catalytic subunxt [Sulfolobus solfataricus P2]

MPTGARILVDSLKRΞGVKWFGIPGLΞNMQIYDAFVΞDLANGΞLRHVLMRHEQA AAHAADGYARASGVPG VCTATSGPGTTNLTTGLITAYWDSSPVIAITGNVPRSVMGKMAFQΞADAMGVFENVTKY VIGIKRIDEIP QWIKNAFYIATTGRPGPVWDIPRDIFYEKMEEIKWPEKPLVKGYRDFPTRIDRLALKKAA EILINAERP

GARFSDRTFTSYDEMVETRKKFIMVNIDPTDGΞKAIKVDVGLYGNAKIILRΞLIK AIITLGQKRDKΞAWI KRVKEYKΞYYSQFYYTKΞNGKLKPWRIMRTIRQSLPRDAIVTTGVGQHQMWAEVFWEV LEPRTFLTSSGM GTMGFGLPAAMGAKLARPDKIWDLDGDGSFLMTGTNLATAVDΞKIPVISVIFDNRTLGL VRQVQDLFFG

KQVILRDPRKSS

Ketαl-acid reductoisomerase (1.1.1.86)

>gi | 15897495 | ref |NP_342100.1 | ketol-acid reductoisomerase [Sulfolobus solfataricus P2]

MKCTSKIYTDNDANLDLIKGKRIAVLGYGSQGRAWAQNLRDSGLNWVGLΞDLGKSW ELAKSDGITPLHT

KDAVKDADIIIFLVPDMVQRTLWLESVQPYMKKGAELVFAHGFNIHYKIiIDPPKDS DVYMIAPKGPGPTV

REYYKAGGGVPALVAVHQDVSGTALHKALAIAKGIGΆTRAGVIPTTFKEETETDLF GEQVILVGGIMELM

RAAFETLVEEGYQPEVAYFETINELKMLVDLVYEKGISGMLKAVSDTAKYGGMTVGK FVIDESVRKRMKE ALQRIKSGKFAΞΞWVEEYGRGMPTWNGLSNVQNSLEΞKI GNQLRDLVQKGKPKS

AScohoi Dehydrogenase (1.1 ,1.1 )

>gi |20502556 j db] [BAB91411.11 alcohol dehydrogenase [Flavobacteπum fngidimaris]

MLPKTMKAAVIRΞFGSLLKIEEVEVKRPGRNEILVKVIASGVCHTDLHAVEGDWPV KPKMPLIPGHBAVG YWAVGQEVKNVKEGDAVGVPWLYSACGGCDQCITGWETLCDTQQNGGYSVDGGFAEYVIA DARYVGLLP SNVNFMEMAPILCAGVTVYKGLKETEVKPGEWVAISGIGGLGHVAVQYAKAMGMHVAAID VADDKLDLAK KLGADLWNAKNQNPGEFLKKEVGGMHGALITAVΞPIAFKQGLETLRRKGTMALNGLPPG NFDLSΪFDTV LNRITIRGSIVGTRKDMKΞAIEFAVEGKVKATVTPAKLENINEVFDKMKKGQIΞGRWL EIAKA

Alpha-acetoiactate decarboxylase (4.1.1.5)

>gi I 113592 i sp j P23616.1 | ALDC_BREBE RecName: Full=Alpha-acetolactate decarboxylase; Short=ALDC; Flags: Precursor

MKKNIITΞITSLALVAGLΞLTAFAATTATVPAPPAKQΞΞKPAVAANPAPKHVL FQYSTINALMLGQFΞGD

LTLKDLKLRGDMGLGTINDLDGEMIQMGTKFYQIDSTGKLSΞLPΞΞVKTPFAVT THFEPKΞKTTLTNVQD

YNQLTKMLEEKFΞNKNVFYAVKLTGTFKMVKARTVPKQTRPYPQLTEVTKKQSEFE FKNVKGTLIGFYTP

NYAAALNVPGFHLHFITEDKTΞGGHVLNLQFDNANLEIΞPIHEFDVQLPHTDDFA HSDLTQVTTSQVHQA

ESERK

Alcohole dehydrogenase (1.1.1.1 bzw. 1.1.1.4)

>gi 1 18978332 ' re f | NP_5 7 96 8 9 . 1 1 aldose reductase [ Pyrococcus furiosus DSM

363 8 ]

MKRWAFNDLKRIGDDKVTAIGMGTWGIGGRΞTPDYSRDKEΞIΞAIRYGLELGMN LIDTAEFYGAGKAΞE

IVGΞAIKEFEREDIFIVΞKVWPTHFGYEEAKKAARASAKRLGTYIDLYLLHWPVD DFKKIΞETLHALEDL

VDEGVIRYIGVSNFNLELLQRSQEVMRKYEIVANQVKYSVKDRWPΞTTGLLDYMKR EGIALMAYTPLΞKG

TLARNECLAKIGEKYGKTAAQVALNYLIWEENWAIPKASNΪCEHLKENFGAMGWRL ΞEEDREMARRCV

Dio! dehydratase (4.2.1.28)

>gi 116334194 j re f \ YP__795721 . 1 | propanedio l dehydratase , small subunrt [Lactobac il lus brevi s ATCC 367 ] MSΞIDDLVAKIVQQIGGTEAADQTTATPTSTATQTQHAALΞKQDYPLYSKRPBLVHSP ΞGKALNDITLDKF VLNDDIKANDLRITPDTLRMQGEVANDAGRDAVQRNFQRASELTSIPDDRLLΞMYNALR PYRSTKAELLA

ISAΞLKDKYHAPVNAGWFAEAADYYESRKKLKGDN

>gi j 11633419S j ref I YP_795722.1 | did dehydratase medium subunxt

[Lactobacillus brevis ATCC 367]

MAQEIDENLLRNIIRDVIAETQTGDTPIΞFKADAPAASSATTATAAPVNGDGPEPE KPVDWFKHVGVAKP GYSRDEWΣAVAPAFAEVNDHNLTGISHKEILRQMVAGIEEEGLKARIVKVYRTSDVSFC GAEGDHLSGS GIAIAIQSKGTTIIHQKDQEPLSNLELFPQAPVLDGDTYRAIGKNAAEYAKGMSPSPVPT VNDQMARVQY QALSALMHIKETKQVVMGKPAEQXEVNFN >gi | 116334196 | ref |YP_795723.1 j propanediol dehydratase, large subunit

[Lactobacillus brevis ATCC 367]

MKRQKRFESLEKRPIHLDGFVKEWPEEGFVAMMGPNDPKPSIKIENGKVTEMDSKPA ADFDI ₁ IDLYIAKY GIKLENAEKVMAMDSTKIANMLCDPNVPRKDIIEITTAMTPAKAEEVISKLNFAEMIMAT QKMRPRRTPA TQCHVTNIRDNPVQIAADAADAALRGFPΞQΞTTTAVARYAPLNAIΞLMVGAQTGRPG VITQCSVΞEAΞEL SLGMRGFTGYAETISVYGTDKVFTDGDDTPWSKGFLASCYASRGLKMR.FTSGSGSEVMM GYTEGKSMLYL ESRCIFITKASGVQGLQNGGVSCIGIPGSVPSGIRSVLGENLLCKMLDLECASANDQAFS HSDMRRTERL LGQFIAGTDYISSGYΞSTPNYDNTFAGSNTDGLDYDDYYVMΞRDLAINGGIHPVDEQT 11KARNKAARAL QGVFEDLGLPKITDEEVEAATYANTSKDMPΞRNMVEDMKAAQDLMDRGITGVDIVKALF NHGFKDVAQAV LDLQKQKVCGDFLQTSAIFDSIOTHVISAVNDANDYQGPGTGYRLEΞDTEΞWΞRIKN LPFAIDPQNMQL